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Sample records for imaging microscopy flim

  1. Studying membrane properties using Fluorescence Lifetime Imaging Microscopy (FLIM)

    NARCIS (Netherlands)

    Stöckl, M.T.; Bizzarri, R.; Subramaniam, Vinod; Mely, Y.; Duportail, G.

    2012-01-01

    Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to investigate the structure and composition of biological membranes. A wide variety of fluorescent probes suitable for FLIM experiments have been described. These compounds differ strongly in the details of their incorporation into

  2. phiFLIM: a new method to avoid aliasing in frequency domain fluorescence lifetime imaging microscopy.

    NARCIS (Netherlands)

    van Munster, E.B.; Gadella, Th.W.J.

    2004-01-01

    In conventional wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM), excitation light is intensity-modulated at megahertz frequencies. Emitted fluorescence is recorded by a CCD camera through an image intensifier, which is modulated at the same frequency. From images recorded

  3. Nuclear uptake of ultrasmall gold-doxorubicin conjugates imaged by fluorescence lifetime imaging microscopy (FLIM) and electron microscopy

    Science.gov (United States)

    Zhang, Xuan; Shastry, Sathvik; Bradforth, Stephen E.; Nadeau, Jay L.

    2014-11-01

    Fluorescence lifetime imaging microscopy (FLIM) has been used to image free and encapsulated doxorubicin (Dox) uptake into cells, since interaction of Dox with DNA leads to a characteristic lifetime change. However, none of the reported Dox conjugates were able to enter cell nuclei. In this work, we use FLIM to show nuclear uptake of 2.7 nm mean diameter Au nanoparticles conjugated to Dox. The pattern of labelling differed substantially from what was seen with free Dox, with slower nuclear entry and stronger cytoplasmic labelling at all time points. As the cells died, the pattern of labelling changed further as intracellular structures disintegrated, consistent with association of Au-Dox to membranes. The patterns of Au distribution and intracellular structure changes were confirmed using electron microscopy, and indicate different mechanisms of cytotoxicity with stable Au-Dox conjugates compared to Dox alone. Such conjugates are promising tools for overcoming resistance in Dox-resistant cancers.Fluorescence lifetime imaging microscopy (FLIM) has been used to image free and encapsulated doxorubicin (Dox) uptake into cells, since interaction of Dox with DNA leads to a characteristic lifetime change. However, none of the reported Dox conjugates were able to enter cell nuclei. In this work, we use FLIM to show nuclear uptake of 2.7 nm mean diameter Au nanoparticles conjugated to Dox. The pattern of labelling differed substantially from what was seen with free Dox, with slower nuclear entry and stronger cytoplasmic labelling at all time points. As the cells died, the pattern of labelling changed further as intracellular structures disintegrated, consistent with association of Au-Dox to membranes. The patterns of Au distribution and intracellular structure changes were confirmed using electron microscopy, and indicate different mechanisms of cytotoxicity with stable Au-Dox conjugates compared to Dox alone. Such conjugates are promising tools for overcoming resistance in

  4. Two-Photon Microscopy (TPM) and Fluorescence Lifetime Imaging Microscopy (FLIM) of Retinal Pigment Epithelium (RPE) of Mice In Vivo.

    Science.gov (United States)

    Miura, Yoko

    2018-01-01

    Retinal pigment epithelium (RPE), a monolayer of epithelial cells located between the neural retina and the choroid, plays a significant role in the maintenance of retinal function. Its in vivo imaging is still technically challenging in human eye. With the mouse eye, there is a possibility to look into the RPE through the sclera using two-photon microscopy (TPM). TPM is a two photon-excited nonlinear fluorescence microscopy that enables the observation of deep tissues up to several hundred micrometers. Since the simultaneous absorption of two photons occurs only at the focal plane, spatial resolution of the TPM is quite high, such that pinhole as used in a confocal microscope is not necessary. TPM enables observation of autofluorescence at the cellular level, and thus may provide new insights into the fluorescent molecules in/around RPE cells.The combination of TPM with fluorescence lifetime imaging microscopy (FLIM) may expand the breadth of information about cells and tissues. Fluorescence lifetime is a fluorophore-specific property, which is independent of fluorescence intensity and changes with the alteration of molecular environment. FLIM may have therefore the potentials to distinguish different fluorophores and to indicate the change in the environment of a fluorophore. Some energy metabolisms-related intracellular fluorophores, such as NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), show characteristic fluorescence lifetimes that shift under different molecular environments, and thus their fluorescence lifetime have been used to indicate cell energy metabolic states. These nonlabeling imaging methods offer us the opportunity to engage in the study of the RPE in vivo as well as in vitro both in morphological as well as metabolic aspects.

  5. Fluorescence Lifetime Imaging Microscopy (FLIM) as a Tool to Investigate Hypoxia-Induced Protein-Protein Interaction in Living Cells.

    Science.gov (United States)

    Schützhold, Vera; Fandrey, Joachim; Prost-Fingerle, Katrin

    2018-01-01

    Fluorescence resonance energy transfer (FRET) is widely used as a method to investigate protein-protein interactions in living cells. A FRET pair donor fluorophore in close proximity to an appropriate acceptor fluorophore transfers emission energy to the acceptor, resulting in a shorter lifetime of the donor fluorescence. When the respective FRET donor and acceptor are fused with two proteins of interest, a reduction in donor lifetime, as detected by fluorescence lifetime imaging microscopy (FLIM), can be taken as proof of close proximity between the fluorophores and therefore interaction between the proteins of interest. Here, we describe the usage of time-domain FLIM-FRET in hypoxia-related research when we record the interaction of the hypoxia-inducible factor-1 (HIF-1) subunits HIF-1α and HIF-1β in living cells in a temperature- and CO 2 -controlled environment under the microscope.

  6. Referencing techniques for high-speed confocal fluorescence lifetime imaging microscopy (FLIM) based on analog mean-delay (AMD) method

    Science.gov (United States)

    Kim, Byungyeon; Lee, Minsuk; Park, Byungjun; Lee, Seungrag; Won, Youngjae

    2017-02-01

    Analog mean-delay (AMD) method is a new powerful alternative method in determining the lifetime of a fluorescence molecule for high-speed confocal fluorescence lifetime imaging microscopy (FLIM). Even though the photon economy and the lifetime precision of the AMD method are proven to be as good as the state-of-the-art time-correlated single photon counting (TC-SPC) method, there have been some speculations and concerns about the accuracy of this method. In the AMD method, the temporal waveform of an emitted fluorescence signal is directly recorded with a slow digitizer whose bandwidth is much lower than the temporal resolution of lifetime to be measured. We found that the drifts and the fluctuations of the absolute zero position in a measured temporal waveform are the major problems in the AMD method. As a referencing technique, we already proposed dual-channel waveform measurement scheme that may suppress these errors. In this study, we have demonstrated real-time confocal AMD-FLIM system with dual-channel waveform measurement technique.

  7. Wide-field time-domain fluorescence lifetime imaging microscopy (FLIM): Molecular snapshots of metabolic function in biological systems

    Science.gov (United States)

    Sud, Dhruv

    2008-12-01

    Steady-state fluorescence imaging is routinely employed to obtain physiological information but is susceptible to artifacts such as absorption and photobleaching. FLIM provides an additional source of contrast oblivious to these but is affected by factors such as pH, gases, and temperature. Here we focused on developing a resolution-enhanced FLIM system for quantitative oxygen sensing. Oxygen is one of the most critical components of metabolic machinery and affects growth, differentiation, and death. FLIM-based oxygen sensing provides a valuable tool for biologists without the need of alternate technologies. We also developed novel computational approaches to improve spatial resolution of FLIM images, extending its potential for thick tissue studies. We designed a wide-field time-domain UV-vis-NIR FLIM system with high temporal resolution (50 ps), large temporal dynamic range (750 ps -- 1 mus), short data acquisition/processing times (15 s) and noise-removal capability. Lifetime calibration of an oxygen-sensitive, ruthenium dye (RTDP) enabled in vivo oxygen level measurements (resolution = 8 muM, range = 1 -- 300 muM). Combining oxygen sensing with endogenous imaging allowed for the study of two key molecules (NADH and oxygen) consumed at the termini of the oxidative phosphorylation pathway in Barrett's adenocarcinoma columnar (SEG-1) cells and Esophageal normal squamous cells (HET-1). Starkly higher intracellular oxygen and NADH levels in living SEG-1 vs. HET-1 cells were detected by FLIM and attributed to altered metabolic pathways in malignant cells. We performed FLIM studies in microfluidic bioreactors seeded with mouse myoblasts. For these systems, oxygen concentrations play an important role in cell behavior and gene expression. Oxygen levels decreased with increasing cell densities and were consistent with simulated model outcomes. In single bioreactor loops, FLIM detected spatial heterogeneity in oxygen levels as high as 20%. We validated our calibration

  8. Atherosclerosis staging: imaging using FLIM technique

    Science.gov (United States)

    Sicchieri, Leticia B.; Barioni, Marina Berardi; Silva, Mônica Nascimento; Monteiro, Andrea Moreira; Figueiredo Neto, Antonio Martins; Ito, Amando S.; Courrol, Lilia C.

    2014-03-01

    In this work it was used fluorescence lifetime imaging (FLIM) to analyze biochemical composition of atherosclerotic plaque. For this purpose an animal experimentation was done with New Zealand rabbits divided into two groups: a control group of 4 rabbits that received a regular diet for 0, 20, 40 and 60 days; and an experimental group of 9 rabbits, divided in 3 subgroups, that were fed with 1% cholesterol diet for 20, 40 and 60 days respectively. The aortas slices stained with europium chlortetracycline were analyzed by FLIM exciting samples at 440 nm. The results shown an increase in the lifetime imaging of rabbits fed with cholesterol. It was observed that is possible to detect the metabolic changes associated with atherosclerosis at an early stage using FLIM technique exciting the tissue around 440 nm and observing autofluorescence lifetime. Lifetimes longer than 1.75 ns suggest the presence of porphyrins in the tissue and consequently, inflammation and the presence of macrophages.

  9. Imaging cytochrome C oxidase and FoF1-ATP synthase in mitochondrial cristae of living human cells by FLIM and superresolution microscopy

    Science.gov (United States)

    Foertsch, Franziska; Ilchenko, Mykhailo; Heitkamp, Thomas; Noßmann, Silke; Hoffmann, Birgit; Starke, Ilka; Mrowka, Ralf; Biskup, Christoph; Börsch, Michael

    2017-02-01

    Cytochrome C oxidase and FoF1-ATP synthase constitute complex IV and V, respectively, of the five membrane-bound enzymes in mitochondria comprising the respiratory chain. These enzymes are located in the inner mitochondrial membrane (IMM), which exhibits large invaginations called cristae. According to recent electron cryotomography, FoF1-ATP synthases are located predominantly at the rim of the cristae, while cytochrome C oxidases are likely distributed in planar membrane areas of the cristae. Previous FLIM measurements (K. Busch and coworkers) of complex II and III unravelled differences in the local environment of the membrane enzymes in the cristae. Here, we tagged complex IV and V with mNeonGreen and investigated their mitochondrial nano-environment by FLIM and superresolution microscopy in living human cells. Different lifetimes and anisotropy values were found and will be discussed.

  10. High frame-rate TCSPC-FLIM using a novel SPAD-based image sensor

    NARCIS (Netherlands)

    Gersbach, M.; Trimananda, R.; Maruyama, Y.; Fishburn, M.; Cahrbon, E. et al

    2010-01-01

    Imaging techniques based on time-correlated single photon counting (TCSPC), such as fluorescence lifetime imaging microscopy (FLIM), rely on fast single-photon detectors as well as timing electronics in the form of time-to-digital or time-to-analog converters. Conventional systems rely on

  11. Measuring the effect of a Western diet on liver tissue architecture by FLIM autofluorescence and harmonic generation microscopy.

    Science.gov (United States)

    Ranjit, Suman; Dvornikov, Alexander; Dobrinskikh, Evgenia; Wang, Xiaoxin; Luo, Yuhuan; Levi, Moshe; Gratton, Enrico

    2017-07-01

    The phasor approach to auto-fluorescence lifetime imaging was used to identify and characterize a long lifetime species (LLS) (~7.8 ns) in livers of mice fed with a Western diet. The size of the areas containing this LLS species depends on the type of diet and the size distribution shows Western diet has much larger LLS sizes. Combination of third harmonic generation images with FLIM identified the LLS species with fat droplets and the droplet size distribution was estimated. Second harmonic generation microscopy combined with phasor FLIM shows that there is an increase in fibrosis with a Western diet. A new decomposition in three components of the phasor plot shows that a Western diet is correlated with a higher fraction of free NADH, signifying more reducing condition and more glycolytic condition. Multiparametric analysis of phasor distribution shows that from the distribution of phasor points, a Western diet fed versus a low fat diet fed samples of mice livers can be separated. The phasor approach for the analysis of FLIM images of autofluorescence in liver specimens can result in discovery of new fluorescent species and then these new fluorescent species can help assess tissue architecture. Finally integrating FLIM and second and third harmonic analysis provides a measure of the advancement of fibrosis as an effect of diet.

  12. Single pulse two-photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate and an all fiber based setup

    Science.gov (United States)

    Eibl, Matthias; Karpf, Sebastian; Hakert, Hubertus; Weng, Daniel; Pfeiffer, Tom; Kolb, Jan Philip; Huber, Robert

    2017-07-01

    Newly developed microscopy methods have the goal to give researches in bio-molecular science a better understanding of processes ongoing on a cellular level. Especially two-photon excited fluorescence (TPEF) microscopy is a readily applied and widespread modality. Compared to one photon fluorescence imaging, it is possible to image not only the surface but also deeper lying structures. Together with fluorescence lifetime imaging (FLIM), which provides information on the chemical composition of a specimen, deeper insights on a molecular level can be gained. However, the need for elaborate light sources for TPEF and speed limitations for FLIM hinder an even wider application. In this contribution, we present a way to overcome this limitations by combining a robust and inexpensive fiber laser for nonlinear excitation with a fast analog digitization method for rapid FLIM imaging. The applied sub nanosecond pulsed laser source is perfectly suited for fiber delivery as typically limiting non-linear effects like self-phase or cross-phase modulation (SPM, XPM) are negligible. Furthermore, compared to the typically applied femtosecond pulses, our longer pulses produce much more fluorescence photons per single shot. In this paper, we show that this higher number of fluorescence photons per pulse combined with a high analog bandwidth detection makes it possible to not only use a single pulse per pixel for TPEF imaging but also to resolve the exponential time decay for FLIM. To evaluate our system, we acquired FLIM images of a dye solution with single exponential behavior to assess the accuracy of our lifetime determination and also FLIM images of a plant stem at a pixel rate of 1 MHz to show the speed performance of our single pulse two-photon FLIM (SP-FLIM) system.

  13. Tunable PIE and synchronized gating detections by FastFLIM for quantitative microscopy measurements of fast dynamics of single molecules

    Science.gov (United States)

    Sun, Yuansheng; Coskun, Ulas; Ferreon, Allan Chris; Barbieri, Beniamino; Liao, Shih-Chu Jeff

    2016-03-01

    The crosstalk between two fluorescent species causes problems in fluorescence microscopy imaging, especially for quantitative measurements such as co-localization, Förster resonance energy transfer (FRET), fluorescence cross correlation spectroscopy (FCCS). In laser scanning confocal microscopy, the lasers can be switched on and off by acousto-optic tunable filters (AOTF) in the microsecond scale for alternative line scanning in order to avoid the crosstalk while minimizing the time delay between two lasers on the same pixel location. In contrast, the pulsed interleaved excitation (PIE) technique synchronizes two pulsed lasers of different wavelengths in the nanosecond scale to enable measuring superfast dynamics of two fluorescent species simultaneously and yet quantitatively without the crosstalk contamination. This feature is critical for many cell biology applications, e.g. accurate determination of stoichiometry in FRET measurements for studying protein-protein interactions or cell signal events, detection of weaker bindings in FCCS by eliminating the false cross correlation due to the crosstalk. The PIE has been used with the time correlated single photon counting (TCSPC) electronics. Here, we describe a novel PIE development using the digital frequency domain (DFD) technique -- FastFLIM, which provides tunable PIE setups and synchronized gating detections, tailored and optimized to specific applications. A few PIE setups by FastFLIM and measurement examples are described. Combined with the sensitivity of Alba and Q2 systems, the PIE allowed us to quantitatively measure the fast dynamics of single molecules.

  14. FLIM-FRET image analysis of tryptophan in prostate cancer cells

    Science.gov (United States)

    Periasamy, Ammasi; Alam, Shagufta R.; Svindrych, Zdenek; Wallrabe, Horst

    2017-07-01

    A region of interest (ROI) based quantitative FLIM-FRET image analysis is developed to quantitate the autofluorescence signals of the essential amino acid tryptophan as a biomarker to investigate the metabolism in prostate cancer cells.

  15. Refractive index sensing using Fluorescence Lifetime Imaging (FLIM)

    International Nuclear Information System (INIS)

    Jones, Carolyn; Suhling, Klaus

    2006-01-01

    The fluorescence lifetime is a function of the refractive index of the fluorophore's environment, for example in the case of the biologically important green fluorescent protein (GFP). In order to address the question whether this effect can be exploited to image the local environment of specific proteins in cell biology, we need to determine the distance over which the fluorophore's lifetime is sensitive to the refractive index. To this end, we employ Fluorescence Lifetime Imaging (FLIM) of fluorescein in NaOH buffer at an interface. This approach allows us to map the fluorescence lifetime as a function of distance from a buffer/air and buffer/oil interface. Preliminary data show that the fluorescence lifetime of fluorescein increases near a buffer/air interface and decreases near a buffer/oil interface. The range over which this fluorescence lifetime change occurs is found to be of the order several μm which is consistent with a theoretical model based on the full width at half maximum of the emission spectrum proposed by Toptygin

  16. Label-free SHG imaging and spectral FLIM of corneas using a sub-15 fs laser microscope

    Science.gov (United States)

    Batista, Ana; Breunig, Hans Georg; Uchugonova, Aisada; Seitz, Berthold; Morgado, António Miguel; König, Karsten

    2014-02-01

    Alterations to the corneal cell metabolism or to the structural organization of collagen fibrils occur in several corneal and systemic pathologies. In this work we resort to multiphoton microscopy corneal imaging to achieve a characterization of the corneal state. Using fluorescence lifetime imaging microscopy (FLIM) the assessment of the metabolic state of corneal cells is possible, whereas second harmonic generation (SHG) imaging can be used to assess corneal structural alterations. A sub-15 fs near-infrared laser source with a broad excitation spectrum was used for SHG imaging and FLIM. The broad spectrum allows simultaneous excitation of both metabolic co-factors. The signals were collected by a photomultiplier tubes (PMT) detector with 16 simultaneous recording channels, which allowed the separation of fluorophores autofluorescence based on their emission wavelengths. We were able to successfully image ex-vivo human and porcine cornea at multiple depths. Simultaneous NADH and flavin autofluorescence, SHG of collagen fibrils, and stroma autofluorescence imaging was performed which may in future allow an improved characterization of the metabolic and structural alterations of the corneal tissue due to pathophysiological conditions. This would be an important step towards a better understanding of corneal dystrophies and systemic metabolic disorders.

  17. Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging.

    Science.gov (United States)

    Pelegati, Vitor B; Adur, Javier; De Thomaz, André A; Almeida, Diogo B; Baratti, Mariana O; Andrade, Liliana A L A; Bottcher-Luiz, Fátima; Cesar, Carlos L

    2012-10-01

    In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two-photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy-to-operate platform capable to perform two-photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples. Copyright © 2012 Wiley Periodicals, Inc.

  18. A Full Parallel Event Driven Readout Technique for Area Array SPAD FLIM Image Sensors

    Directory of Open Access Journals (Sweden)

    Kaiming Nie

    2016-01-01

    Full Text Available This paper presents a full parallel event driven readout method which is implemented in an area array single-photon avalanche diode (SPAD image sensor for high-speed fluorescence lifetime imaging microscopy (FLIM. The sensor only records and reads out effective time and position information by adopting full parallel event driven readout method, aiming at reducing the amount of data. The image sensor includes four 8 × 8 pixel arrays. In each array, four time-to-digital converters (TDCs are used to quantize the time of photons’ arrival, and two address record modules are used to record the column and row information. In this work, Monte Carlo simulations were performed in Matlab in terms of the pile-up effect induced by the readout method. The sensor’s resolution is 16 × 16. The time resolution of TDCs is 97.6 ps and the quantization range is 100 ns. The readout frame rate is 10 Mfps, and the maximum imaging frame rate is 100 fps. The chip’s output bandwidth is 720 MHz with an average power of 15 mW. The lifetime resolvability range is 5–20 ns, and the average error of estimated fluorescence lifetimes is below 1% by employing CMM to estimate lifetimes.

  19. A Full Parallel Event Driven Readout Technique for Area Array SPAD FLIM Image Sensors.

    Science.gov (United States)

    Nie, Kaiming; Wang, Xinlei; Qiao, Jun; Xu, Jiangtao

    2016-01-27

    This paper presents a full parallel event driven readout method which is implemented in an area array single-photon avalanche diode (SPAD) image sensor for high-speed fluorescence lifetime imaging microscopy (FLIM). The sensor only records and reads out effective time and position information by adopting full parallel event driven readout method, aiming at reducing the amount of data. The image sensor includes four 8 × 8 pixel arrays. In each array, four time-to-digital converters (TDCs) are used to quantize the time of photons' arrival, and two address record modules are used to record the column and row information. In this work, Monte Carlo simulations were performed in Matlab in terms of the pile-up effect induced by the readout method. The sensor's resolution is 16 × 16. The time resolution of TDCs is 97.6 ps and the quantization range is 100 ns. The readout frame rate is 10 Mfps, and the maximum imaging frame rate is 100 fps. The chip's output bandwidth is 720 MHz with an average power of 15 mW. The lifetime resolvability range is 5-20 ns, and the average error of estimated fluorescence lifetimes is below 1% by employing CMM to estimate lifetimes.

  20. Quantitative FLIM-FRET Microscopy to Monitor Nanoscale Chromatin Compaction In Vivo Reveals Structural Roles of Condensin Complexes

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    David Llères

    2017-02-01

    Full Text Available How metazoan genomes are structured at the nanoscale in living cells and tissues remains unknown. Here, we adapted a quantitative FRET (Förster resonance energy transfer-based fluorescence lifetime imaging microscopy (FLIM approach to assay nanoscale chromatin compaction in living organisms. Caenorhabditis elegans was chosen as a model system. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 (HP1 and SETDB1 H3-lysine-9 methyltransferase homologs in vivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes.

  1. Clinical multiphoton FLIM tomography

    Science.gov (United States)

    König, Karsten

    2012-03-01

    This paper gives an overview on current clinical high resolution multiphoton fluorescence lifetime imaging in volunteers and patients. Fluorescence lifetime imaging (FLIM) in Life Sciences was introduced in Jena/Germany in 1988/89 based on a ZEISS confocal picosecond dye laser scanning microscope equipped with a single photon counting unit. The porphyrin distribution in living cells and living tumor-bearing mice was studied with high spatial, temporal, and spectral resolution. Ten years later, time-gated cameras were employed to detect dental caries in volunteers based on one-photon excitation of autofluorescent bacteria with long fluorescence lifetimes. Nowadays, one-photon FLIM based on picosecond VIS laser diodes are used to study ocular diseases in humans. Already one decade ago, first clinical twophoton FLIM images in humans were taken with the certified clinical multiphoton femtosecond laser tomograph DermaInspectTM. Multiphoton tomographs with FLIM modules are now operating in hospitals at Brisbane, Tokyo, Berlin, Paris, London, Modena and other European cities. Multiple FLIM detectors allow spectral FLIM with a temporal resolution down to 20 ps (MCP) / 250 ps (PMT) and a spectral resolution of 10 nm. Major FLIM applications include the detection of intradermal sunscreen and tattoo nanoparticles, the detection of different melanin types, the early diagnosis of dermatitis and malignant melanoma, as well as the measurement of therapeutic effects in pateints suffering from dermatitis. So far, more than 1,000 patients and volunteers have been investigated with the clinical multiphoton FLIM tomographs DermaInspectTM and MPTflexTM.

  2. Toward intravascular morphological and biochemical imaging of atherosclerosis with optical coherence tomography (OCT) and fluorescence lifetime imaging (FLIM) (Conference Presentation)

    Science.gov (United States)

    Chen, Xi; Kim, Wihan; Serafino, Michael; Walton, Brian; Jo, Javier A.; Applegate, Brian E.

    2017-02-01

    We have shown in an ex vivo human coronary artery study that the biochemical information derived from FLIM interpreted in the context of the morphological information from OCT enables a detailed classification of human coronary plaques associated with atherosclerosis. The identification of lipid-rich plaques prone to erosion or rupture and associated with sudden coronary events can impact current clinical practice as well as future development of targeted therapies for "vulnerable" plaques. In order to realize clinical translation of intravascular OCT/FLIM we have had to develop several key technologies. A multimodal catheter endoscope capable of delivering near UV excitation for FLIM and shortwave IR for OCT has been fabricated using a ball lens design with a double clad fiber. The OCT illumination and the FLIM excitation propogate down the inner core while the large outer multimode core captures the fluorescence emission. To enable intravascular pullback imaging with this endoscope we have developed an ultra-wideband fiber optic rotary joint using the same double clad fiber. The rotary joint is based on a lensless design where two cleaved fibers, one fixed and one rotating, are brought into close proximity but not touching. Using water as the lubricant enabled operation over the near UV-shortwave IR range. Transmission over this bandwidth has been measured to be near 100% at rotational frequencies up to 147 Hz. The entire system has been assembled and placed on a mobile cart suitable for cath lab based imaging. System development, performance, and early ex vivo imaging results will be discussed.

  3. Monitoring biosensor activity in living cells with fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Hum, Julia M; Siegel, Amanda P; Pavalko, Fredrick M; Day, Richard N

    2012-11-07

    Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing "FRET standard" fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail.

  4. Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy

    NARCIS (Netherlands)

    van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K; Roos, Dirk; Otto, Cees

    2008-01-01

    We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91(phox), which are both subunits of the phagocyte NADPH

  5. pawFLIM: reducing bias and uncertainty to enable lower photon count in FLIM experiments

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    Silberberg, Mauro; Grecco, Hernán E.

    2017-06-01

    Förster resonant energy transfer measured by fluorescence lifetime imaging microscopy (FRET-FLIM) is the method of choice for monitoring the spatio-temporal dynamics of protein interactions in living cells. To obtain an accurate estimate of the molecular fraction of interacting proteins requires a large number of photons, which usually precludes the observation of a fast process, particularly with time correlated single photon counting (TCSPC) based FLIM. In this work, we propose a novel method named pawFLIM (phasor analysis via wavelets) that allows the denoising of FLIM datasets by adaptively and selectively adjusting the desired compromise between spatial and molecular resolution. The method operates by applying a weighted translational-invariant Haar-wavelet transform denoising algorithm to phasor images. This results in significantly less bias and mean square error than other existing methods. We also present a new lifetime estimator (named normal lifetime) with a smaller mean squared error and overall bias as compared to frequency domain phase and modulation lifetimes. Overall, we present an approach that will enable the observation of the dynamics of biological processes at the molecular level with better temporal and spatial resolution.

  6. CARS microscopy for imaging

    International Nuclear Information System (INIS)

    Arzumanyan Grigory; Voskanyan Karine

    2013-01-01

    Optical microscopy grows in its importance with the development of modern nanotechnology, biotechnology, methods of diagnostics and treatment of most dangerous diseases for mankind. There are several important goals of optical microscopy for biomedical studies among which the next three may be distinguished: fast imaging with high lateral spatial resolution, 3-D sectioning capability and high contrast for chemical selectivity. To meet these specific requirements, various types of both linear and nonlinear optical microscopy were elaborated. (authors)

  7. Rapid diagnosis and intraoperative margin assessment of human lung cancer with fluorescence lifetime imaging microscopy

    Directory of Open Access Journals (Sweden)

    Mengyan Wang

    2017-12-01

    Full Text Available A method of rapidly differentiating lung tumor from healthy tissue is extraordinarily needed for both the diagnosis and the intraoperative margin assessment. We assessed the ability of fluorescence lifetime imaging microscopy (FLIM for differentiating human lung cancer and normal tissues with the autofluorescence, and also elucidated the mechanism in tissue studies and cell studies. A 15-patient testing group was used to compare FLIM results with traditional histopathology diagnosis. Based on the endogenous fluorescence lifetimes of the testing group, a criterion line was proposed to distinguish normal and cancerous tissues. Then by blinded examined 41 sections from the validation group of other 16 patients, the sensitivity and specificity of FLIM were determined. The cellular metabolism was studied with specific perturbations of oxidative phosphorylation and glycolysis in cell studies. The fluorescence lifetime of cancerous lung tissues is consistently lower than normal tissues, and this is due to the both decrease of reduced nicotinamide adenine dinucleotide (NADH and flavin adenine dinucleotide (FAD lifetimes. A criterion line of lifetime at 1920 ps can be given for differentiating human lung cancer and normal tissues.The sensitivity and specificity of FLIM for lung cancer diagnosis were determined as 92.9% and 92.3%. These findings suggest that NADH and FAD can be used to rapidly diagnose lung cancer. FLIM is a rapid, accurate and highly sensitive technique in the judgment during lung cancer surgery and it can be potential in earlier cancer detection.

  8. Solid-State Camera System for Fluorescence Lifetime Microscopy

    NARCIS (Netherlands)

    Zhao, Q.

    2014-01-01

    Fluorescence microscopy is a well-established platform for biology and biomedical research (Chapter 2). Based on this platform, fluorescence lifetime imaging microscopy (FLIM) has been developed to measure fluorescence lifetimes, which are independent of fluorophore concentration and excitation

  9. Rapid global fitting of large fluorescence lifetime imaging microscopy datasets.

    Directory of Open Access Journals (Sweden)

    Sean C Warren

    Full Text Available Fluorescence lifetime imaging (FLIM is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset. This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis

  10. Microsphere imaging with confocal microscopy and two photon microscopy

    International Nuclear Information System (INIS)

    Chun, Hyung Su; An, Kyung Won; Lee, Jai Hyung

    2002-01-01

    We have acquired images of polystyrene and fused-silica microsphere by using conventional optical microscopy, confocal microscopy and two-photon microscopy, and performed comparative analysis of these images. Different from conventional optical microscopy, confocal and two-photon microscopy had good optical sectioning capability. In addition, confocal microscopy and two-photon microscopy had better lateral resolution than conventional optical microscopy. These results are attributed to confocality and nonlinearity of confocal microscopy and two photon microscopy, respectively.

  11. Fluorescence intensity decay shape analysis microscopy (FIDSAM) for quantitative and sensitive live-cell imaging

    Science.gov (United States)

    Peter, Sébastien; Elgass, Kirstin; Sackrow, Marcus; Caesar, Katharina; Born, Anne-Kathrin; Maniura, Katharina; Harter, Klaus; Meixner, Alfred J.; Schleifenbaum, Frank

    2010-02-01

    Fluorescence microscopy became an invaluable tool in cell biology in the past 20 years. However, the information that lies in these studies is often corrupted by a cellular fluorescence background known as autofluorescence. Since the unspecific background often overlaps with most commonly used labels in terms of fluorescence spectra and fluorescence lifetime, the use of spectral filters in the emission beampath or timegating in fluorescence lifetime imaging (FLIM) is often no appropriate means for distinction between signal and background. Despite the prevalence of fluorescence techniques only little progress has been reported in techniques that specifically suppress autofluorescence or that clearly discriminate autofluorescence from label fluorescence. Fluorescence intensity decay shape analysis microscopy (FIDSAM) is a novel technique which is based on the image acquisition protocol of FLIM. Whereas FLIM spatially resolved maps the average fluorescence lifetime distribution in a heterogeneous sample such as a cell, FIDSAM enhances the dynamic image contrast by determination of the autofluorescence contribution by comparing the fluorescence decay shape to a reference function. The technique therefore makes use of the key difference between label and autofluorescence, i.e. that for label fluorescence only one emitting species contributes to fluorescence intensity decay curves whereas many different species of minor intensity contribute to autofluorescence. That way, we were able to suppress autofluorescence contributions from chloroplasts in Arabidopsis stoma cells and from cell walls in Arabidopsis hypocotyl cells to background level. Furthermore, we could extend the method to more challenging labels such as the cyan fluorescent protein CFP in human fibroblasts.

  12. Penetration of silver nanoparticles into porcine skin ex vivo using fluorescence lifetime imaging microscopy, Raman microscopy, and surface-enhanced Raman scattering microscopy.

    Science.gov (United States)

    Zhu, Yongjian; Choe, Chun-Sik; Ahlberg, Sebastian; Meinke, Martina C; Alexiev, Ulrike; Lademann, Juergen; Darvin, Maxim E

    2015-05-01

    In order to investigate the penetration depth of silver nanoparticles (Ag NPs) inside the skin, porcine ears treated with Ag NPs are measured by two-photon tomography with a fluorescence lifetime imaging microscopy (TPT-FLIM) technique, confocal Raman microscopy (CRM), and surface-enhanced Raman scattering (SERS) microscopy. Ag NPs are coated with poly-N-vinylpyrrolidone and dispersed in pure water solutions. After the application of Ag NPs, porcine ears are stored in the incubator for 24 h at a temperature of 37°C. The TPT-FLIM measurement results show a dramatic decrease of the Ag NPs' signal intensity from the skin surface to a depth of 4 μm. Below 4 μm, the Ag NPs' signal continues to decline, having completely disappeared at 12 to 14 μm depth. CRM shows that the penetration depth of Ag NPs is 11.1 ± 2.1 μm. The penetration depth measured with a highly sensitive SERS microscopy reaches 15.6 ± 8.3 μm. Several results obtained with SERS show that the penetration depth of Ag NPs can exceed the stratum corneum (SC) thickness, which can be explained by both penetration of trace amounts of Ag NPs through the SC barrier and by the measurements inside the hair follicle, which cannot be excluded in the experiment.

  13. Penetration of silver nanoparticles into porcine skin ex vivo using fluorescence lifetime imaging microscopy, Raman microscopy, and surface-enhanced Raman scattering microscopy

    Science.gov (United States)

    Zhu, Yongjian; Choe, Chun-Sik; Ahlberg, Sebastian; Meinke, Martina C.; Alexiev, Ulrike; Lademann, Juergen; Darvin, Maxim E.

    2015-05-01

    In order to investigate the penetration depth of silver nanoparticles (Ag NPs) inside the skin, porcine ears treated with Ag NPs are measured by two-photon tomography with a fluorescence lifetime imaging microscopy (TPT-FLIM) technique, confocal Raman microscopy (CRM), and surface-enhanced Raman scattering (SERS) microscopy. Ag NPs are coated with poly-N-vinylpyrrolidone and dispersed in pure water solutions. After the application of Ag NPs, porcine ears are stored in the incubator for 24 h at a temperature of 37°C. The TPT-FLIM measurement results show a dramatic decrease of the Ag NPs' signal intensity from the skin surface to a depth of 4 μm. Below 4 μm, the Ag NPs' signal continues to decline, having completely disappeared at 12 to 14 μm depth. CRM shows that the penetration depth of Ag NPs is 11.1±2.1 μm. The penetration depth measured with a highly sensitive SERS microscopy reaches 15.6±8.3 μm. Several results obtained with SERS show that the penetration depth of Ag NPs can exceed the stratum corneum (SC) thickness, which can be explained by both penetration of trace amounts of Ag NPs through the SC barrier and by the measurements inside the hair follicle, which cannot be excluded in the experiment.

  14. Microscopy and Image Analysis.

    Science.gov (United States)

    McNamara, George; Difilippantonio, Michael; Ried, Thomas; Bieber, Frederick R

    2017-07-11

    This unit provides an overview of light microscopy, including objectives, light sources, filters, film, and color photography for fluorescence microscopy and fluorescence in situ hybridization (FISH). We believe there are excellent opportunities for cytogeneticists, pathologists, and other biomedical readers, to take advantage of specimen optical clearing techniques and expansion microscopy-we briefly point to these new opportunities. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  15. Improving accuracy and precision in biological applications of fluorescence lifetime imaging microscopy

    Science.gov (United States)

    Chang, Ching-Wei

    The quantitative understanding of cellular and molecular responses in living cells is important for many reasons, including identifying potential molecular targets for treatments of diseases like cancer. Fluorescence lifetime imaging microscopy (FLIM) can quantitatively measure these responses in living cells by producing spatially resolved images of fluorophore lifetime, and has advantages over intensity-based measurements. However, in live-cell microscopy applications using high-intensity light sources such as lasers, maintaining biological viability remains critical. Although high-speed, time-gated FLIM significantly reduces light delivered to live cells, making measurements at low light levels remains a challenge affecting quantitative FLIM results. We can significantly improve both accuracy and precision in gated FLIM applications. We use fluorescence resonance energy transfer (FRET) with fluorescent proteins to detect molecular interactions in living cells: the use of FLIM, better fluorophores, and temperature/CO2 controls can improve live-cell FRET results with higher consistency, better statistics, and less non-specific FRET (for negative control comparisons, p-value = 0.93 (physiological) vs. 9.43E-05 (non-physiological)). Several lifetime determination methods are investigated to optimize gating schemes. We demonstrate a reduction in relative standard deviation (RSD) from 52.57% to 18.93% with optimized gating in an example under typical experimental conditions. We develop two novel total variation (TV) image denoising algorithms, FWTV ( f-weighted TV) and UWTV (u-weighted TV), that can achieve significant improvements for real imaging systems. With live-cell images, they improve the precision of local lifetime determination without significantly altering the global mean lifetime values (high-light cases (RSD = 12.76% at total photon counts (TC) = 100 vs. RSD = 23.03% at TC = 400). Therefore, high-intensity excitation of living cells can be avoided

  16. Scanning Tunneling Microscopy - image interpretation

    International Nuclear Information System (INIS)

    Maca, F.

    1998-01-01

    The basic ideas of image interpretation in Scanning Tunneling Microscopy are presented using simple quantum-mechanical models and supplied with examples of successful application. The importance is stressed of a correct interpretation of this brilliant experimental surface technique

  17. Combined nonlinear laser imaging (two-photon excitation fluorescence, second and third-harmonic generation, and fluorescence lifetime imaging microscopies) in ovarian tumors

    Science.gov (United States)

    Adur, J.; Pelegati, V. B.; de Thomaz, A. A.; Bottcher-Luiz, F.; Andrade, L. A. L. A.; Almeida, D. B.; Carvalho, H. F.; Cesar, C. L.

    2012-03-01

    We applied Two-photon Excited Fluorescence (TPEF), Second/Third Harmonic Generation (SHG and THG) and Fluorescence Lifetime Imaging (FLIM) Non Linear Optics (NLO) Laser-Scanning Microscopy within the same imaging platform to evaluate their use as a diagnostic tool in ovarian tumors. We assess of applicability of this multimodal approach to perform a pathological evaluation of serous and mucinous tumors in human samples. The combination of TPEF-SHG-THG imaging provided complementary information about the interface epithelium/stromal, such as the transformation of epithelium surface (THG) and the overall fibrillar tissue architecture (SHG). The fact that H&E staining is the standard method used in clinical pathology and that the stored samples are usually fixed makes it important a re-evaluation of these samples with NLO microscopy to compare new results with a library of already existing samples. FLIM, however, depends on the chemical environment around the fluorophors that was completely changed after fixation; therefore it only makes sense in unstained samples. Our FLIM results in unstained samples demonstrate that it is possible to discriminate healthy epithelia from serous or mucinous epithelia. Qualitative and quantitative analysis of the different imaging modalities used showed that multimodal nonlinear microscopy has the potential to differentiate between cancerous and healthy ovarian tissue.

  18. Image scanning microscopy: an overview.

    Science.gov (United States)

    Ward, E N; Pal, R

    2017-05-01

    For almost a century, the resolution of optical microscopy was thought to be limited by Abbé's law describing the diffraction limit of light. At the turn of the millennium, aided by new technologies and fluorophores, the field of optical microscopy finally surpassed the diffraction barrier: a milestone achievement that has been recognized by the 2014 Nobel Prize in Chemistry. Many super-resolution methods rely on the unique photophysical properties of the fluorophores to improve resolution, posing significant limitations on biological imaging, such as multicoloured staining, live-cell imaging and imaging thick specimens. Structured Illumination Microscopy (SIM) is one branch of super-resolution microscopy that requires no such special properties of the applied fluorophores, making it more versatile than other techniques. Since its introduction in biological imaging, SIM has proven to be a popular tool in the biologist's arsenal for following biological interaction and probing structures of nanometre scale. SIM continues to see much advancement in design and implementation, including the development of Image Scanning Microscopy (ISM), which uses patterned excitation via either predefined arrays or raster-scanned single point-spread functions (PSF). This review aims to give a brief overview of the SIM and ISM processes and subsequent developments in the image reconstruction process. Drawing from this, and incorporating more recent achievements in light shaping (i.e. pattern scanning and super-resolution beam shaping), this study also intends to suggest potential future directions for this ever-expanding field. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

  19. Time-Resolved Fluorescence Spectroscopy and Fluorescence Lifetime Imaging Microscopy for Characterization of Dendritic Polymer Nanoparticles and Applications in Nanomedicine

    Directory of Open Access Journals (Sweden)

    Alexander Boreham

    2016-12-01

    Full Text Available The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief and for monitoring of disease progression. One route of realizing this approach is through carefully constructed nanoparticles. Due to the small size inherent to the nanoparticles a proper characterization is not trivial. This review highlights the application of time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy (FLIM for the analysis of nanoparticles, covering aspects ranging from molecular properties to particle detection in tissue samples. The latter technique is particularly important as FLIM allows for distinguishing of target molecules from the autofluorescent background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle or its interactions with other biomolecules. Thus, these techniques offer highly suitable tools in the fields of particle development, such as organic chemistry, and in the fields of particle application, such as in experimental dermatology or pharmaceutical research.

  20. Time-Resolved Fluorescence Spectroscopy and Fluorescence Lifetime Imaging Microscopy for Characterization of Dendritic Polymer Nanoparticles and Applications in Nanomedicine.

    Science.gov (United States)

    Boreham, Alexander; Brodwolf, Robert; Walker, Karolina; Haag, Rainer; Alexiev, Ulrike

    2016-12-24

    The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief and for monitoring of disease progression. One route of realizing this approach is through carefully constructed nanoparticles. Due to the small size inherent to the nanoparticles a proper characterization is not trivial. This review highlights the application of time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy (FLIM) for the analysis of nanoparticles, covering aspects ranging from molecular properties to particle detection in tissue samples. The latter technique is particularly important as FLIM allows for distinguishing of target molecules from the autofluorescent background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle or its interactions with other biomolecules. Thus, these techniques offer highly suitable tools in the fields of particle development, such as organic chemistry, and in the fields of particle application, such as in experimental dermatology or pharmaceutical research.

  1. Monitor RNA synthesis in live cell nuclei by using two-photon excited fluorescence lifetime imaging microscopy

    Science.gov (United States)

    Peng, Xiao; Lin, Danying; Wang, Yan; Qi, Jing; Yan, Wei; Qu, Junle

    2015-03-01

    Probing of local molecular environment in cells is of significant value in creating a fundamental understanding of cellular processes and molecular profiles of diseases, as well as studying drug cell interactions. In order to investigate the dynamically changing in subcellular environment during RNA synthesis, we applied two-photon excited fluorescence lifetime imaging microscopy (FLIM) method to monitor the green fluorescent protein (GFP) fused nuclear protein ASF/SF2. The fluorescence lifetime of fluorophore is known to be in inverse correlation with a local refractive index, and thus fluorescence lifetimes of GFP fusions provide real-time information of the molecular environment of ASF/SF2- GFP. The FLIM results showed continuous and significant fluctuations of fluorescence lifetimes of the fluorescent protein fusions in live HeLa cells under physiological conditions. The fluctuations of fluorescence lifetime values indicated the variations of activities of RNA polymerases. Moreover, treatment with pharmacological drugs inhibiting RNA polymerase activities led to irreversible decreases of fluorescence lifetime values. In summary, our study of FLIM imaging of GFP fusion proteins has provided a sensitive and real-time method to investigate RNA synthesis in live cell nuclei.

  2. The Use of Two-Photon FRET-FLIM to Study Protein Interactions During Nuclear Envelope Fusion In Vivo and In Vitro.

    Science.gov (United States)

    Byrne, Richard D; Larijani, Banafshé; Poccia, Dominic L

    2016-01-01

    FRET-FLIM techniques have wide application in the study of protein and protein-lipid interactions in cells. We have pioneered an imaging platform for accurate detection of functional states of proteins and their interactions in fixed cells. This platform, two-site-amplified Förster resonance energy transfer (a-FRET), allows greater signal generation while retaining minimal noise thus enabling application of fluorescence lifetime imaging microscopy (FLIM) to be routinely deployed in different types of cells and tissue. We have used the method described here, time-resolved FRET monitored by two-photon FLIM, to demonstrate the direct interaction of Phospholipase Cγ (PLCγ) by Src Family Kinase 1 (SFK1) during nuclear envelope formation and during male and female pronuclear membrane fusion in fertilized sea urchin eggs. We describe here a generic method that can be applied to monitor any proteins of interest.

  3. A flow bioreactor system compatible with real-time two-photon fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Shen, Nian; Riedl, Julia A; Carvajal Berrio, Daniel A; Davis, Zachary; Monaghan, Michael G; Layland, Shannon L; Hinderer, Svenja; Schenke-Layland, Katja

    2018-02-02

    Bioreactors are essential cell and tissue culture tools that allow the introduction of biophysical signals into in vitro cultures. One major limitation is the need to interrupt experiments and sacrifice samples at certain time points for analyses. To address this issue, we designed a bioreactor that combines high-resolution contact-free imaging and continuous flow in a closed system that is compatible with various types of microscopes. The high throughput fluid flow bioreactor was combined with two-photon fluorescence lifetime imaging microscopy (2P-FLIM) and validated. The hydrodynamics of the bioreactor chamber were characterized using COMSOL. The simulation of shear stress indicated that the bioreactor system provides homogeneous and reproducible flow conditions. The designed bioreactor was used to investigate the effects of low shear stress on human umbilical vein endothelial cells (HUVECs). In a scratch assay, we observed decreased migration of HUVECs under shear stress conditions. Furthermore, metabolic activity shifts from glycolysis to oxidative phosphorylation-dependent mechanisms in HUVECs cultured under low shear stress conditions were detected using 2P-FLIM. Future applications for this bioreactor range from observing cell fate development in real-time to monitoring the environmental effects on cells or metabolic changes due to drug applications.

  4. NICHD Microscopy and Imaging Core (MIC)

    Data.gov (United States)

    Federal Laboratory Consortium — The NICHD Microscopy and Imaging Core (MIC) is designed as a multi-user research facility providing training and instrumentation for high resolution microscopy and...

  5. Probing energy metabolism and microviscosity in cancer using FLIM

    Science.gov (United States)

    Shirmanova, Marina V.; Lukina, Maria M.; Shimolina, Lyubov'E.; Kuimova, Marina K.; Dudenkova, Varvara V.; Shcheslavskiy, Vladislav I.; Zagaynova, Elena V.

    2017-07-01

    Fluorescence lifetime imaging microscopy (FLIM) is a promising non-invasive highly sensitive technique for probing multiple physiological and physicochemical parameters in living cells and tissues. The present study is focused on the investigation of bioenergetics and microscopic viscosity of cultured cancer cells and animal tumors using FLIM during natural growth and chemotherapy. Fluorescence lifetime measurements of the metabolic cofactor NAD(P)H revealed a decrease of the relative amplitude of free NAD(P)H after cisplatin treatment, indicating a change towards a more oxidative metabolic state. Microviscosity mapping performed with the use of fluorescent molecular rotor BODIPY-2 showed a pronounced increase in the plasma membrane viscosity in cancer cells exposed to cisplatin. Although biochemical mechanisms underlying the metabolic and viscosity alterations during chemotherapy have yet to be clarified, our data suggest that the cisplatin-induced changes in cellular metabolism and membrane viscosity play a role in the cytotoxicity of the drug. The results of the study contribute to an understanding of mechanisms of cisplatin action and will be useful for development new approach for assessing response to a therapy.

  6. Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging.

    Science.gov (United States)

    Zhao, Qiaole; Schelen, Ben; Schouten, Raymond; van den Oever, Rein; Leenen, René; van Kuijk, Harry; Peters, Inge; Polderdijk, Frank; Bosiers, Jan; Raspe, Marcel; Jalink, Kees; Geert Sander de Jong, Jan; van Geest, Bert; Stoop, Karel; Young, Ian Ted

    2012-12-01

    We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device design in a frequency-domain FLIM system. The first stage of evaluation for the camera has been carried out. Camera characteristics such as noise distribution, dark current influence, camera gain, sampling density, sensitivity, linearity of photometric response, and optical transfer function have been studied through experiments. We are able to do lifetime measurement using our modulated, electron-multiplied fluorescence lifetime imaging microscope (MEM-FLIM) camera for various objects, e.g., fluorescein solution, fixed green fluorescent protein (GFP) cells, and GFP-actin stained live cells. A detailed comparison of a conventional microchannel plate (MCP)-based FLIM system and the MEM-FLIM system is presented. The MEM-FLIM camera shows higher resolution and a better image quality. The MEM-FLIM camera provides a new opportunity for performing frequency-domain FLIM.

  7. Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging

    Science.gov (United States)

    Zhao, Qiaole; Schelen, Ben; Schouten, Raymond; van den Oever, Rein; Leenen, René; van Kuijk, Harry; Peters, Inge; Polderdijk, Frank; Bosiers, Jan; Raspe, Marcel; Jalink, Kees; de Jong, Jan Geert Sander; van Geest, Bert; Stoop, Karel; Young, Ian Ted

    2012-12-01

    We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device design in a frequency-domain FLIM system. The first stage of evaluation for the camera has been carried out. Camera characteristics such as noise distribution, dark current influence, camera gain, sampling density, sensitivity, linearity of photometric response, and optical transfer function have been studied through experiments. We are able to do lifetime measurement using our modulated, electron-multiplied fluorescence lifetime imaging microscope (MEM-FLIM) camera for various objects, e.g., fluorescein solution, fixed green fluorescent protein (GFP) cells, and GFP-actin stained live cells. A detailed comparison of a conventional microchannel plate (MCP)-based FLIM system and the MEM-FLIM system is presented. The MEM-FLIM camera shows higher resolution and a better image quality. The MEM-FLIM camera provides a new opportunity for performing frequency-domain FLIM.

  8. Interferometric Synthetic Aperture Microscopy: Computed Imaging for Scanned Coherent Microscopy

    Directory of Open Access Journals (Sweden)

    Stephen A. Boppart

    2008-06-01

    Full Text Available Three-dimensional image formation in microscopy is greatly enhanced by the use of computed imaging techniques. In particular, Interferometric Synthetic Aperture Microscopy (ISAM allows the removal of out-of-focus blur in broadband, coherent microscopy. Earlier methods, such as optical coherence tomography (OCT, utilize interferometric ranging, but do not apply computed imaging methods and therefore must scan the focal depth to acquire extended volumetric images. ISAM removes the need to scan the focus by allowing volumetric image reconstruction from data collected at a single focal depth. ISAM signal processing techniques are similar to the Fourier migration methods of seismology and the Fourier reconstruction methods of Synthetic Aperture Radar (SAR. In this article ISAM is described and the close ties between ISAM and SAR are explored. ISAM and a simple strip-map SAR system are placed in a common mathematical framework and compared to OCT and radar respectively. This article is intended to serve as a review of ISAM, and will be especially useful to readers with a background in SAR.

  9. Image calibration in fluorescence microscopy.

    NARCIS (Netherlands)

    Zwier, J.M.; van Rooij, G.J.; Hofstraat, J.W.; Brakenhoff, G.J.

    2004-01-01

    A fluorescence image calibration method is presented based on the use of standardized uniformly fluorescing reference layers. It is demonstrated to be effective for the correction of non-uniform imaging characteristics across the image (shading correction) as well as for relating fluorescence

  10. Scanning transmission electron microscopy imaging and analysis

    CERN Document Server

    Pennycook, Stephen J

    2011-01-01

    Provides the first comprehensive treatment of the physics and applications of this mainstream technique for imaging and analysis at the atomic level Presents applications of STEM in condensed matter physics, materials science, catalysis, and nanoscience Suitable for graduate students learning microscopy, researchers wishing to utilize STEM, as well as for specialists in other areas of microscopy Edited and written by leading researchers and practitioners

  11. Multiphoton microscopy imaging of developing tooth germs

    Directory of Open Access Journals (Sweden)

    Pei-Yu Pan

    2014-01-01

    Conclusion: In this study, a novel multiphoton microscopy database of images from developing tooth germs in mice was set up. We confirmed that multiphoton laser microscopy is a powerful tool for investigating the development of tooth germ and is worthy for further application in the study of tooth regeneration.

  12. Modulated electron-multiplied fluorescence lifetime imaging microscope: All-solid-state camera for fluorescence lifetime imaging

    OpenAIRE

    Zhao, Q.; Schelen, B.; Schouten, R.

    2012-01-01

    We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device design in a frequency-domain FLIM system. The first stage of evaluation for the camera has been carried out. Camera characteristics such as noise distribution, dark current influence, camera gain, sam...

  13. A high throughput spectral image microscopy system

    Science.gov (United States)

    Gesley, M.; Puri, R.

    2018-01-01

    A high throughput spectral image microscopy system is configured for rapid detection of rare cells in large populations. To overcome flow cytometry rates and use of fluorophore tags, a system architecture integrates sample mechanical handling, signal processors, and optics in a non-confocal version of light absorption and scattering spectroscopic microscopy. Spectral images with native contrast do not require the use of exogeneous stain to render cells with submicron resolution. Structure may be characterized without restriction to cell clusters of differentiation.

  14. FLIM data analysis of NADH and Tryptophan autofluorescence in prostate cancer cells

    Science.gov (United States)

    O'Melia, Meghan J.; Wallrabe, Horst; Svindrych, Zdenek; Rehman, Shagufta; Periasamy, Ammasi

    2016-03-01

    Fluorescence lifetime imaging microscopy (FLIM) is one of the most sensitive techniques to measure metabolic activity in living cells, tissues and whole animals. We used two- and three-photon fluorescence excitation together with time-correlated single photon counting (TCSPC) to acquire FLIM signals from normal and prostate cancer cell lines. FLIM requires complex data fitting and analysis; we explored different ways to analyze the data to match diverse cellular morphologies. After non-linear least square fitting of the multi-photon TCSPC images by the SPCImage software (Becker & Hickl), all image data are exported and further processed in ImageJ. Photon images provide morphological, NAD(P)H signal-based autofluorescent features, for which regions of interest (ROIs) are created. Applying these ROIs to all image data parameters with a custom ImageJ macro, generates a discrete, ROI specific database. A custom Excel (Microsoft) macro further analyzes the data with charts and statistics. Applying this highly automated assay we compared normal and cancer prostate cell lines with respect to their glycolytic activity by analyzing the NAD(P)H-bound fraction (a2%), NADPH/NADH ratio and efficiency of energy transfer (E%) for Tryptophan (Trp). Our results show that this assay is able to differentiate the effects of glucose stimulation and Doxorubicin in these prostate cell lines by tracking the changes in a2% of NAD(P)H, NADPH/NADH ratio and the changes in Trp E%. The ability to isolate a large, ROI-based data set, reflecting the heterogeneous cellular environment and highlighting even subtle changes -- rather than whole cell averages - makes this assay particularly valuable.

  15. Highly sensitive and quantitative FRET-FLIM imaging in single dendritic spines using improved non-radiative YFP.

    Science.gov (United States)

    Murakoshi, Hideji; Lee, Seok-Jin; Yasuda, Ryohei

    2008-08-01

    Two-photon fluorescence lifetime imaging microscopy (TPFLIM) enables the quantitative measurements of fluorescence resonance energy transfer (FRET) in small subcellular compartments in light scattering tissue. We evaluated and optimized the FRET pair of mEGFP (monomeric EGFP with the A206K mutation) and REACh (non-radiative YFP variants) for TPFLIM. We characterized several mutants of REACh in terms of their "darkness," and their ability to act as a FRET acceptor for mEGFP in HeLa cells and hippocampal neurons. Since the commonly used monomeric mutation A206K increases the brightness of REACh, we introduced a different monomeric mutation (F223R) which does not affect the brightness. Also, we found that the folding efficiency of original REACh, as measured by the fluorescence lifetime of a mEGFP-REACh tandem dimer, was low and variable from cell to cell. Introducing two folding mutations (F46L, Q69M) into REACh increased the folding efficiency by approximately 50%, and reduced the variability of FRET signal. Pairing mEGFP with the new REACh (super-REACh, or sREACh) improved the signal-to-noise ratio compared to the mEGFP-mRFP or mEGFP-original REACh pair by approximately 50%. Using this new pair, we demonstrated that the fraction of actin monomers in filamentous and globular forms in single dendritic spines can be quantitatively measured with high sensitivity. Thus, the mEGFP-sREACh pair is suited for quantitative FRET measurement by TPFLIM, and enables us to measure protein-protein interactions in individual dendritic spines in brain slices with high sensitivity.

  16. Microscopy image segmentation tool: Robust image data analysis

    International Nuclear Information System (INIS)

    Valmianski, Ilya; Monton, Carlos; Schuller, Ivan K.

    2014-01-01

    We present a software package called Microscopy Image Segmentation Tool (MIST). MIST is designed for analysis of microscopy images which contain large collections of small regions of interest (ROIs). Originally developed for analysis of porous anodic alumina scanning electron images, MIST capabilities have been expanded to allow use in a large variety of problems including analysis of biological tissue, inorganic and organic film grain structure, as well as nano- and meso-scopic structures. MIST provides a robust segmentation algorithm for the ROIs, includes many useful analysis capabilities, and is highly flexible allowing incorporation of specialized user developed analysis. We describe the unique advantages MIST has over existing analysis software. In addition, we present a number of diverse applications to scanning electron microscopy, atomic force microscopy, magnetic force microscopy, scanning tunneling microscopy, and fluorescent confocal laser scanning microscopy

  17. Optically sectioned imaging by oblique plane microscopy

    Science.gov (United States)

    Kumar, Sunil; Lin, Ziduo; Lyon, Alex R.; MacLeod, Ken T.; Dunsby, Chris

    2011-03-01

    Oblique Plane Microscopy (OPM) is a light sheet microscopy technique that combines oblique illumination with correction optics that tilt the focal plane of the collection system. OPM can be used to image conventionally mounted specimens on coverslips or tissue culture dishes and has low out-of-plane photobleaching and phototoxicity. No moving parts are required to achieve an optically sectioned image and so high speed optically sectioned imaging is possible. The first OPM results obtained using a high NA water immersion lens on a commercially available inverted microscope frame are presented, together with a measurement of the achievable optical resolution.

  18. Transmission Electron Microscopy Physics of Image Formation

    CERN Document Server

    Kohl, Helmut

    2008-01-01

    Transmission Electron Microscopy: Physics of Image Formation presents the theory of image and contrast formation, and the analytical modes in transmission electron microscopy. The principles of particle and wave optics of electrons are described. Electron-specimen interactions are discussed for evaluating the theory of scattering and phase contrast. Also discussed are the kinematical and dynamical theories of electron diffraction and their applications for crystal-structure analysis and imaging of lattices and their defects. X-ray microanalysis and electron energy-loss spectroscopy are treated as analytical methods. Specimen damage and contamination by electron irradiation limits the resolution for biological and some inorganic specimens. This fifth edition includes discussion of recent progress, especially in the area of aberration correction and energy filtering; moreover, the topics introduced in the fourth edition have been updated. Transmission Electron Microscopy: Physics of Image Formation is written f...

  19. Scanning Electron Microscopy Sample Preparation and Imaging.

    Science.gov (United States)

    Nguyen, Jenny Ngoc Tran; Harbison, Amanda M

    2017-01-01

    Scanning electron microscopes allow us to reach magnifications of 20-130,000× and resolve compositional and topographical images with intense detail. These images are created by bombarding a sample with electrons in a focused manner to generate a black and white image from the electrons that bounce off of the sample. The electrons are detected using positively charged detectors. Scanning electron microscopy permits three-dimensional imaging of desiccated specimens or wet cells and tissues by using variable pressure chambers. SEM ultrastructural analysis and intracellular imaging supplement light microscopy for molecular profiling of prokaryotes, plants, and mammals. This chapter demonstrates how to prepare and image samples that are (a) desiccated and conductive, (b) desiccated and nonconductive but coated with an electron conductive film using a gold sputter coater, and (c) wet and maintained in a hydrated state using a Deben Coolstage.

  20. Image formation and image analysis in electron microscopy

    International Nuclear Information System (INIS)

    Heel, M. van.

    1981-01-01

    This thesis covers various aspects of image formation and image analysis in electron microscopy. The imaging of relatively strong objects in partially coherent illumination, the coherence properties of thermionic emission sources and the detection of objects in quantum noise limited images are considered. IMAGIC, a fast, flexible and friendly image analysis software package is described. Intelligent averaging of molecular images is discussed. (C.F.)

  1. Single cell FRET analysis for the identification of optimal FRET-pairs in Bacillus subtilis using a prototype MEM-FLIM system.

    Directory of Open Access Journals (Sweden)

    Ruud G J Detert Oude Weme

    Full Text Available Protein-protein interactions can be studied in vitro, e.g. with bacterial or yeast two-hybrid systems or surface plasmon resonance. In contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. One approach to study protein-protein interactions in vivo is via Förster Resonance Energy Transfer (FRET. Here, FRET efficiency of selected FRET-pairs was studied at the single cell level using sensitized emission and Frequency Domain-Fluorescence Lifetime Imaging Microscopy (FD-FLIM. For FRET-FLIM, a prototype Modulated Electron-Multiplied FLIM system was used, which is, to the best of our knowledge, the first account of Frequency Domain FLIM to analyze FRET in single bacterial cells. To perform FRET-FLIM, we first determined and benchmarked the best fluorescent protein-pair for FRET in Bacillus subtilis using a novel BglBrick-compatible integration vector. We show that GFP-tagRFP is an excellent donor-acceptor pair for B. subtilis in vivo FRET studies. As a proof of concept, selected donor and acceptor fluorescent proteins were fused using a linker that contained a tobacco etch virus (TEV-protease recognition sequence. Induction of TEV-protease results in loss of FRET efficiency and increase in fluorescence lifetime. The loss of FRET efficiency after TEV induction can be followed in time in single cells via time-lapse microscopy. This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.

  2. Microscopy imaging device with advanced imaging properties

    Science.gov (United States)

    Ghosh, Kunal; Burns, Laurie; El Gamal, Abbas; Schnitzer, Mark J.; Cocker, Eric; Ho, Tatt Wei

    2015-11-24

    Systems, methods and devices are implemented for microscope imaging solutions. One embodiment of the present disclosure is directed toward an epifluorescence microscope. The microscope includes an image capture circuit including an array of optical sensor. An optical arrangement is configured to direct excitation light of less than about 1 mW to a target object in a field of view of that is at least 0.5 mm.sup.2 and to direct epi-fluorescence emission caused by the excitation light to the array of optical sensors. The optical arrangement and array of optical sensors are each sufficiently close to the target object to provide at least 2.5 .mu.m resolution for an image of the field of view.

  3. Microscopy imaging device with advanced imaging properties

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Kunal; Burns, Laurie; El Gamal, Abbas; Schnitzer, Mark J.; Cocker, Eric; Ho, Tatt Wei

    2017-04-25

    Systems, methods and devices are implemented for microscope imaging solutions. One embodiment of the present disclosure is directed toward an epifluorescence microscope. The microscope includes an image capture circuit including an array of optical sensor. An optical arrangement is configured to direct excitation light of less than about 1 mW to a target object in a field of view of that is at least 0.5 mm.sup.2 and to direct epi-fluorescence emission caused by the excitation light to the array of optical sensors. The optical arrangement and array of optical sensors are each sufficiently close to the target object to provide at least 2.5 .mu.m resolution for an image of the field of view.

  4. Microscopy imaging device with advanced imaging properties

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Kunal; Burns, Laurie; El Gamal, Abbas; Schnitzer, Mark J.; Cocker, Eric; Ho, Tatt Wei

    2016-10-25

    Systems, methods and devices are implemented for microscope imaging solutions. One embodiment of the present disclosure is directed toward an epifluorescence microscope. The microscope includes an image capture circuit including an array of optical sensor. An optical arrangement is configured to direct excitation light of less than about 1 mW to a target object in a field of view of that is at least 0.5 mm.sup.2 and to direct epi-fluorescence emission caused by the excitation light to the array of optical sensors. The optical arrangement and array of optical sensors are each sufficiently close to the target object to provide at least 2.5 .mu.m resolution for an image of the field of view.

  5. Microscopy imaging device with advanced imaging properties

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Kunal; Burns, Laurie; El Gamal, Abbas; Schnitzer, Mark J.; Cocker, Eric; Ho, Tatt Wei

    2016-11-22

    Systems, methods and devices are implemented for microscope imaging solutions. One embodiment of the present disclosure is directed toward an epifluorescence microscope. The microscope includes an image capture circuit including an array of optical sensor. An optical arrangement is configured to direct excitation light of less than about 1 mW to a target object in a field of view of that is at least 0.5 mm.sup.2 and to direct epi-fluorescence emission caused by the excitation light to the array of optical sensors. The optical arrangement and array of optical sensors are each sufficiently close to the target object to provide at least 2.5 .mu.m resolution for an image of the field of view.

  6. Elements of image processing in localization microscopy

    International Nuclear Information System (INIS)

    Rees, Eric J; Erdelyi, Miklos; Schierle, Gabriele S Kaminski; Kaminski, Clemens F; Knight, Alex

    2013-01-01

    Localization microscopy software generally contains three elements: a localization algorithm to determine fluorophore positions on a specimen, a quality control method to exclude imprecise localizations, and a visualization technique to reconstruct an image of the specimen. Such algorithms may be designed for either sparse or partially overlapping (dense) fluorescence image data, and making a suitable choice of software depends on whether an experiment calls for simplicity and resolution (favouring sparse methods), or for rapid data acquisition and time resolution (requiring dense methods). We discuss the factors involved in this choice. We provide a full set of MATLAB routines as a guide to localization image processing, and demonstrate the usefulness of image simulations as a guide to the potential artefacts that can arise when processing over-dense experimental fluorescence images with a sparse localization algorithm. (special issue article)

  7. Biostatistical analysis of quantitative immunofluorescence microscopy images.

    Science.gov (United States)

    Giles, C; Albrecht, M A; Lam, V; Takechi, R; Mamo, J C

    2016-12-01

    Semiquantitative immunofluorescence microscopy has become a key methodology in biomedical research. Typical statistical workflows are considered in the context of avoiding pseudo-replication and marginalising experimental error. However, immunofluorescence microscopy naturally generates hierarchically structured data that can be leveraged to improve statistical power and enrich biological interpretation. Herein, we describe a robust distribution fitting procedure and compare several statistical tests, outlining their potential advantages/disadvantages in the context of biological interpretation. Further, we describe tractable procedures for power analysis that incorporates the underlying distribution, sample size and number of images captured per sample. The procedures outlined have significant potential for increasing understanding of biological processes and decreasing both ethical and financial burden through experimental optimization. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

  8. Multiwavelength FLIM: new concept for fluorescence diagnosis

    Science.gov (United States)

    Rück, Angelika; Lorenz, S.; Hauser, Carmen; Mosch, S.; Kalinina, S.

    2012-03-01

    Fluorescence guided tumor resection is very well accepted in the case of bladder cancer and brain tumor, respectively. However, false positive results are one of the major problems, which will make the discrimination between tumor tissue and inflammation difficult. In contrast fluorescence lifetime imaging (FLIM) and especially spectral resolved FLIM (SLIM) can significantly improve the analysis. The fluorescence decay of a fluorophore in many cases does not show a simple monoexponential profile. A very complex situation arises, when more than one compound has to be analyzed. This could be the case when endogenous fluorophores of living cells and tissues have to be discriminated to identify oxidative metabolic changes. Other examples are PDT, when different photosensitizer metabolites are observed simultaneously. In those cases a considerable improvement could be achieved when time-resolved and spectral-resolved techniques are simultaneously incorporated. Within this presentation the principles of spectral and time-resolved fluorescence imaging will be discussed. Successful applications as autofluorescence and 5-ALA induced porphyrin fluorescence will be described in more detail.

  9. Direct imaging detectors for electron microscopy

    Science.gov (United States)

    Faruqi, A. R.; McMullan, G.

    2018-01-01

    Electronic detectors used for imaging in electron microscopy are reviewed in this paper. Much of the detector technology is based on the developments in microelectronics, which have allowed the design of direct detectors with fine pixels, fast readout and which are sufficiently radiation hard for practical use. Detectors included in this review are hybrid pixel detectors, monolithic active pixel sensors based on CMOS technology and pnCCDs, which share one important feature: they are all direct imaging detectors, relying on directly converting energy in a semiconductor. Traditional methods of recording images in the electron microscope such as film and CCDs, are mentioned briefly along with a more detailed description of direct electronic detectors. Many applications benefit from the use of direct electron detectors and a few examples are mentioned in the text. In recent years one of the most dramatic advances in structural biology has been in the deployment of the new backthinned CMOS direct detectors to attain near-atomic resolution molecular structures with electron cryo-microscopy (cryo-EM). The development of direct detectors, along with a number of other parallel advances, has seen a very significant amount of new information being recorded in the images, which was not previously possible-and this forms the main emphasis of the review.

  10. Image segmentation for integrated multiphoton microscopy and reflectance confocal microscopy imaging of human skin in vivo.

    Science.gov (United States)

    Chen, Guannan; Lui, Harvey; Zeng, Haishan

    2015-02-01

    Non-invasive cellular imaging of the skin in vivo can be achieved in reflectance confocal microscopy (RCM) and multiphoton microscopy (MPM) modalities to yield complementary images of the skin based on different optical properties. One of the challenges of in vivo microscopy is the delineation (i.e., segmentation) of cellular and subcellular architectural features. In this work we present a method for combining watershed and level-set models for segmentation of multimodality images obtained by an integrated MPM and RCM imaging system from human skin in vivo. Firstly, a segmentation model based on watershed is introduced for obtaining the accurate structure of cell borders from the RCM image. Secondly,, a global region based energy level-set model is constructed for extracting the nucleus of each cell from the MPM image. Thirdly, a local region-based Lagrange Continuous level-set approach is used for segmenting cytoplasm from the MPM image. Experimental results demonstrated that cell borders from RCM image and boundaries of cytoplasm and nucleus from MPM image can be obtained by our segmentation method with better accuracy and effectiveness. We are planning to use this method to perform quantitative analysis of MPM and RCM images of in vivo human skin to study the variations of cellular parameters such as cell size, nucleus size and other mophormetric features with skin pathologies.

  11. Fast algorithms for the analysis of spectral FLIM data

    Science.gov (United States)

    Gregor, I.; Krämer, B.; Koberling, F.; Erdmann, R.; Enderlein, J.; Wahl, M.; Fore, S.

    2011-03-01

    The combination of simultaneous spectral detection together with Fluorescence Lifetime Imaging (sFLIM) allows collecting the complete information inherent to the fluorescence signal. Their fingerprint of lifetime and spectral properties identify the fluorescent labels unambiguously. Multiple labels can be investigated in parallel and separated from inherent auto-fluorescence of the sample. In addition, spectral FLIM FRET has the prospect to allow simultaneous detection of multiple FRET signals with quantitative analysis of FRET-efficiency and degree of binding. Spectral FLIM measurements generate huge amount of data. Suitable analysis procedures must be found to condense the inherent information to answer the scientific questions in a straightforward way. Different analysis techniques have been evaluated for a diversity of applications as multiplex labeling, quantitative determination of environmental parameters and distance measurements via FLIM FRET. In order to reach highest sensitivity in single photon detection, different detector types are investigated and developed. SPAD arrays equipped with micro-lenses promise superior detection efficiency while the integration of a spectrograph with a PMT array is easier to realize and allows for a higher number of detection channels. High detection speed can be realized through parallel TCSPC channels. In order to overcome the limits of the USB 2.0 interface, new interface solutions have been realized for the multichannel TCSPC unit HydraHarp 400.

  12. Confocal microscopy imaging of the biofilm matrix

    DEFF Research Database (Denmark)

    Schlafer, Sebastian; Meyer, Rikke L

    2017-01-01

    The extracellular matrix is an integral part of microbial biofilms and an important field of research. Confocal laser scanning microscopy is a valuable tool for the study of biofilms, and in particular of the biofilm matrix, as it allows real-time visualization of fully hydrated, living specimens....... Confocal microscopes are held by many research groups, and a number of methods for qualitative and quantitative imaging of the matrix have emerged in recent years. This review provides an overview and a critical discussion of techniques used to visualize different matrix compounds, to determine...... the concentration of solutes and the diffusive properties of the biofilm matrix....

  13. Fluorescence resonance energy transfer imaging of CFP/YFP labeled NDH in cyanobacterium cell

    International Nuclear Information System (INIS)

    Ji Dongmei; Lv Wei; Huang Zhengxi; Xia Andong; Xu Min; Ma Weimin; Mi Hualing; Ogawa Teruo

    2007-01-01

    The laser confocal scanning microscopy combined with time-correlated single photon counting imaging technique to obtain fluorescence intensity and fluorescence lifetime images for fluorescence resonance energy transfer measurement is reported. Both the fluorescence lifetime imaging microscopy (FLIM) and intensity images show inhomogeneous cyan fluorescent protein and yellow fluorescent protein (CFP /YFP) expression or inhomogeneous energy transfer between CFP and YFP over whole cell. The results presented in this work show that FLIM could be a potential method to reveal the structure-function behavior of NAD(P)H dehydrogenase complexes in living cell

  14. The phasor-FLIM fingerprints reveal shifts from OXPHOS to enhanced glycolysis in Huntington Disease

    Science.gov (United States)

    Sameni, Sara; Syed, Adeela; Marsh, J. Lawrence; Digman, Michelle A.

    2016-10-01

    Huntington disease (HD) is an autosomal neurodegenerative disorder caused by the expansion of Polyglutamine (polyQ) in exon 1 of the Huntingtin protein. Glutamine repeats below 36 are considered normal while repeats above 40 lead to HD. Impairment in energy metabolism is a common trend in Huntington pathogenesis; however, this effect is not fully understood. Here, we used the phasor approach and Fluorescence Lifetime Imaging Microscopy (FLIM) to measure changes between free and bound fractions of NADH as a indirect measure of metabolic alteration in living cells. Using Phasor-FLIM, pixel maps of metabolic alteration in HEK293 cell lines and in transgenic Drosophila expressing expanded and unexpanded polyQ HTT exon1 in the eye disc were developed. We found a significant shift towards increased free NADH, indicating an increased glycolytic state for cells and tissues expressing the expanded polyQ compared to unexpanded control. In the nucleus, a further lifetime shift occurs towards higher free NADH suggesting a possible synergism between metabolic dysfunction and transcriptional regulation. Our results indicate that metabolic dysfunction in HD shifts to increased glycolysis leading to oxidative stress and cell death. This powerful label free method can be used to screen native HD tissue samples and for potential drug screening.

  15. Picosecond fluorescence lifetime imaging microscope for imaging of living glioma cells

    Science.gov (United States)

    Fang, Qiyin; Wang, Jingjing; Sun, Yinghua; Vernier, Thomas; Papaioannou, Thanassis; Jo, Javier; Thu, Mya M.; Gundersen, Martin A.; Marcu, Laura

    2005-03-01

    In this communication, we report the imaging of living glioma cells using fluorescence lifetime imaging (FLIM) technique. The growing interests in developing novel techniques for diagnosis and minimally invasive therapy of brain tumor have led to microscopic studies of subcellular structures and intracellular processes in glioma cells. Fluorescence microscopy has been used with a number of exogenous molecular probes specific for certain intracellular structures such as mitochondria, peripheral benzodiazepine receptor (PBR), and calcium concentration. When probes with overlapping emission spectra being used, separate samples are required to image each probe individually under conventional fluorescence microscopy. We have developed a wide-field FLIM microscope that uses fluorescence lifetime as an additional contrast for resolving multiple markers in the same essay. The FLIM microscope consists of a violet diode laser and a nitrogen-pumped dye laser to provide tunable sub-nanosecond excitation from UV to NIR. The detection system is based on a time-gated ICCD camera with minimum 80 ps gate width. The performance of the system was evaluated using fluorescence dyes with reported lifetime values. Living rat glioma C6 cells were stained with JC-1 and Rhodamine 123. FLIM images were acquired and their lifetimes in living cells were found in good agreements with values measured in solutions by a time-domain fluorescence spectrometer. These results indicate that imaging of glioma cells using FLIM can resolve multiple spectrally-overlapping probes and provide quantitative functional information about the intracellular environment.

  16. Nonlinear Polarimetric Microscopy for Biomedical Imaging

    Science.gov (United States)

    Samim, Masood

    A framework for the nonlinear optical polarimetry and polarimetric microscopy is developed. Mathematical equations are derived in terms of linear and nonlinear Stokes Mueller formalism, which comprehensively characterize the polarization properties of the incoming and outgoing radiations, and provide structural information about the organization of the investigated materials. The algebraic formalism developed in this thesis simplifies many predictions for a nonlinear polarimetry study and provides an intuitive understanding of various polarization properties for radiations and the intervening medium. For polarimetric microscopy experiments, a custom fast-scanning differential polarization microscope is developed, which is also capable of real-time three-dimensional imaging. The setup is equipped with a pair of high-speed resonant and galvanometric scanning mirrors, and supplemented by advanced adaptive optics and data acquisition modules. The scanning mirrors when combined with the adaptive optics deformable mirror enable fast 3D imaging. Deformable membrane mirrors and genetic algorithm optimization routines are employed to improve the imaging conditions including correcting the optical aberrations, maximizing signal intensities, and minimizing point-spread-functions of the focal volume. A field-programmable-gate array (FPGA) chip is exploited to rapidly acquire and process the multidimensional data. Using the nonlinear optical polarimetry framework and the home-built polarization microscope, a few biologically important tissues are measured and analyzed to gain insight as to their structure and dynamics. The structure and distribution of muscle sarcomere myosins, connective tissue collagen, carbohydrate-rich starch, and fruit fly eye retinal molecules are characterized with revealing polarization studies. In each case, using the theoretical framework, polarization sensitive data are analyzed to decipher the molecular orientations and nonlinear optical

  17. The use of fluorescence microscopy to visualise homotypic interactions of tomato spotted wilt virus nucleocapsid protein in living cells

    NARCIS (Netherlands)

    Snippe, M.; Borst, J.W.; Goldbach, R.W.; Kormelink, R.J.M.

    2005-01-01

    Fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) were employed to study homotypic protein¿protein interactions in living cells. To this end, the nucleocapsid (N) protein of tomato spotted wilt virus (TSWV) was expressed as a fusion protein with either

  18. A framework for creating realistic synthetic fluorescence microscopy image sequences

    CSIR Research Space (South Africa)

    Mabaso, M

    2016-02-01

    Full Text Available Fluorescence microscopy imaging is an important tool in modern biological research, allowing insights into the processes of biological systems. Automated image analysis algorithms help in extracting information from these images. Validation...

  19. Sub-Angstrom microscopy through incoherent imaging and image reconstruction

    International Nuclear Information System (INIS)

    Pennycook, S.J.; Jesson, D.E.; Chisholm, M.F.; Ferridge, A.G.; Seddon, M.J.

    1992-03-01

    Z-contrast scanning transmission electron microscopy (STEM) with a high-angle annular detector breaks the coherence of the imaging process, and provides an incoherent image of a crystal projection. Even in the presence of strong dynamical diffraction, the image can be accurately described as a convolution between an object function, sharply peaked at the projected atomic sites, and the probe intensity profile. Such an image can be inverted intuitively without the need for model structures, and therefore provides the important capability to reveal unanticipated interfacial arrangements. It represents a direct image of the crystal projection, revealing the location of the atomic columns and their relative high-angle scattering power. Since no phase is associated with a peak in the object function or the contrast transfer function, extension to higher resolution is also straightforward. Image restoration techniques such as maximum entropy, in conjunction with the 1.3 Angstrom probe anticipated for a 300 kV STEM, appear to provide a simple and robust route to the achievement of sub-Angstrom resolution electron microscopy

  20. Performance evaluation of spot detection algorithms in fluorescence microscopy images

    CSIR Research Space (South Africa)

    Mabaso, M

    2012-10-01

    Full Text Available Detection of messenger Ribonucleic Acid (mRNA) spots in fluorescence microscopy images is of great importance for biologists seeking better understanding of cell functionality. Fluorescence microscopy and specific staining methods make biological...

  1. Imaging Cytoskeleton Components by Electron Microscopy.

    Science.gov (United States)

    Svitkina, Tatyana

    2016-01-01

    The cytoskeleton is a complex of detergent-insoluble components of the cytoplasm playing critical roles in cell motility, shape generation, and mechanical properties of a cell. Fibrillar polymers-actin filaments, microtubules, and intermediate filaments-are major constituents of the cytoskeleton, which constantly change their organization during cellular activities. The actin cytoskeleton is especially polymorphic, as actin filaments can form multiple higher order assemblies performing different functions. Structural information about cytoskeleton organization is critical for understanding its functions and mechanisms underlying various forms of cellular activity. Because of the nanometer-scale thickness of cytoskeletal fibers, electron microscopy (EM) is a key tool to determine the structure of the cytoskeleton. This article describes application of rotary shadowing (or metal replica) EM for visualization of the cytoskeleton. The procedure is applicable to thin cultured cells growing on glass coverslips and consists of detergent extraction of cells to expose their cytoskeleton, chemical fixation to provide stability, ethanol dehydration and critical point drying to preserve three-dimensionality, rotary shadowing with platinum to create contrast, and carbon coating to stabilize replicas. This technique provides easily interpretable three-dimensional images, in which individual cytoskeletal fibers are clearly resolved, and individual proteins can be identified by immunogold labeling. More importantly, replica EM is easily compatible with live cell imaging, so that one can correlate the dynamics of a cell or its components, e.g., expressed fluorescent proteins, with high resolution structural organization of the cytoskeleton in the same cell.

  2. Modulated electron-multiplied fluorescence lifetime imaging microscope : All-solid-state camera for fluorescence lifetime imaging

    NARCIS (Netherlands)

    Zhao, Q.; Schelen, B.; Schouten, R.

    2012-01-01

    We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device

  3. Functional cardiac imaging by random access microscopy

    Directory of Open Access Journals (Sweden)

    Claudia eCrocini

    2014-10-01

    Full Text Available Advances in the development of voltage sensitive dyes and Ca2+ sensors in combination with innovative microscopy techniques allowed researchers to perform functional measurements with an unprecedented spatial and temporal resolution. At the moment, one of the shortcomings of available technologies is their incapability of imaging multiple fast phenomena while controlling the biological determinants involved. In the near future, ultrafast deflectors can be used to rapidly scan laser beams across the sample, performing optical measurements of action potential and Ca2+ release from multiple sites within cardiac cells and tissues. The same scanning modality could also be used to control local Ca2+ release and membrane electrical activity by activation of caged compounds and light-gated ion channels. With this approach, local Ca2+ or voltage perturbations could be induced, simulating arrhythmogenic events, and their impact on physiological cell activity could be explored. The development of this optical methodology will provide fundamental insights in cardiac disease, boosting new therapeutic strategies, and, more generally, it will represent a new approach for the investigation of the physiology of excitable cells.

  4. Understanding the optics to aid microscopy image segmentation.

    Science.gov (United States)

    Yin, Zhaozheng; Li, Kang; Kanade, Takeo; Chen, Mei

    2010-01-01

    Image segmentation is essential for many automated microscopy image analysis systems. Rather than treating microscopy images as general natural images and rushing into the image processing warehouse for solutions, we propose to study a microscope's optical properties to model its image formation process first using phase contrast microscopy as an exemplar. It turns out that the phase contrast imaging system can be relatively well explained by a linear imaging model. Using this model, we formulate a quadratic optimization function with sparseness and smoothness regularizations to restore the "authentic" phase contrast images that directly correspond to specimen's optical path length without phase contrast artifacts such as halo and shade-off. With artifacts removed, high quality segmentation can be achieved by simply thresholding the restored images. The imaging model and restoration method are quantitatively evaluated on two sequences with thousands of cells captured over several days.

  5. Restoration of uneven illumination in light sheet microscopy images.

    Science.gov (United States)

    Uddin, Mohammad Shorif; Lee, Hwee Kuan; Preibisch, Stephan; Tomancak, Pavel

    2011-08-01

    Light microscopy images suffer from poor contrast due to light absorption and scattering by the media. The resulting decay in contrast varies exponentially across the image along the incident light path. Classical space invariant deconvolution approaches, while very effective in deblurring, are not designed for the restoration of uneven illumination in microscopy images. In this article, we present a modified radiative transfer theory approach to solve the contrast degradation problem of light sheet microscopy (LSM) images. We confirmed the effectiveness of our approach through simulation as well as real LSM images.

  6. Differential levels of metabolic activity in isolated versus confluent/partially confluent HeLa cells are analyzed by autofluorescent NAD(P)H using multi-photon FLIM microscopy

    Science.gov (United States)

    Chandler, Andrea; Chandler, Aaron; Wallrabe, Horst; Periasamy, Ammasi

    2017-02-01

    NAD(P)H is a known biomarker for cellular metabolism; a higher ratio of enzyme-bound NAD(P)H to free/unbound NAD(P)H indicates an increase in metabolic activity. Free NADH has a shorter fluorescence lifetime (τ1), the bound version (τ2) a longer lifetime. FLIM's unique capability to establish inter alia the relative fractions of τ1 (a1%) and τ2 (a2%) in each pixel, determines the level of metabolic activity. The relative abundances of bound NAD(P)H were analyzed for single cells, confluent and partially confluent cells within 3 Fields-of-View (FoVs). A gradient of increasing a 2% levels of bound NAD(P)H from single, partially confluent to confluent cells was observed.

  7. Time-Domain Fluorescence Lifetime Imaging Techniques Suitable for Solid-State Imaging Sensor Arrays

    Directory of Open Access Journals (Sweden)

    Robert K. Henderson

    2012-05-01

    Full Text Available We have successfully demonstrated video-rate CMOS single-photon avalanche diode (SPAD-based cameras for fluorescence lifetime imaging microscopy (FLIM by applying innovative FLIM algorithms. We also review and compare several time-domain techniques and solid-state FLIM systems, and adapt the proposed algorithms for massive CMOS SPAD-based arrays and hardware implementations. The theoretical error equations are derived and their performances are demonstrated on the data obtained from 0.13 μm CMOS SPAD arrays and the multiple-decay data obtained from scanning PMT systems. In vivo two photon fluorescence lifetime imaging data of FITC-albumin labeled vasculature of a P22 rat carcinosarcoma (BD9 rat window chamber are used to test how different algorithms perform on bi-decay data. The proposed techniques are capable of producing lifetime images with enough contrast.

  8. Signal-dependent turnover of the bacterial flagellar switch protein FliM

    Science.gov (United States)

    Delalez, Nicolas J.; Wadhams, George H.; Rosser, Gabriel; Xue, Quan; Brown, Mostyn T.; Dobbie, Ian M.; Berry, Richard M.; Leake, Mark C.; Armitage, Judith P.

    2010-01-01

    Most biological processes are performed by multiprotein complexes. Traditionally described as static entities, evidence is now emerging that their components can be highly dynamic, exchanging constantly with cellular pools. The bacterial flagellar motor contains ∼13 different proteins and provides an ideal system to study functional molecular complexes. It is powered by transmembrane ion flux through a ring of stator complexes that push on a central rotor. The Escherichia coli motor switches direction stochastically in response to binding of the response regulator CheY to the rotor switch component FliM. Much is known of the static motor structure, but we are just beginning to understand the dynamics of its individual components. Here we measure the stoichiometry and turnover of FliM in functioning flagellar motors, by using high-resolution fluorescence microscopy of E. coli expressing genomically encoded YPet derivatives of FliM at physiological levels. We show that the ∼30 FliM molecules per motor exist in two discrete populations, one tightly associated with the motor and the other undergoing stochastic turnover. This turnover of FliM molecules depends on the presence of active CheY, suggesting a potential role in the process of motor switching. In many ways the bacterial flagellar motor is as an archetype macromolecular assembly, and our results may have further implications for the functional relevance of protein turnover in other large molecular complexes. PMID:20498085

  9. Platinum replica electron microscopy: Imaging the cytoskeleton globally and locally.

    Science.gov (United States)

    Svitkina, Tatyana M

    2017-05-01

    Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the "comfort zones" of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Image correction in magneto-optical microscopy

    DEFF Research Database (Denmark)

    Paturi, P.; Larsen, B.H.; Jacobsen, B.A.

    2003-01-01

    An image-processing procedure that assures correct determination of the magnetic field distribution of magneto-optical images is presented. The method remedies image faults resulting from sources that are proportional to the incident light intensity, such as different types of defects...

  11. Reconstruction of Undersampled Atomic Force Microscopy Images

    DEFF Research Database (Denmark)

    Jensen, Tobias Lindstrøm; Arildsen, Thomas; Østergaard, Jan

    2013-01-01

    , or a special case of compressed sensing. We argue that the preferred approach depends upon the type of image. Of the methods proposed for AFM, images containing high frequencies should be reconstructed using basis pursuit from data collected in a spiral pattern. Images without too much high frequency content...

  12. Image Resolution in Scanning Transmission Electron Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Pennycook, S. J.; Lupini, A.R.

    2008-06-26

    Digital images captured with electron microscopes are corrupted by two fundamental effects: shot noise resulting from electron counting statistics and blur resulting from the nonzero width of the focused electron beam. The generic problem of computationally undoing these effects is called image reconstruction and for decades has proved to be one of the most challenging and important problems in imaging science. This proposal concerned the application of the Pixon method, the highest-performance image-reconstruction algorithm yet devised, to the enhancement of images obtained from the highest-resolution electron microscopes in the world, now in operation at Oak Ridge National Laboratory.

  13. Translation Microscopy (TRAM) for super-resolution imaging

    Science.gov (United States)

    Qiu, Zhen; Wilson, Rhodri S; Liu, Yuewei; R Dun, Alison; Saleeb, Rebecca S; Liu, Dongsheng; Rickman, Colin; Frame, Margaret; Duncan, Rory R; Lu, Weiping

    2016-01-01

    Super-resolution microscopy is transforming our understanding of biology but accessibility is limited by its technical complexity, high costs and the requirement for bespoke sample preparation. We present a novel, simple and multi-color super-resolution microscopy technique, called translation microscopy (TRAM), in which a super-resolution image is restored from multiple diffraction-limited resolution observations using a conventional microscope whilst translating the sample in the image plane. TRAM can be implemented using any microscope, delivering up to 7-fold resolution improvement. We compare TRAM with other super-resolution imaging modalities, including gated stimulated emission deletion (gSTED) microscopy and atomic force microscopy (AFM). We further developed novel ‘ground-truth’ DNA origami nano-structures to characterize TRAM, as well as applying it to a multi-color dye-stained cellular sample to demonstrate its fidelity, ease of use and utility for cell biology. PMID:26822455

  14. Microscopy

    Science.gov (United States)

    Patricia A. Moss; Les Groom

    2001-01-01

    Microscopy is the study and interpretation of images produced by a microscope. "Interpretation" is the keyword, because the microscope enables one to see structures that are too small or too close together to be resolved by the unaided eye. (The human eye cannot separate two points or lines that are closer together than 0.1 mm.) it is important to...

  15. Classifying and segmenting microscopy images with deep multiple instance learning.

    Science.gov (United States)

    Kraus, Oren Z; Ba, Jimmy Lei; Frey, Brendan J

    2016-06-15

    High-content screening (HCS) technologies have enabled large scale imaging experiments for studying cell biology and for drug screening. These systems produce hundreds of thousands of microscopy images per day and their utility depends on automated image analysis. Recently, deep learning approaches that learn feature representations directly from pixel intensity values have dominated object recognition challenges. These tasks typically have a single centered object per image and existing models are not directly applicable to microscopy datasets. Here we develop an approach that combines deep convolutional neural networks (CNNs) with multiple instance learning (MIL) in order to classify and segment microscopy images using only whole image level annotations. We introduce a new neural network architecture that uses MIL to simultaneously classify and segment microscopy images with populations of cells. We base our approach on the similarity between the aggregation function used in MIL and pooling layers used in CNNs. To facilitate aggregating across large numbers of instances in CNN feature maps we present the Noisy-AND pooling function, a new MIL operator that is robust to outliers. Combining CNNs with MIL enables training CNNs using whole microscopy images with image level labels. We show that training end-to-end MIL CNNs outperforms several previous methods on both mammalian and yeast datasets without requiring any segmentation steps. Torch7 implementation available upon request. oren.kraus@mail.utoronto.ca. © The Author 2016. Published by Oxford University Press.

  16. Pyramidal nanowire tip for atomic force microscopy and thermal imaging

    NARCIS (Netherlands)

    Burouni, N.; Sarajlic, Edin; Siekman, Martin Herman; Abelmann, Leon; Tas, Niels Roelof

    2012-01-01

    We present a novel 3D nanowire pyramid as scanning microscopy probe for thermal imaging and atomic force microscopy. This probe is fabricated by standard micromachining and conventional optical contact lithography. The probe features an AFM-type cantilever with a sharp pyramidal tip composed of four

  17. Image processing for drift compensation in fluorescence microscopy

    DEFF Research Database (Denmark)

    Petersen, Steffen; Thiagarajan, Viruthachalam; Coutinho, Isabel

    2013-01-01

    Fluorescence microscopy is characterized by low background noise, thus a fluorescent object appears as an area of high signal/noise. Thermal gradients may result in apparent motion of the object, leading to a blurred image. Here, we have developed an image processing methodology that may remove....../reduce blur significantly for any type of microscopy. A total of ~100 images were acquired with a pixel size of 30 nm. The acquisition time for each image was approximately 1second. We can quantity the drift in X and Y using the sub pixel accuracy computed centroid location of an image object in each frame....... We can measure drifts down to approximately 10 nm in size and a drift-compensated image can therefore be reconstructed on a grid of the same size using the “Shift and Add” approach leading to an image of identical size asthe individual image. We have also reconstructed the image using a 3 fold larger...

  18. Ferritin protein imaging and detection by magnetic force microscopy.

    Science.gov (United States)

    Hsieh, Chiung-Wen; Zheng, Bin; Hsieh, Shuchen

    2010-03-14

    Magnetic force microscopy was used to image and detect ferritin proteins and the strength of the magnetic signal is discussed, revealing a large workable lift height between the magnetic tip and the ferritin sample.

  19. Revealing the cellular metabolism and microstructural changes in vivo in senescing Acer saccharum leaves using two-photon FLIM and full-field OCM

    Science.gov (United States)

    Chakraborty, Sandeep; Anna, Tulsi; Kuo, Wen-Chuan; Chiou, Arthur

    2016-10-01

    Seasonal as well as climate changes have immense effect on bud burst, leaf color and leaf abscission. Autumn phenology of leaves is clearly distinguishable in deciduous plant leaves where the leaf color changes from green to red (leaf senescence). In this work, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) and full-field optical coherence microscopy (FF-OCM) were applied to study mitochondrial activity and microstructural changes, respectively, in the senescence of Acer saccharum (Sugar maple) leaves. Fluorescence lifetime of reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] was recorded using 2P-FLIM to quantify the cellular metabolic changes. Compared to the green leaves, the red leaves showed a 19% increase (P fluorescence lifetime of NAD(P)H, and a 52% decrease (p protein-bound NAD(P)H ratio. This infers a significant change in mitochondrial metabolic regulation in red leaves in contrast to green leaves. Additionally, en-face sectional images at 0.8 μm axial resolutions of the green and the red color Acer saccharum leaves via FF-OCM using white light emitting diode (WLED) showed a well-defined microstructure of epicuticular waxy layer in green leaves as compared to red leaves where disintegrated microstructure was observed. Our approach can potentially be used to correlate mitochondrial activity with epicuticular microstructural changes in senescing leaves and other biological tissues.

  20. QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY

    Directory of Open Access Journals (Sweden)

    Merete Krog Raarup

    2011-05-01

    Full Text Available This paper discusses recent advances in confocal laser scanning microscopy (CLSM for imaging of 3D structure as well as quantitative characterization of biomolecular interactions and diffusion behaviour by means of one- and two-photon excitation. The use of CLSM for improved stereological length estimation in thick (up to 0.5 mm tissue is proposed. The techniques of FRET (Fluorescence Resonance Energy Transfer, FLIM (Fluorescence Lifetime Imaging Microscopy, FCS (Fluorescence Correlation Spectroscopy and FRAP (Fluorescence Recovery After Photobleaching are introduced and their applicability for quantitative imaging of biomolecular (co-localization and trafficking in live cells described. The advantage of two-photon versus one-photon excitation in relation to these techniques is discussed.

  1. Super-resolution Microscopy in Plant Cell Imaging.

    Science.gov (United States)

    Komis, George; Šamajová, Olga; Ovečka, Miroslav; Šamaj, Jozef

    2015-12-01

    Although the development of super-resolution microscopy methods dates back to 1994, relevant applications in plant cell imaging only started to emerge in 2010. Since then, the principal super-resolution methods, including structured-illumination microscopy (SIM), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and stimulated emission depletion microscopy (STED), have been implemented in plant cell research. However, progress has been limited due to the challenging properties of plant material. Here we summarize the basic principles of existing super-resolution methods and provide examples of applications in plant science. The limitations imposed by the nature of plant material are reviewed and the potential for future applications in plant cell imaging is highlighted. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. High-resolution imaging by scanning electron microscopy of semithin sections in correlation with light microscopy.

    Science.gov (United States)

    Koga, Daisuke; Kusumi, Satoshi; Shodo, Ryusuke; Dan, Yukari; Ushiki, Tatsuo

    2015-12-01

    In this study, we introduce scanning electron microscopy (SEM) of semithin resin sections. In this technique, semithin sections were adhered on glass slides, stained with both uranyl acetate and lead citrate, and observed with a backscattered electron detector at a low accelerating voltage. As the specimens are stained in the same manner as conventional transmission electron microscopy (TEM), the contrast of SEM images of semithin sections was similar to TEM images of ultrathin sections. Using this technique, wide areas of semithin sections were also observed by SEM, without the obstruction of grids, which was inevitable for traditional TEM. This study also applied semithin section SEM to correlative light and electron microscopy. Correlative immunofluorescence microscopy and immune-SEM were performed in semithin sections of LR white resin-embedded specimens using a FluoroNanogold-labeled secondary antibody. Because LR white resin is hydrophilic and electron stable, this resin is suitable for immunostaining and SEM observation. Using correlative microscopy, the precise localization of the primary antibody was demonstrated by fluorescence microscopy and SEM. This method has great potential for studies examining the precise localization of molecules, including Golgi- and ER-associated proteins, in correlation with LM and SEM. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Photobleaching correction in fluorescence microscopy images

    International Nuclear Information System (INIS)

    Vicente, Nathalie B; Diaz Zamboni, Javier E; Adur, Javier F; Paravani, Enrique V; Casco, Victor H

    2007-01-01

    Fluorophores are used to detect molecular expression by highly specific antigen-antibody reactions in fluorescence microscopy techniques. A portion of the fluorophore emits fluorescence when irradiated with electromagnetic waves of particular wavelengths, enabling its detection. Photobleaching irreversibly destroys fluorophores stimulated by radiation within the excitation spectrum, thus eliminating potentially useful information. Since this process may not be completely prevented, techniques have been developed to slow it down or to correct resulting alterations (mainly, the decrease in fluorescent signal). In the present work, the correction by photobleaching curve was studied using E-cadherin (a cell-cell adhesion molecule) expression in Bufo arenarum embryos. Significant improvements were observed when applying this simple, inexpensive and fast technique

  4. Image processing for drift compensation in fluorescence microscopy

    DEFF Research Database (Denmark)

    Petersen, Steffen B.; Thiagarajan, Viruthachalam; Coutinho, Isabel

    2013-01-01

    Fluorescence microscopy is characterized by low background noise, thus a fluorescent object appears as an area of high signal/noise. Thermal gradients may result in apparent motion of the object, leading to a blurred image. Here, we have developed an image processing methodology that may remove...

  5. Recent progress in NMR microscopy towards cellular imaging

    International Nuclear Information System (INIS)

    Cho, Z.H.; Ahn, C.B.; Juh, S.C.; Friedenberg, R.M.; Fraser, S.E.; Jacobs, R.E.

    1990-01-01

    Recent advances in NMR microscopy based on fundamental physical parameters and experimental factors are discussed. We consider fundamental resolution limits due to molecular diffusion and the experimental system bandwidth, as well as practical resolution limits arising from poor signal-to-noise ratio due to small imaging voxel size and finite line broadening due to signal attenuation brought about by diffusion. Several microscopic imaging pulse sequences are presented and applied to elucidating cellular imaging problems such as the cell lineage patterns in Xenopus laevis embryos. Experimental results obtained with 7.0 T NMR microscopy system are presented. (author)

  6. Transmission electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1989-01-01

    The aim of this book is to present the theory of image and contrast formation and the analytical modes in transmission electron microscopy The principles of particle and wave optics of electrons are described Electron-specimen interactions are discussed for evaluating the theory of scattering and phase contrast Also discussed are the kinematical and dynamical theories of electron diffraction and their applications for crystal structure determination and imaging of lattice defects X-ray microanalysis and energy-loss spectroscopy are treated as analytical methods The second edition includes discussion of recent progress, especially in the areas of energy-loss spectroscopy, crystal-lattice imaging and reflection electron microscopy

  7. Scanning Tunneling Microscopy methods for spectroscopic imaging of subsurface interfaces

    Science.gov (United States)

    Bell, L. D.; Kaiser, W. J.

    1988-01-01

    A new method for spatially-resolved, spectroscopic investigation of subsurface interface structure has been developed. The method, Ballistic Electron Emission Microscopy (BEEM), is based on Scanning Tunneling Microscopy (STM) techniques. BEEM combines STM vacuum tunneling with unique ballistic electron spectroscopy capabilities. BEEM enables, for the first time, direct imaging of subsurface interface electronic properties with nanometer spatial resolution. STM topographic images of surface structure and BEEM images of subsurface properties are obtained simultaneously. BEEM capabilities are demonstrated by investigation of important metal-semiconductor interfaces.

  8. Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with in silico virtual microscopy

    International Nuclear Information System (INIS)

    Flaberg, Emilie; Sabelström, Per; Strandh, Christer; Szekely, Laszlo

    2008-01-01

    Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture. Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale in silico image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM). We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates. The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes

  9. Scanning electron microscopy image representativeness: morphological data on nanoparticles.

    Science.gov (United States)

    Odziomek, Katarzyna; Ushizima, Daniela; Oberbek, Przemyslaw; Kurzydłowski, Krzysztof Jan; Puzyn, Tomasz; Haranczyk, Maciej

    2017-01-01

    A sample of a nanomaterial contains a distribution of nanoparticles of various shapes and/or sizes. A scanning electron microscopy image of such a sample often captures only a fragment of the morphological variety present in the sample. In order to quantitatively analyse the sample using scanning electron microscope digital images, and, in particular, to derive numerical representations of the sample morphology, image content has to be assessed. In this work, we present a framework for extracting morphological information contained in scanning electron microscopy images using computer vision algorithms, and for converting them into numerical particle descriptors. We explore the concept of image representativeness and provide a set of protocols for selecting optimal scanning electron microscopy images as well as determining the smallest representative image set for each of the morphological features. We demonstrate the practical aspects of our methodology by investigating tricalcium phosphate, Ca 3 (PO 4 ) 2 , and calcium hydroxyphosphate, Ca 5 (PO 4 ) 3 (OH), both naturally occurring minerals with a wide range of biomedical applications. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

  10. Imaging the chemical activity of single nanoparticles with optical microscopy.

    Science.gov (United States)

    Wang, Wei

    2018-04-03

    Nanomaterials exhibit structural and functional heterogeneity among individual nanoparticles, thus requiring a capability to study single nanoparticles. While electron microscopes often provide static images of their chemical composition, morphology and structure, imaging the chemical activity of single nanoparticles is highly desirable for exploring the structure-activity relationship via a bottom-up strategy, to understand their microscopic reaction mechanisms and kinetics, and to identify a minority subpopulation with extraordinary activity. Recently, various optical microscopes have been emerging as powerful techniques towards this goal, owing to their non-invasive nature, excellent sensitivity, diversified spectroscopic principles and sufficient spatial and temporal resolution. In this review, we first introduce the motivational concept and the strength of using optical microscopy to study the chemical activity of single nanoparticles. In the second section, five types of commonly used optical microscopy, fluorescence microscopy, dark-field microscopy, surface plasmon resonance microscopy, Raman microscopy and photothermal microscopy are described, with an emphasis on their applicable nanomaterials and mechanisms for application. Recent achievements of these techniques in nanosensing, nanoelectrochemistry and nanocatalysis are surveyed and summarized in the subsequent sections, respectively. We finally conclude with our perspective on the remaining challenges and the future trends in this field.

  11. Low Dimensional Representation of Fisher Vectors for Microscopy Image Classification.

    Science.gov (United States)

    Song, Yang; Li, Qing; Huang, Heng; Feng, Dagan; Chen, Mei; Cai, Weidong

    2017-08-01

    Microscopy image classification is important in various biomedical applications, such as cancer subtype identification, and protein localization for high content screening. To achieve automated and effective microscopy image classification, the representative and discriminative capability of image feature descriptors is essential. To this end, in this paper, we propose a new feature representation algorithm to facilitate automated microscopy image classification. In particular, we incorporate Fisher vector (FV) encoding with multiple types of local features that are handcrafted or learned, and we design a separation-guided dimension reduction method to reduce the descriptor dimension while increasing its discriminative capability. Our method is evaluated on four publicly available microscopy image data sets of different imaging types and applications, including the UCSB breast cancer data set, MICCAI 2015 CBTC challenge data set, and IICBU malignant lymphoma, and RNAi data sets. Our experimental results demonstrate the advantage of the proposed low-dimensional FV representation, showing consistent performance improvement over the existing state of the art and the commonly used dimension reduction techniques.

  12. Imaging hydrated microbial extracellular polymers: Comparative analysis by electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Dohnalkova, A.C.; Marshall, M. J.; Arey, B. W.; Williams, K. H.; Buck, E. C.; Fredrickson, J. K.

    2011-01-01

    Microbe-mineral and -metal interactions represent a major intersection between the biosphere and geosphere but require high-resolution imaging and analytical tools for investigating microscale associations. Electron microscopy has been used extensively for geomicrobial investigations and although used bona fide, the traditional methods of sample preparation do not preserve the native morphology of microbiological components, especially extracellular polymers. Herein, we present a direct comparative analysis of microbial interactions using conventional electron microscopy approaches of imaging at room temperature and a suite of cryogenic electron microscopy methods providing imaging in the close-to-natural hydrated state. In situ, we observed an irreversible transformation of the hydrated bacterial extracellular polymers during the traditional dehydration-based sample preparation that resulted in their collapse into filamentous structures. Dehydration-induced polymer collapse can lead to inaccurate spatial relationships and hence could subsequently affect conclusions regarding nature of interactions between microbial extracellular polymers and their environment.

  13. Imaging Cytometry of Human Leukocytes with Third Harmonic Generation Microscopy

    Science.gov (United States)

    Wu, Cheng-Ham; Wang, Tzung-Dau; Hsieh, Chia-Hung; Huang, Shih-Hung; Lin, Jong-Wei; Hsu, Szu-Chun; Wu, Hau-Tieng; Wu, Yao-Ming; Liu, Tzu-Ming

    2016-11-01

    Based on third-harmonic-generation (THG) microscopy and a k-means clustering algorithm, we developed a label-free imaging cytometry method to differentiate and determine the types of human leukocytes. According to the size and average intensity of cells in THG images, in a two-dimensional scatter plot, the neutrophils, monocytes, and lymphocytes in peripheral blood samples from healthy volunteers were clustered into three differentiable groups. Using these features in THG images, we could count the number of each of the three leukocyte types both in vitro and in vivo. The THG imaging-based counting results agreed well with conventional blood count results. In the future, we believe that the combination of this THG microscopy-based imaging cytometry approach with advanced texture analysis of sub-cellular features can differentiate and count more types of blood cells with smaller quantities of blood.

  14. Transmission electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1993-01-01

    "Transmission Electron Microscopy" presents the theory of image and contrastformation, and the analytical modes in transmission electron microscopy Theprinciples of particle and wave optics of electrons are described Electron-specimen interactions are discussed for evaluating the theory of scattering and phase contrast Also analysed are the kinetical and dynamical theories of electron diffraction and their applications for crystal-structure determination and imaging of lattices and their defects X-ray microanalysis and electron energy-loss spectroscopy are treated as analytical methods The third edition includes a brief discussionof Schottky emission guns, some clarification of minor details, and references to the recent literature

  15. Quantitative Image Restoration in Bright Field Optical Microscopy.

    Science.gov (United States)

    Gutiérrez-Medina, Braulio; Sánchez Miranda, Manuel de Jesús

    2017-11-07

    Bright field (BF) optical microscopy is regarded as a poor method to observe unstained biological samples due to intrinsic low image contrast. We introduce quantitative image restoration in bright field (QRBF), a digital image processing method that restores out-of-focus BF images of unstained cells. Our procedure is based on deconvolution, using a point spread function modeled from theory. By comparing with reference images of bacteria observed in fluorescence, we show that QRBF faithfully recovers shape and enables quantify size of individual cells, even from a single input image. We applied QRBF in a high-throughput image cytometer to assess shape changes in Escherichia coli during hyperosmotic shock, finding size heterogeneity. We demonstrate that QRBF is also applicable to eukaryotic cells (yeast). Altogether, digital restoration emerges as a straightforward alternative to methods designed to generate contrast in BF imaging for quantitative analysis. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  16. Three-dimensional nanoscale imaging by plasmonic Brownian microscopy

    Science.gov (United States)

    Labno, Anna; Gladden, Christopher; Kim, Jeongmin; Lu, Dylan; Yin, Xiaobo; Wang, Yuan; Liu, Zhaowei; Zhang, Xiang

    2017-12-01

    Three-dimensional (3D) imaging at the nanoscale is a key to understanding of nanomaterials and complex systems. While scanning probe microscopy (SPM) has been the workhorse of nanoscale metrology, its slow scanning speed by a single probe tip can limit the application of SPM to wide-field imaging of 3D complex nanostructures. Both electron microscopy and optical tomography allow 3D imaging, but are limited to the use in vacuum environment due to electron scattering and to optical resolution in micron scales, respectively. Here we demonstrate plasmonic Brownian microscopy (PBM) as a way to improve the imaging speed of SPM. Unlike photonic force microscopy where a single trapped particle is used for a serial scanning, PBM utilizes a massive number of plasmonic nanoparticles (NPs) under Brownian diffusion in solution to scan in parallel around the unlabeled sample object. The motion of NPs under an evanescent field is three-dimensionally localized to reconstruct the super-resolution topology of 3D dielectric objects. Our method allows high throughput imaging of complex 3D structures over a large field of view, even with internal structures such as cavities that cannot be accessed by conventional mechanical tips in SPM.

  17. Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy

    Science.gov (United States)

    2013-01-01

    Background Delignification pretreatments of biomass and methods to assess their efficacy are crucial for biomass-to-biofuels research and technology. Here, we applied confocal and fluorescence lifetime imaging microscopy (FLIM) using one- and two-photon excitation to map the lignin distribution within bagasse fibers pretreated with acid and alkali. The evaluated spectra and decay times are correlated with previously calculated lignin fractions. We have also investigated the influence of the pretreatment on the lignin distribution in the cell wall by analyzing the changes in the fluorescence characteristics using two-photon excitation. Eucalyptus fibers were also analyzed for comparison. Results Fluorescence spectra and variations of the decay time correlate well with the delignification yield and the lignin distribution. The decay dependences are considered two-exponential, one with a rapid (τ1) and the other with a slow (τ2) decay time. The fastest decay is associated to concentrated lignin in the bagasse and has a low sensitivity to the treatment. The fluorescence decay time became longer with the increase of the alkali concentration used in the treatment, which corresponds to lignin emission in a less concentrated environment. In addition, the two-photon fluorescence spectrum is very sensitive to lignin content and accumulation in the cell wall, broadening with the acid pretreatment and narrowing with the alkali one. Heterogeneity of the pretreated cell wall was observed. Conclusions Our results reveal lignin domains with different concentration levels. The acid pretreatment caused a disorder in the arrangement of lignin and its accumulation in the external border of the cell wall. The alkali pretreatment efficiently removed lignin from the middle of the bagasse fibers, but was less effective in its removal from their surfaces. Our results evidenced a strong correlation between the decay times of the lignin fluorescence and its distribution within the cell

  18. Super-Resolution Real Imaging in Microsphere-Assisted Microscopy.

    Directory of Open Access Journals (Sweden)

    Hok Sum Sam Lai

    Full Text Available Microsphere-assisted microscopy has received a lot of attention recently due to its simplicity and its capability to surpass the diffraction limit. However, to date, sub-diffraction-limit features have only been observed in virtual images formed through the microspheres. We show that it is possible to form real, super-resolution images using high-refractive index microspheres. Also, we report on how changes to a microsphere's refractive index and size affect image formation and planes. The relationship between the focus position and the additional magnification factor is also investigated using experimental and theoretical methods. We demonstrate that such a real imaging mode, combined with the use of larger microspheres, can enlarge sub-diffraction-limit features up to 10 times that of wide-field microscopy's magnification with a field-of-view diameter of up to 9 μm.

  19. Cytology 3D structure formation based on optical microscopy images

    International Nuclear Information System (INIS)

    Pronichev, A N; Polyakov, E V; Zaitsev, S M; Shabalova, I P; Djangirova, T V

    2017-01-01

    The article the article is devoted to optimization of the parameters of imaging of biological preparations in optical microscopy using a multispectral camera in visible range of electromagnetic radiation. A model for the image forming of virtual preparations was proposed. The optimum number of layers was determined for the object scan in depth and holistic perception of its switching according to the results of the experiment. (paper)

  20. Watershed Merge Tree Classification for Electron Microscopy Image Segmentation

    OpenAIRE

    Liu, Ting; Jurrus, Elizabeth; Seyedhosseini, Mojtaba; Ellisman, Mark; Tasdizen, Tolga

    2012-01-01

    Automated segmentation of electron microscopy (EM) images is a challenging problem. In this paper, we present a novel method that utilizes a hierarchical structure and boundary classification for 2D neuron segmentation. With a membrane detection probability map, a watershed merge tree is built for the representation of hierarchical region merging from the watershed algorithm. A boundary classifier is learned with non-local image features to predict each potential merge in the tree, upon which...

  1. Cytology 3D structure formation based on optical microscopy images

    Science.gov (United States)

    Pronichev, A. N.; Polyakov, E. V.; Shabalova, I. P.; Djangirova, T. V.; Zaitsev, S. M.

    2017-01-01

    The article the article is devoted to optimization of the parameters of imaging of biological preparations in optical microscopy using a multispectral camera in visible range of electromagnetic radiation. A model for the image forming of virtual preparations was proposed. The optimum number of layers was determined for the object scan in depth and holistic perception of its switching according to the results of the experiment.

  2. Confocal microscopy for astrocyte in vivo imaging: Recycle and reuse in microscopy

    Science.gov (United States)

    Pérez-Alvarez, Alberto; Araque, Alfonso; Martín, Eduardo D.

    2013-01-01

    In vivo imaging is one of the ultimate and fundamental approaches for the study of the brain. Two-photon laser scanning microscopy (2PLSM) constitutes the state-of-the-art technique in current neuroscience to address questions regarding brain cell structure, development and function, blood flow regulation and metabolism. This technique evolved from laser scanning confocal microscopy (LSCM), which impacted the field with a major improvement in image resolution of live tissues in the 1980s compared to widefield microscopy. While nowadays some of the unparalleled features of 2PLSM make it the tool of choice for brain studies in vivo, such as the possibility to image deep within a tissue, LSCM can still be useful in this matter. Here we discuss the validity and limitations of LSCM and provide a guide to perform high-resolution in vivo imaging of the brain of live rodents with minimal mechanical disruption employing LSCM. We describe the surgical procedure and experimental setup that allowed us to record intracellular calcium variations in astrocytes evoked by sensory stimulation, and to monitor intact neuronal dendritic spines and astrocytic processes as well as blood vessel dynamics. Therefore, in spite of certain limitations that need to be carefully considered, LSCM constitutes a useful, convenient, and affordable tool for brain studies in vivo. PMID:23658537

  3. Embryonic Heart Morphogenesis from Confocal Microscopy Imaging and Automatic Segmentation

    Directory of Open Access Journals (Sweden)

    Hongda Mao

    2013-01-01

    Full Text Available Embryonic heart morphogenesis (EHM is a complex and dynamic process where the heart transforms from a single tube into a four-chambered pump. This process is of great biological and clinical interest but is still poorly understood for two main reasons. On the one hand, the existing imaging modalities for investigating EHM suffered from either limited penetration depth or limited spatial resolution. On the other hand, current works typically adopted manual segmentation, which was tedious, subjective, and time consuming considering the complexity of developing heart geometry and the large size of images. In this paper, we propose to utilize confocal microscopy imaging with tissue optical immersion clearing technique to image the heart at different stages of development for EHM study. The imaging method is able to produce high spatial resolution images and achieve large penetration depth at the same time. Furthermore, we propose a novel convex active contour model for automatic image segmentation. The model has the ability to deal with intensity fall-off in depth which is characterized by confocal microscopy images. We acquired the images of embryonic quail hearts from day 6 to day 14 of incubation for EHM study. The experimental results were promising and provided us with an insight view of early heart growth pattern and also paved the road for data-driven heart growth modeling.

  4. Scanning electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1998-01-01

    Scanning Electron Microscopy provides a description of the physics of electron-probe formation and of electron-specimen interations The different imaging and analytical modes using secondary and backscattered electrons, electron-beam-induced currents, X-ray and Auger electrons, electron channelling effects, and cathodoluminescence are discussed to evaluate specific contrasts and to obtain quantitative information

  5. Imaging and manipulation of single viruses by atomic force microscopy

    NARCIS (Netherlands)

    Baclayon, M.; Wuite, G.J.L.; Roos, W.H.

    2010-01-01

    The recent developments in virus research and the application of functional viral particles in nanotechnology and medicine rely on sophisticated imaging and manipulation techniques at nanometre resolution in liquid, air and vacuum. Atomic force microscopy (AFM) is a tool that combines these

  6. Introduction: Advances in Optical Coherence Tomography, Photoacoustic Imaging, and Microscopy

    OpenAIRE

    Li, Xingde; Beard, Paul C.; Georgakoudi, Irene

    2010-01-01

    The editors introduce the Biomedical Optics Express feature issue, “Advances in Optical Coherence Tomography, Photoacoustic Imaging, and Microscopy,” which combines three technical areas from the 2010 Optical Society of America (OSA), Biomedical Optics (BIOMED) Topical Meeting held on 11–14 April in Miami, Florida, and includes contributions from conference attendees.

  7. Volume scanning electron microscopy for imaging biological ultrastructure.

    Science.gov (United States)

    Titze, Benjamin; Genoud, Christel

    2016-11-01

    Electron microscopy (EM) has been a key imaging method to investigate biological ultrastructure for over six decades. In recent years, novel volume EM techniques have significantly advanced nanometre-scale imaging of cells and tissues in three dimensions. Previously, this had depended on the slow and error-prone manual tasks of cutting and handling large numbers of sections, and imaging them one-by-one with transmission EM. Now, automated volume imaging methods mostly based on scanning EM (SEM) allow faster and more reliable acquisition of serial images through tissue volumes and achieve higher z-resolution. Various software tools have been developed to manipulate the acquired image stacks and facilitate quantitative analysis. Here, we introduce three volume SEM methods: serial block-face electron microscopy (SBEM), focused ion beam SEM (FIB-SEM) and automated tape-collecting ultramicrotome SEM (ATUM-SEM). We discuss and compare their capabilities, provide an overview of the full volume SEM workflow for obtaining 3D datasets and showcase different applications for biological research. © 2016 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

  8. Multidirectional Image Sensing for Microscopy Based on a Rotatable Robot.

    Science.gov (United States)

    Shen, Yajing; Wan, Wenfeng; Zhang, Lijun; Yong, Li; Lu, Haojian; Ding, Weili

    2015-12-15

    Image sensing at a small scale is essentially important in many fields, including microsample observation, defect inspection, material characterization and so on. However, nowadays, multi-directional micro object imaging is still very challenging due to the limited field of view (FOV) of microscopes. This paper reports a novel approach for multi-directional image sensing in microscopes by developing a rotatable robot. First, a robot with endless rotation ability is designed and integrated with the microscope. Then, the micro object is aligned to the rotation axis of the robot automatically based on the proposed forward-backward alignment strategy. After that, multi-directional images of the sample can be obtained by rotating the robot within one revolution under the microscope. To demonstrate the versatility of this approach, we view various types of micro samples from multiple directions in both optical microscopy and scanning electron microscopy, and panoramic images of the samples are processed as well. The proposed method paves a new way for the microscopy image sensing, and we believe it could have significant impact in many fields, especially for sample detection, manipulation and characterization at a small scale.

  9. Multidirectional Image Sensing for Microscopy Based on a Rotatable Robot

    Directory of Open Access Journals (Sweden)

    Yajing Shen

    2015-12-01

    Full Text Available Image sensing at a small scale is essentially important in many fields, including microsample observation, defect inspection, material characterization and so on. However, nowadays, multi-directional micro object imaging is still very challenging due to the limited field of view (FOV of microscopes. This paper reports a novel approach for multi-directional image sensing in microscopes by developing a rotatable robot. First, a robot with endless rotation ability is designed and integrated with the microscope. Then, the micro object is aligned to the rotation axis of the robot automatically based on the proposed forward-backward alignment strategy. After that, multi-directional images of the sample can be obtained by rotating the robot within one revolution under the microscope. To demonstrate the versatility of this approach, we view various types of micro samples from multiple directions in both optical microscopy and scanning electron microscopy, and panoramic images of the samples are processed as well. The proposed method paves a new way for the microscopy image sensing, and we believe it could have significant impact in many fields, especially for sample detection, manipulation and characterization at a small scale.

  10. Real-time fluorescence lifetime imaging system with a 32 × 32 0.13?m CMOS low dark-count single-photon avalanche diode array

    NARCIS (Netherlands)

    Li, D.U.; Arlt, J.; Richardson, J.; Walker, R.; Buts, A.; Stoppa, D.; Charbon, E.; Henderson, R.

    2010-01-01

    A compact real-time fluorescence lifetime imaging microscopy (FLIM) system based on an array of low dark count 0.13?m CMOS singlephoton avalanche diodes (SPADs) is demonstrated. Fast background-insensitive fluorescence lifetime determination is achieved by use of a recently proposed algorithm called

  11. Bright-field quantitative phase microscopy (BFQPM) for accurate phase imaging using conventional microscopy hardware

    Science.gov (United States)

    Jenkins, Micah; Gaylord, Thomas K.

    2015-03-01

    Most quantitative phase microscopy methods require the use of custom-built or modified microscopic configurations which are not typically available to most bio/pathologists. There are, however, phase retrieval algorithms which utilize defocused bright-field images as input data and are therefore implementable in existing laboratory environments. Among these, deterministic methods such as those based on inverting the transport-of-intensity equation (TIE) or a phase contrast transfer function (PCTF) are particularly attractive due to their compatibility with Köhler illuminated systems and numerical simplicity. Recently, a new method has been proposed, called multi-filter phase imaging with partially coherent light (MFPI-PC), which alleviates the inherent noise/resolution trade-off in solving the TIE by utilizing a large number of defocused bright-field images spaced equally about the focal plane. Despite greatly improving the state-ofthe- art, the method has many shortcomings including the impracticality of high-speed acquisition, inefficient sampling, and attenuated response at high frequencies due to aperture effects. In this report, we present a new method, called bright-field quantitative phase microscopy (BFQPM), which efficiently utilizes a small number of defocused bright-field images and recovers frequencies out to the partially coherent diffraction limit. The method is based on a noiseminimized inversion of a PCTF derived for each finite defocus distance. We present simulation results which indicate nanoscale optical path length sensitivity and improved performance over MFPI-PC. We also provide experimental results imaging live bovine mesenchymal stem cells at sub-second temporal resolution. In all, BFQPM enables fast and accurate phase imaging with unprecedented spatial resolution using widely available bright-field microscopy hardware.

  12. Imaging bacterial spores by soft-x-ray microscopy

    International Nuclear Information System (INIS)

    Stead, A.D.; Ford, T.W.; Judge, J.

    1997-01-01

    Bacterial spores are able to survive dehydration, but neither the physiological nor structural basis of this have been fully elucidated. Furthermore, once hydrated, spores often require activation before they will germinate. Several treatments can be used to activate spores, but in the case of Bacillus subtlis the most effective is heat treatment. The physiological mechanism associated with activation is also not understood, but some workers suggest that the loss of calcium from the spores may be critical. However, just prior to germination, the spores change from being phase bright to phase dark when viewed by light microscopy. Imaging spores by soft x-ray microscopy is possible without fixation. Thus, in contrast to electron microscopy, it is possible to compare the structure of dehydrated and hydrated spores in a manner not possible previously. A further advantage is that it is possible to monitor individual spores by phase contrast light microscopy immediately prior to imaging with soft x-rays; whereas, with both electron microscopy and biochemical studies, it is a population of spores being studied without knowledge of the phase characteristics of individual spores. This study has therefore tried to compare dehydrated and hydrated spores and to determine if there is a mass loss from individual spores as they pass the transition from being phase bright to phase dark

  13. Imaging bacterial spores by soft-x-ray microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Stead, A.D.; Ford, T.W. [Univ. of London, Surrey (United Kingdom); Judge, J. [Unilever plc, Sharnbrook (United Kingdom)] [and others

    1997-04-01

    Bacterial spores are able to survive dehydration, but neither the physiological nor structural basis of this have been fully elucidated. Furthermore, once hydrated, spores often require activation before they will germinate. Several treatments can be used to activate spores, but in the case of Bacillus subtlis the most effective is heat treatment. The physiological mechanism associated with activation is also not understood, but some workers suggest that the loss of calcium from the spores may be critical. However, just prior to germination, the spores change from being phase bright to phase dark when viewed by light microscopy. Imaging spores by soft x-ray microscopy is possible without fixation. Thus, in contrast to electron microscopy, it is possible to compare the structure of dehydrated and hydrated spores in a manner not possible previously. A further advantage is that it is possible to monitor individual spores by phase contrast light microscopy immediately prior to imaging with soft x-rays; whereas, with both electron microscopy and biochemical studies, it is a population of spores being studied without knowledge of the phase characteristics of individual spores. This study has therefore tried to compare dehydrated and hydrated spores and to determine if there is a mass loss from individual spores as they pass the transition from being phase bright to phase dark.

  14. In-line digital holographic imaging in volume holographic microscopy.

    Science.gov (United States)

    Zhai, Xiaomin; Lin, Wei-Tang; Chen, Hsi-Hsun; Wang, Po-Hao; Yeh, Li-Hao; Tsai, Jui-Chang; Singh, Vijay Raj; Luo, Yuan

    2015-12-01

    A dual-plane in-line digital holographic imaging method incorporating volume holographic microscopy (VHM) is presented to reconstruct objects in a single shot while eliminating zero-order and twin-image diffracted waves. The proposed imaging method is configured such that information from different axial planes is acquired simultaneously using multiplexed volume holographic imaging gratings, as used in VHM, and recorded as in-line holograms where the corresponding reference beams are generated in the fashion of Gabor's in-line holography. Unlike conventional VHM, which can take axial intensity information only at focal depths, the proposed method digitally reconstructs objects at any axial position. Further, we demonstrate the proposed imaging technique's ability to effectively eliminate zero-order and twin images for single-shot three-dimensional object reconstruction.

  15. Transmission scanning electron microscopy: Defect observations and image simulations.

    Science.gov (United States)

    Callahan, Patrick G; Stinville, Jean-Charles; Yao, Eric R; Echlin, McLean P; Titus, Michael S; De Graef, Marc; Gianola, Daniel S; Pollock, Tresa M

    2018-03-01

    The new capabilities of a FEG scanning electron microscope (SEM) equipped with a scanning transmission electron microscopy (STEM) detector for defect characterization have been studied in parallel with transmission electron microscopy (TEM) imaging. Stacking faults and dislocations have been characterized in strontium titanate, a polycrystalline nickel-base superalloy and a single crystal cobalt-base material. Imaging modes that are similar to conventional TEM (CTEM) bright field (BF) and dark field (DF) and STEM are explored, and some of the differences due to the different accelerating voltages highlighted. Defect images have been simulated for the transmission scanning electron microscopy (TSEM) configuration using a scattering matrix formulation, and diffraction contrast in the SEM is discussed in comparison to TEM. Interference effects associated with conventional TEM, such as thickness fringes and bending contours are significantly reduced in TSEM by using a convergent probe, similar to a STEM imaging modality, enabling individual defects to be imaged clearly even in high dislocation density regions. Beyond this, TSEM provides significant advantages for high throughput and dynamic in-situ characterization. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Image Formation in Second-Harmonic Near-Field Microscopy

    DEFF Research Database (Denmark)

    Bozhevolnyi, Sergey I.; Lozovski, Valeri Z.; Pedersen, Kjeld

    1999-01-01

    A macroscopic self-consistent approach that enables one to rigorously describe image formation in scanning near-field optical second-harmonic generation microscopy is developed. The self-consistent second-harmonic field is determined by taking into account both the linear and nonlinear...... results calculated for a rectangular object are presented and compared with experimental observations. It is demonstrated that the optical contrast and the spatial resolution are significantly better in the second-harmonic images than in the images obtained at the fundamental frequency....

  17. Probing superconductors. Spectroscopic-imaging scanning tunneling microscopy

    International Nuclear Information System (INIS)

    Hanaguri, Tetsuo

    2011-01-01

    Discovery of high-temperature superconductivity in a cuprate triggered developments of various spectroscopic tools which have been utilized to elucidate electronic states of this mysterious compound. Particularly, angle-resolved photoemission spectroscopy and scanning-tunneling microscopy/spectroscopy are improved considerably. It is now possible to map the superconducting gap in both momentum and real spaces using these two techniques. Here we review spectroscopic-imaging scanning tunneling microscopy which is able to explore momentum-space phase structure of the superconducting gap, as well as real-space structure. Applications of this technique to a cuprate and an iron-based superconductor are discussed. (author)

  18. Low energy electron point source microscopy: beyond imaging.

    Science.gov (United States)

    Beyer, André; Gölzhäuser, Armin

    2010-09-01

    Low energy electron point source (LEEPS) microscopy has the capability to record in-line holograms at very high magnifications with a fairly simple set-up. After the holograms are numerically reconstructed, structural features with the size of about 2 nm can be resolved. The achievement of an even higher resolution has been predicted. However, a number of obstacles are known to impede the realization of this goal, for example the presence of electric fields around the imaged object, electrostatic charging or radiation induced processes. This topical review gives an overview of the achievements as well as the difficulties in the efforts to shift the resolution limit of LEEPS microscopy towards the atomic level. A special emphasis is laid on the high sensitivity of low energy electrons to electrical fields, which limits the structural determination of the imaged objects. On the other hand, the investigation of the electrical field around objects of known structure is very useful for other tasks and LEEPS microscopy can be extended beyond the task of imaging. The determination of the electrical resistance of individual nanowires can be achieved by a proper analysis of the corresponding LEEPS micrographs. This conductivity imaging may be a very useful application for LEEPS microscopes.

  19. Analysis of image reconstruction artifacts in structured illumination microscopy

    Science.gov (United States)

    Pospíšil, Jakub; Fliegel, Karel; Klíma, Miloš

    2017-09-01

    Structured Illumination Microscopy (SIM) is a super-resolution technique which enables to enhance the resolution of optical microscopes beyond the diffraction limit. The final super-resolution image quality strongly depends on the performance of SIM image reconstruction. Standard SIM methods require precise knowledge of the illumination pattern and assume the sample to be stationary during the acquisition of illumination patterned images. In the case of imaging live cells, the movements of the cell result in the occurrence of image reconstruction artifacts. To reduce this kind of artifacts the short acquisition time is needed. However, short exposure time causes low signal-to-noise ratio (SNR). Moreover, a drift of the specimen may distort the illumination pattern properties in each image. This issue together with the low SNR makes the estimation of reconstruction parameters a challenging task. Inaccurate assessment of spatial frequency, phase shift or orientation of the illumination pattern leads to incorrect separation and shift of spectral components in Fourier space. This results in unwanted image reconstruction artifacts and hampers the resolution enhancement in practice. In this paper, we analyze possible artifacts in super-resolution images reconstructed using super-resolution SIM technique (SR-SIM). An overview of typical image reconstruction artifact types is presented. Distinguishing image artifacts from newly resolved sample features is essential for future SIM applications in cell biology.

  20. Reconstruction of atomic force microscopy image using compressed sensing.

    Science.gov (United States)

    Han, Guoqiang; Lin, Bo; Lin, Yuling

    2018-02-01

    Atomic Force Microscopy (AFM) is one of the most popular and advanced tools for ultra high-resolution imaging and nanomanipulation of nano-scale matter. But AFM imaging typically takes a long time. High-speed and high-precision AFM measurement has attracted wide attention in recent several years. In traditional AFM, simple reduction in the number of measurement points may lose essential sample topography information. To resolve such problems, an AFM image reconstruction method based on Compressed Sensing (CS) theory is applied to reduce image acquisition time without cutting down the image quality. The benefit of using CS approach in AFM is shortening the imaging time, minimizing the interaction with the sample, and finally avoiding sample damage in AFM. Three kinds of testing samples with high and low frequency components were examined by a scanning electron microscope (SEM) and by AFM. An orthogonal Matching Pursuit (OMP) algorithm is employed to reconstruct an AFM image with different sampling rates. Subsequently the reconstruction results of sample topography images are analyzed and evaluated. Using the CS approach in AFM can greatly improve the AFM imaging process. Experimental results show that the obtained reconstructed images have different resolution and quality, depending on the surface morphology of the sample and sampling rates. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Humidity effects on scanning polarization force microscopy imaging

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Yue, E-mail: shenyue@isl.ac.cn [Key Laboratory of Comprehensive and Highly Efficient Utilization of Salt Lake Resources, Key Laboratory of Salt Lake Resources Chemistry of Qinghai Province, Qinghai Institute of Salt Lakes, Chinese Academy of Sciences, Xining, Qinghai 810008 (China); Key Laboratory of Interfacial Physics and Technology of Chinese Academy of Sciences, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800 (China); Zhou, Yuan, E-mail: zhouy@isl.ac.cn [Key Laboratory of Comprehensive and Highly Efficient Utilization of Salt Lake Resources, Key Laboratory of Salt Lake Resources Chemistry of Qinghai Province, Qinghai Institute of Salt Lakes, Chinese Academy of Sciences, Xining, Qinghai 810008 (China); Sun, Yanxia; Zhang, Lijuan [Key Laboratory of Comprehensive and Highly Efficient Utilization of Salt Lake Resources, Key Laboratory of Salt Lake Resources Chemistry of Qinghai Province, Qinghai Institute of Salt Lakes, Chinese Academy of Sciences, Xining, Qinghai 810008 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Wang, Ying; Hu, Jun; Zhang, Yi [Key Laboratory of Interfacial Physics and Technology of Chinese Academy of Sciences, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800 (China)

    2017-08-01

    Highlights: • The humidity dramatically affects the contrast of scanning polarization force microscopy (SPFM) imaging on mica surface. • This influence roots in the sensitive dielectric constant of mica surface to the humidity change. • A strategy of controllable and repeatable imaging the local dielectric properties of nanomaterials with SPFM is proposed. - Abstract: Scanning polarization force microscopy (SPFM) is a useful surface characterization technique to visually characterize and distinguish nanomaterial with different local dielectric properties at nanometer scale. In this paper, taking the individual one-atom-thick graphene oxide (GO) and reduced graphene oxide (rGO) sheets on mica as examples, we described the influences of environmental humidity on SPFM imaging. We found that the apparent heights (AHs) or contrast of SPFM imaging was influenced significantly by relative humidity (RH) at a response time of a few seconds. And this influence rooted in the sensitive dielectric constant of mica surface to the RH change. While dielectric properties of GO and rGO sheets were almost immune to the humidity change. In addition, we gave the method to determine the critical humidity at which the contrast conversion happened under different conditions. And this is important to the contrast control and repeatable imaging of SPFM through RH adjusting. These findings suggest a strategy of controllable and repeatable imaging the local dielectric properties of nanomaterials with SPFM, which is critically important for further distinguishment, manipulation, electronic applications, etc.

  2. Exploring the Potential of Airyscan Microscopy for Live Cell Imaging

    Directory of Open Access Journals (Sweden)

    Kseniya Korobchevskaya

    2017-07-01

    Full Text Available Biological research increasingly demands the use of non-invasive and ultra-sensitive imaging techniques. The Airyscan technology was recently developed to bridge the gap between conventional confocal and super-resolution microscopy. This technique combines confocal imaging with a 0.2 Airy Unit pinhole, deconvolution and the pixel-reassignment principle in order to enhance both the spatial resolution and signal-to-noise-ratio without increasing the excitation power and acquisition time. Here, we present a detailed study evaluating the performance of Airyscan as compared to confocal microscopy by imaging a variety of reference samples and biological specimens with different acquisition and processing parameters. We found that the processed Airyscan images at default deconvolution settings have a spatial resolution similar to that of conventional confocal imaging with a pinhole setting of 0.2 Airy Units, but with a significantly improved signal-to-noise-ratio. Further gains in the spatial resolution could be achieved by the use of enhanced deconvolution filter settings, but at a steady loss in the signal-to-noise ratio, which at more extreme settings resulted in significant data loss and image distortion.

  3. Local crystallography analysis for atomically resolved scanning tunneling microscopy images

    International Nuclear Information System (INIS)

    Lin, Wenzhi; Li, Qing; Belianinov, Alexei; Gai, Zheng; Baddorf, Arthur P; Pan, Minghu; Jesse, Stephen; Kalinin, Sergei V; Sales, Brian C; Sefat, Athena

    2013-01-01

    Scanning probe microscopy has emerged as a powerful and flexible tool for atomically resolved imaging of surface structures. However, due to the amount of information extracted, in many cases the interpretation of such data is limited to being qualitative and semi-quantitative in nature. At the same time, much can be learned from local atom parameters, such as distances and angles, that can be analyzed and interpreted as variations of local chemical bonding, or order parameter fields. Here, we demonstrate an iterative algorithm for indexing and determining atomic positions that allows the analysis of inhomogeneous surfaces. This approach is further illustrated by local crystallographic analysis of several real surfaces, including highly ordered pyrolytic graphite and an Fe-based superconductor FeTe 0.55 Se 0.45 . This study provides a new pathway to extract and quantify local properties for scanning probe microscopy images. (paper)

  4. Effective cellulose nanocrystal imaging using transmission electron microscopy.

    Science.gov (United States)

    Stinson-Bagby, Kelly L; Roberts, Rose; Foster, E Johan

    2018-04-15

    Characterization of cellulose nanocrystals (CNCs) is often complex and tedious. With their increased use for biological materials, polymer reinforcing agents, and other applications, better characterization methods of CNCs are needed to ensure product quality. However, because of their small size, hydrogen bonding, and low electron density, individual CNCs are difficult to image with high resolution and magnification using electron microscopy. Methods to help counter these challenges include staining for increased contrast and techniques to increase dispersion. This work tested several stains, dispersing agents, and sample supports to find a consistent method of individualizing CNCs and providing good contrast for imaging in transmission electron microscopy (TEM). The most consistent method found uses a low concentration of CNCs, bovine serum albumin as a dispersing agent, and Nanovan ® as the contrasting stain on a silicon monoxide-coated Formvar TEM grid. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. Transmission electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1997-01-01

    Transmission Electron Microscopy presents the theory of image and contrast formation, and the analytical modes in transmission electron microscopy. The principles of particle and wave optics of electrons are described. Electron-specimen interactions are discussed for evaluating the theory of scattering and phase contrast. Also discussed are the kinematical and dynamical theories of electron diffraction and their applications for crystal-structure analysis and imaging of lattices and their defects. X-ray micronanalysis and electron energy-loss spectroscopy are treated as analytical methods. Specimen damage and contamination by electron irradiation limits the resolution for biological and some inorganic specimens. This fourth edition includes discussion of recent progress, especially in the area of Schottky emission guns, convergent-beam electron diffraction, electron tomography, holography and the high resolution of crystal lattices.

  6. Hybrid Imaging for Extended Depth of Field Microscopy

    Science.gov (United States)

    Zahreddine, Ramzi Nicholas

    An inverse relationship exists in optical systems between the depth of field (DOF) and the minimum resolvable feature size. This trade-off is especially detrimental in high numerical aperture microscopy systems where resolution is pushed to the diffraction limit resulting in a DOF on the order of 500 nm. Many biological structures and processes of interest span over micron scales resulting in significant blurring during imaging. This thesis explores a two-step computational imaging technique known as hybrid imaging to create extended DOF (EDF) microscopy systems with minimal sacrifice in resolution. In the first step a mask is inserted at the pupil plane of the microscope to create a focus invariant system over 10 times the traditional DOF, albeit with reduced contrast. In the second step the contrast is restored via deconvolution. Several EDF pupil masks from the literature are quantitatively compared in the context of biological microscopy. From this analysis a new mask is proposed, the incoherently partitioned pupil with binary phase modulation (IPP-BPM), that combines the most advantageous properties from the literature. Total variation regularized deconvolution models are derived for the various noise conditions and detectors commonly used in biological microscopy. State of the art algorithms for efficiently solving the deconvolution problem are analyzed for speed, accuracy, and ease of use. The IPP-BPM mask is compared with the literature and shown to have the highest signal-to-noise ratio and lowest mean square error post-processing. A prototype of the IPP-BPM mask is fabricated using a combination of 3D femtosecond glass etching and standard lithography techniques. The mask is compared against theory and demonstrated in biological imaging applications.

  7. Comparative analysis of imaging configurations and objectives for Fourier microscopy.

    Science.gov (United States)

    Kurvits, Jonathan A; Jiang, Mingming; Zia, Rashid

    2015-11-01

    Fourier microscopy is becoming an increasingly important tool for the analysis of optical nanostructures and quantum emitters. However, achieving quantitative Fourier space measurements requires a thorough understanding of the impact of aberrations introduced by optical microscopes that have been optimized for conventional real-space imaging. Here we present a detailed framework for analyzing the performance of microscope objectives for several common Fourier imaging configurations. To this end, we model objectives from Nikon, Olympus, and Zeiss using parameters that were inferred from patent literature and confirmed, where possible, by physical disassembly. We then examine the aberrations most relevant to Fourier microscopy, including the alignment tolerances of apodization factors for different objective classes, the effect of magnification on the modulation transfer function, and vignetting-induced reductions of the effective numerical aperture for wide-field measurements. Based on this analysis, we identify an optimal objective class and imaging configuration for Fourier microscopy. In addition, the Zemax files for the objectives and setups used in this analysis have been made publicly available as a resource for future studies.

  8. Localized charge imaging with scanning Kelvin probe microscopy.

    Science.gov (United States)

    Orihuela, M F; Somoza, A M; Colchero, J; Ortuño, M; Palacios-Lidón, E

    2017-01-13

    In this work, we propose an intuitive and easily implementable approach to model and interpret scanning Kelvin probe microscopy images of insulating samples with localized charges. The method, based on the image charges method, has been validated by a systematic comparison of its predictions with experimental measurements performed on charge domains of different sizes, injected in polymethyl methacrylate discontinuous films. The agreement between predictions and experimental lateral profiles, as well as with spectroscopy tip-sample distance curves, supports its consistency. The proposed procedure allows obtaining quantitative information such as total charge and the size of a charge domain and allows estimating the most adequate measurement parameters.

  9. Microstructure imaging of human rectal mucosa using multiphoton microscopy

    Science.gov (United States)

    Liu, N. R.; Chen, G.; Chen, J. X.; Yan, J.; Zhuo, S. M.; Zheng, L. Q.; Jiang, X. S.

    2011-01-01

    Multiphoton microscopy (MPM) has high resolution and sensitivity. In this study, MPM was used to image microstructure of human rectal mucosa. The morphology and distribution of the main components in mucosa layer, absorptive cells and goblet cells in the epithelium, abundant intestinal glands in the lamina propria and smooth muscle fibers in the muscularis mucosa were clearly monitored. The variations of these components were tightly relevant to the pathology in gastrointestine system, especially early rectal cancer. The obtained images will be helpful for the diagnosis of early colorectal cancer.

  10. Imaging of carbon nanomembranes with helium ion microscopy

    Directory of Open Access Journals (Sweden)

    André Beyer

    2015-08-01

    Full Text Available Carbon nanomembranes (CNMs prepared from aromatic self-assembled monolayers constitute a recently developed class of 2D materials. They are made by a combination of self-assembly, radiation-induced cross-linking and the detachment of the cross-linked SAM from its substrate. CNMs can be deposited on arbitrary substrates, including holey and perforated ones, as well as on metallic (transmission electron microscopy grids. Therewith, freestanding membranes with a thickness of 1 nm and macroscopic lateral dimensions can be prepared. Although free-standing CNMs cannot be imaged by light microscopy, charged particle techniques can visualize them. However, CNMs are electrically insulating, which makes them sensitive to charging. We demonstrate that the helium ion microscope (HIM is a good candidate for imaging freestanding CNMs due to its efficient charge compensation tool. Scanning with a beam of helium ions while recording the emitted secondary electrons generates the HIM images. The advantages of HIM are high resolution, high surface sensitivity and large depth of field. The effects of sample charging, imaging of multilayer CNMs as well as imaging artefacts are discussed.

  11. Acoustic and photoacoustic microscopy imaging of single leukocytes

    Science.gov (United States)

    Strohm, Eric M.; Moore, Michael J.; Kolios, Michael C.

    2016-03-01

    An acoustic/photoacoustic microscope was used to create micrometer resolution images of stained cells from a blood smear. Pulse echo ultrasound images were made using a 1000 MHz transducer with 1 μm resolution. Photoacoustic images were made using a fiber coupled 532 nm laser, where energy losses through stimulated Raman scattering enabled output wavelengths from 532 nm to 620 nm. The laser was focused onto the sample using a 20x objective, and the laser spot co-aligned with the 1000 MHz transducer opposite the laser. The blood smear was stained with Wright-Giemsa, a common metachromatic dye that differentially stains the cellular components for visual identification. A neutrophil, lymphocyte and a monocyte were imaged using acoustic and photoacoustic microscopy at two different wavelengths, 532 nm and 600 nm. Unique features in each imaging modality enabled identification of the different cell types. This imaging method provides a new way of imaging stained leukocytes, with applications towards identifying and differentiating cell types, and detecting disease at the single cell level.

  12. Subparticle ultrafast spectrum imaging in 4D electron microscopy.

    Science.gov (United States)

    Yurtsever, Aycan; van der Veen, Renske M; Zewail, Ahmed H

    2012-01-06

    Single-particle imaging of structures has become a powerful methodology in nanoscience and molecular and cell biology. We report the development of subparticle imaging with space, time, and energy resolutions of nanometers, femtoseconds, and millielectron volts, respectively. By using scanning electron probes across optically excited nanoparticles and interfaces, we simultaneously constructed energy-time and space-time maps. Spectrum images were then obtained for the nanoscale dielectric fields, with the energy resolution set by the photon rather than the electron, as demonstrated here with two examples (silver nanoparticles and the metallic copper-vacuum interface). This development thus combines the high spatial resolution of electron microscopy with the high energy resolution of optical techniques and ultrafast temporal response, opening the door to various applications in elemental analysis as well as mapping of interfaces and plasmonics.

  13. Imaging and Quantification of Extracellular Vesicles by Transmission Electron Microscopy.

    Science.gov (United States)

    Linares, Romain; Tan, Sisareuth; Gounou, Céline; Brisson, Alain R

    2017-01-01

    Extracellular vesicles (EVs) are cell-derived vesicles that are present in blood and other body fluids. EVs raise major interest for their diverse physiopathological roles and their potential biomedical applications. However, the characterization and quantification of EVs constitute major challenges, mainly due to their small size and the lack of methods adapted for their study. Electron microscopy has made significant contributions to the EV field since their initial discovery. Here, we describe the use of two transmission electron microscopy (TEM) techniques for imaging and quantifying EVs. Cryo-TEM combined with receptor-specific gold labeling is applied to reveal the morphology, size, and phenotype of EVs, while their enumeration is achieved after high-speed sedimentation on EM grids.

  14. 3D Cell Culture Imaging with Digital Holographic Microscopy

    Science.gov (United States)

    Dimiduk, Thomas; Nyberg, Kendra; Almeda, Dariela; Koshelva, Ekaterina; McGorty, Ryan; Kaz, David; Gardel, Emily; Auguste, Debra; Manoharan, Vinothan

    2011-03-01

    Cells in higher organisms naturally exist in a three dimensional (3D) structure, a fact sometimes ignored by in vitro biological research. Confinement to a two dimensional culture imposes significant deviations from the native 3D state. One of the biggest obstacles to wider use of 3D cultures is the difficulty of 3D imaging. The confocal microscope, the dominant 3D imaging instrument, is expensive, bulky, and light-intensive; live cells can be observed for only a short time before they suffer photodamage. We present an alternative 3D imaging techinque, digital holographic microscopy, which can capture 3D information with axial resolution better than 2 μm in a 100 μm deep volume. Capturing a 3D image requires only a single camera exposure with a sub-millisecond laser pulse, allowing us to image cell cultures using five orders of magnitude less light energy than with confocal. This can be done with hardware costing ~ 1000. We use the instrument to image growth of MCF7 breast cancer cells and p. pastoras yeast. We acknowledge support from NSF GRFP.

  15. Scanning electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1985-01-01

    The aim of this book is to outline the physics of image formation, electron­ specimen interactions, imaging modes, the interpretation of micrographs and the use of quantitative modes "in scanning electron microscopy (SEM). lt forms a counterpart to Transmission Electron Microscopy (Vol. 36 of this Springer Series in Optical Sciences) . The book evolved from lectures delivered at the University of Münster and from a German text entitled Raster-Elektronenmikroskopie (Springer-Verlag), published in collaboration with my colleague Gerhard Pfefferkorn. In the introductory chapter, the principles of the SEM and of electron­ specimen interactions are described, the most important imaging modes and their associated contrast are summarized, and general aspects of eiemental analysis by x-ray and Auger electron emission are discussed. The electron gun and electron optics are discussed in Chap. 2 in order to show how an electron probe of small diameter can be formed, how the elec­ tron beam can be blanked at high fre...

  16. Computational methods for microfluidic microscopy and phase-space imaging

    Science.gov (United States)

    Pegard, Nicolas Christian Richard

    Modern optical devices are made by assembling separate components such as lenses, objectives, and cameras. Traditionally, each part is optimized separately, even though the trade-offs typically limit the performance of the system overall. This component-based approach is particularly unfit to solve the new challenges brought by modern biology: 3D imaging, in vivo environments, and high sample throughput. In the first part of this thesis, we introduce a general method to design integrated optical systems. The laws of wave propagation, the performance of available technology, as well as other design parameters are combined as constraints into a single optimization problem. The solution provides qualitative design rules to improve optical systems as well as quantitative task-specific methods to minimize loss of information. Our results have applications in optical data storage, holography, and microscopy. The second part of this dissertation presents a direct application. We propose a more efficient design for wide-field microscopy with coherent light, based on double transmission through the sample. Historically, speckle noise and aberrations caused by undesired interferences have made coherent illumination unpopular for imaging. We were able to dramatically reduce speckle noise and unwanted interferences using optimized holographic wavefront reconstruction. The resulting microscope not only yields clear coherent images with low aberration---even in thick samples---but also increases contrast and enables optical filtering and in-depth sectioning. In the third part, we develop new imaging techniques that better respond to the needs of modern biology research through implementing optical design optimization. Using a 4D phase-space distribution, we first represent the state and propagation of incoherent light. We then introduce an additional degree of freedom by putting samples in motion in a microfluidic channel, increasing image diversity. From there, we develop a

  17. Imaging ballistic carrier trajectories in graphene using scanning gate microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Morikawa, Sei; Masubuchi, Satoru [Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8505 (Japan); Dou, Ziwei; Wang, Shu-Wei; Smith, Charles G.; Connolly, Malcolm R., E-mail: mrc61@cam.ac.uk [Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge CB3 0HE (United Kingdom); Watanabe, Kenji; Taniguchi, Takashi [National Institute for Materials Science, 1-1 Namiki, Tsukuba 305-0044 (Japan); Machida, Tomoki, E-mail: tmachida@iis.u-tokyo.ac.jp [Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8505 (Japan); Institute for Nano Quantum Information Electronics, University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8505 (Japan)

    2015-12-14

    We use scanning gate microscopy to map out the trajectories of ballistic carriers in high-mobility graphene encapsulated by hexagonal boron nitride and subject to a weak magnetic field. We employ a magnetic focusing geometry to image carriers that emerge ballistically from an injector, follow a cyclotron path due to the Lorentz force from an applied magnetic field, and land on an adjacent collector probe. The local electric field generated by the scanning tip in the vicinity of the carriers deflects their trajectories, modifying the proportion of carriers focused into the collector. By measuring the voltage at the collector while scanning the tip, we are able to obtain images with arcs that are consistent with the expected cyclotron motion. We also demonstrate that the tip can be used to redirect misaligned carriers back to the collector.

  18. Quantitative biological imaging by ptychographic X-ray diffraction microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Giewekemeyer, Klaus; Kalbfleisch, Sebastian; Beerlink, Andre; Salditt, Tim [Institut fuer Roentgenphysik, Georg-August-Universitaet Goettingen (Germany); Thibault, Pierre; Dierolf, Martin; Pfeiffer, Franz [Department Physik (E17), Technische Universitaet Muenchen, Garching (Germany); Kewish, Cameron M. [Paul Scherrer Institut, Villigen PSI (Switzerland)

    2010-07-01

    Mesoscopic structures with specific functions are abundant in many cellular systems and have been well characterized by electron microscopy in the past. However, the quantitative study of the three-dimensional structure and density of subcellular components remains a difficult problem. In this contribution we show how these limitations could be overcome in the future by the application of recently introduced and now rapidly evolving coherent X-ray imaging techniques for quantitative biological imaging on the nanoscale. More specifically, we report on a recent scanning (ptychographic) diffraction experiment on unstained and unsliced freeze-dried cells of the bacterium Deinococcus radiourans using only a pinhole as beam defining optical element. As a result quantitative density projections well below optical resolution have been achieved.

  19. Watershed Merge Tree Classification for Electron Microscopy Image Segmentation.

    Science.gov (United States)

    Liu, Ting; Jurrus, Elizabeth; Seyedhosseini, Mojtaba; Ellisman, Mark; Tasdizen, Tolga

    2012-11-01

    Automated segmentation of electron microscopy (EM) images is a challenging problem. In this paper, we present a novel method that utilizes a hierarchical structure and boundary classification for 2D neuron segmentation. With a membrane detection probability map, a watershed merge tree is built for the representation of hierarchical region merging from the watershed algorithm. A boundary classifier is learned with non-local image features to predict each potential merge in the tree, upon which merge decisions are made with consistency constraints to acquire the final segmentation. Independent of classifiers and decision strategies, our approach proposes a general framework for efficient hierarchical segmentation with statistical learning. We demonstrate that our method leads to a substantial improvement in segmentation accuracy.

  20. Fluorescence microscopy imaging of electroperturbation in mammalian cells.

    Science.gov (United States)

    Sun, Yinghua; Vernier, P Thomas; Behrend, Matthew; Wang, Jingjing; Thu, Mya Mya; Gundersen, Martin; Marcu, Laura

    2006-01-01

    We report the design, integration, and validation of a fluorescence microscopy system for imaging of electroperturbation--the effects of nanosecond, megavolt-per-meter pulsed electric fields on biological cells and tissues. Such effects have potential applications in cancer therapy, gene regulation, and biophysical research by noninvasively disrupting intracellular compartments and inducing apoptosis in malignant cells. As the primary observing platform, an epifluorescence microscope integrating a nanosecond high-voltage pulser and a micrometer electrode chamber enable in situ imaging of the intracellular processes triggered by high electric fields. Using specific fluorescence molecular probes, the dynamic biological responses of Jurkat T lymphocytes to nanosecond electric pulses (nanoelectropulses) are studied with this system, including calcium bursts, the polarized translocation of phosphatidylserine (PS), and nuclear enlargement and chromatin/DNA structural changes.

  1. 3D imaging of neutron tracks using confocal microscopy

    Science.gov (United States)

    Gillmore, Gavin; Wertheim, David; Flowers, Alan

    2016-04-01

    Neutron detection and neutron flux assessment are important aspects in monitoring nuclear energy production. Neutron flux measurements can also provide information on potential biological damage from exposure. In addition to the applications for neutron measurement in nuclear energy, neutron detection has been proposed as a method of enhancing neutrino detectors and cosmic ray flux has also been assessed using ground-level neutron detectors. Solid State Nuclear Track Detectors (or SSNTDs) have been used extensively to examine cosmic rays, long-lived radioactive elements, radon concentrations in buildings and the age of geological samples. Passive SSNTDs consisting of a CR-39 plastic are commonly used to measure radon because they respond to incident charged particles such as alpha particles from radon gas in air. They have a large dynamic range and a linear flux response. We have previously applied confocal microscopy to obtain 3D images of alpha particle tracks in SSNTDs from radon track monitoring (1). As a charged particle traverses through the polymer it creates an ionisation trail along its path. The trail or track is normally enhanced by chemical etching to better expose radiation damage, as the damaged area is more sensitive to the etchant than the bulk material. Particle tracks in CR-39 are usually assessed using 2D optical microscopy. In this study 6 detectors were examined using an Olympus OLS4100 LEXT 3D laser scanning confocal microscope (Olympus Corporation, Japan). The detectors had been etched for 2 hours 50 minutes at 85 °C in 6.25M NaOH. Post etch the plastics had been treated with a 10 minute immersion in a 2% acetic acid stop bath, followed by rinsing in deionised water. The detectors examined had been irradiated with a 2mSv neutron dose from an Am(Be) neutron source (producing roughly 20 tracks per mm2). We were able to successfully acquire 3D images of neutron tracks in the detectors studied. The range of track diameter observed was between 4

  2. Imaging Live Drosophila Brain with Two-Photon Fluorescence Microscopy

    Science.gov (United States)

    Ahmed, Syeed Ehsan

    Two-photon fluorescence microscopy is an imaging technique which delivers distinct benefits for in vivo cellular and molecular imaging. Cyclic adenosine monophosphate (cAMP), a second messenger molecule, is responsible for triggering many physiological changes in neural system. However, the mechanism by which this molecule regulates responses in neuron cells is not yet clearly understood. When cAMP binds to a target protein, it changes the structure of that protein. Therefore, studying this molecular structure change with fluorescence resonance energy transfer (FRET) imaging can shed light on the cAMP functioning mechanism. FRET is a non-radiative dipole-dipole coupling which is sensitive to small distance change in nanometer scale. In this study we have investigated the effect of dopamine in cAMP dynamics in vivo. In our study two-photon fluorescence microscope was used for imaging mushroom bodies inside live Drosophila melanogaster brain and we developed a method for studying the change in cyclic AMP level.

  3. Residual Deconvolutional Networks for Brain Electron Microscopy Image Segmentation.

    Science.gov (United States)

    Fakhry, Ahmed; Zeng, Tao; Ji, Shuiwang

    2017-02-01

    Accurate reconstruction of anatomical connections between neurons in the brain using electron microscopy (EM) images is considered to be the gold standard for circuit mapping. A key step in obtaining the reconstruction is the ability to automatically segment neurons with a precision close to human-level performance. Despite the recent technical advances in EM image segmentation, most of them rely on hand-crafted features to some extent that are specific to the data, limiting their ability to generalize. Here, we propose a simple yet powerful technique for EM image segmentation that is trained end-to-end and does not rely on prior knowledge of the data. Our proposed residual deconvolutional network consists of two information pathways that capture full-resolution features and contextual information, respectively. We showed that the proposed model is very effective in achieving the conflicting goals in dense output prediction; namely preserving full-resolution predictions and including sufficient contextual information. We applied our method to the ongoing open challenge of 3D neurite segmentation in EM images. Our method achieved one of the top results on this open challenge. We demonstrated the generality of our technique by evaluating it on the 2D neurite segmentation challenge dataset where consistently high performance was obtained. We thus expect our method to generalize well to other dense output prediction problems.

  4. Imaging drug delivery to skin with stimulated Raman scattering microscopy.

    Science.gov (United States)

    Saar, Brian G; Contreras-Rojas, L Rodrigo; Xie, X Sunney; Guy, Richard H

    2011-06-06

    Efficient drug delivery to the skin is essential for the treatment of major dermatologic diseases, such as eczema, psoriasis and acne. However, many compounds penetrate the skin barrier poorly and require optimized formulations to ensure their bioavailability. Here, stimulated Raman scattering (SRS) microscopy, a recently developed, label-free chemical imaging tool, is used to acquire high resolution images of multiple chemical components of a topical formulation as it penetrates into mammalian skin. This technique uniquely provides label-free, nondestructive, three-dimensional images with high spatiotemporal resolution. It reveals novel features of (trans)dermal drug delivery in the tissue environment: different rates of drug penetration via hair follicles as compared to the intercellular pathway across the stratum corneum are directly observed, and the precipitation of drug crystals on the skin surface is visualized after the percutaneous penetration of the cosolvent excipient in the formulation. The high speed three-dimensional imaging capability of SRS thus reveals features that cannot be seen with other techniques, providing both kinetic information and mechanistic insight into the (trans)dermal drug delivery process.

  5. Post-processing strategies in image scanning microscopy.

    Science.gov (United States)

    McGregor, J E; Mitchell, C A; Hartell, N A

    2015-10-15

    Image scanning microscopy (ISM) coupled with pixel reassignment offers a resolution improvement of √2 over standard widefield imaging. By scanning point-wise across the specimen and capturing an image of the fluorescent signal generated at each scan position, additional information about specimen structure is recorded and the highest accessible spatial frequency is doubled. Pixel reassignment can be achieved optically in real time or computationally a posteriori and is frequently combined with the use of a physical or digital pinhole to reject out of focus light. Here, we simulate an ISM dataset using a test image and apply standard and non-standard processing methods to address problems typically encountered in computational pixel reassignment and pinholing. We demonstrate that the predicted improvement in resolution is achieved by applying standard pixel reassignment to a simulated dataset and explore the effect of realistic displacements between the reference and true excitation positions. By identifying the position of the detected fluorescence maximum using localisation software and centring the digital pinhole on this co-ordinate before scaling around translated excitation positions, we can recover signal that would otherwise be degraded by the use of a pinhole aligned to an inaccurate excitation reference. This strategy is demonstrated using experimental data from a multiphoton ISM instrument. Finally we investigate the effect that imaging through tissue has on the positions of excitation foci at depth and observe a global scaling with respect to the applied reference grid. Using simulated and experimental data we explore the impact of a globally scaled reference on the ISM image and, by pinholing around the detected maxima, recover the signal across the whole field of view. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Improved sampling and analysis of images in corneal confocal microscopy.

    Science.gov (United States)

    Schaldemose, E L; Fontain, F I; Karlsson, P; Nyengaard, J R

    2017-10-01

    Corneal confocal microscopy (CCM) is a noninvasive clinical method to analyse and quantify corneal nerve fibres in vivo. Although the CCM technique is in constant progress, there are methodological limitations in terms of sampling of images and objectivity of the nerve quantification. The aim of this study was to present a randomized sampling method of the CCM images and to develop an adjusted area-dependent image analysis. Furthermore, a manual nerve fibre analysis method was compared to a fully automated method. 23 idiopathic small-fibre neuropathy patients were investigated using CCM. Corneal nerve fibre length density (CNFL) and corneal nerve fibre branch density (CNBD) were determined in both a manual and automatic manner. Differences in CNFL and CNBD between (1) the randomized and the most common sampling method, (2) the adjusted and the unadjusted area and (3) the manual and automated quantification method were investigated. The CNFL values were significantly lower when using the randomized sampling method compared to the most common method (p = 0.01). There was not a statistical significant difference in the CNBD values between the randomized and the most common sampling method (p = 0.85). CNFL and CNBD values were increased when using the adjusted area compared to the standard area. Additionally, the study found a significant increase in the CNFL and CNBD values when using the manual method compared to the automatic method (p ≤ 0.001). The study demonstrated a significant difference in the CNFL values between the randomized and common sampling method indicating the importance of clear guidelines for the image sampling. The increase in CNFL and CNBD values when using the adjusted cornea area is not surprising. The observed increases in both CNFL and CNBD values when using the manual method of nerve quantification compared to the automatic method are consistent with earlier findings. This study underlines the importance of improving the analysis of the

  7. Parallelized TCSPC for dynamic intravital fluorescence lifetime imaging: quantifying neuronal dysfunction in neuroinflammation.

    Directory of Open Access Journals (Sweden)

    Jan Leo Rinnenthal

    Full Text Available Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain in living animals. However, current technology is still insufficient to elucidate the mechanisms of organ dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. In the context of imaging, Förster resonant energy transfer (FRET is one of the most adequate tools to probe molecular mechanisms of cell function. As a calibration-free technique, fluorescence lifetime imaging (FLIM is superior for quantifying FRET in vivo. Currently, its main limitation is the acquisition speed in the context of deep-tissue 3D and 4D imaging. Here we present a parallelized time-correlated single-photon counting point detector (p-TCSPC (i for dynamic single-beam scanning FLIM of large 3D areas on the range of hundreds of milliseconds relevant in the context of immune-induced pathologies as well as (ii for ultrafast 2D FLIM in the range of tens of milliseconds, a scale relevant for cell physiology. We demonstrate its power in dynamic deep-tissue intravital imaging, as compared to multi-beam scanning time-gated FLIM suitable for fast data acquisition and compared to highly sensitive single-channel TCSPC adequate to detect low fluorescence signals. Using p-TCSPC, 256×256 pixel FLIM maps (300×300 µm(2 are acquired within 468 ms while 131×131 pixel FLIM maps (75×75 µm(2 can be acquired every 82 ms in 115 µm depth in the spinal cord of CerTN L15 mice. The CerTN L15 mice express a FRET-based Ca-biosensor in certain neuronal subsets. Our new technology allows us to perform time-lapse 3D intravital FLIM (4D FLIM in the brain stem of CerTN L15 mice affected by experimental autoimmune encephalomyelitis and, thereby, to truly quantify

  8. Brain tumor classification of microscopy images using deep residual learning

    Science.gov (United States)

    Ishikawa, Yota; Washiya, Kiyotada; Aoki, Kota; Nagahashi, Hiroshi

    2016-12-01

    The crisis rate of brain tumor is about one point four in ten thousands. In general, cytotechnologists take charge of cytologic diagnosis. However, the number of cytotechnologists who can diagnose brain tumors is not sufficient, because of the necessity of highly specialized skill. Computer-Aided Diagnosis by computational image analysis may dissolve the shortage of experts and support objective pathological examinations. Our purpose is to support a diagnosis from a microscopy image of brain cortex and to identify brain tumor by medical image processing. In this study, we analyze Astrocytes that is a type of glia cell of central nerve system. It is not easy for an expert to discriminate brain tumor correctly since the difference between astrocytes and low grade astrocytoma (tumors formed from Astrocyte) is very slight. In this study, we present a novel method to segment cell regions robustly using BING objectness estimation and to classify brain tumors using deep convolutional neural networks (CNNs) constructed by deep residual learning. BING is a fast object detection method and we use pretrained BING model to detect brain cells. After that, we apply a sequence of post-processing like Voronoi diagram, binarization, watershed transform to obtain fine segmentation. For classification using CNNs, a usual way of data argumentation is applied to brain cells database. Experimental results showed 98.5% accuracy of classification and 98.2% accuracy of segmentation.

  9. Quantitative phase imaging with scanning holographic microscopy: an experimental assesment

    Directory of Open Access Journals (Sweden)

    Tada Yoshitaka

    2006-11-01

    Full Text Available Abstract This paper demonstrates experimentally how quantitative phase information can be obtained in scanning holographic microscopy. Scanning holography can operate in both coherent and incoherent modes, simultaneously if desired, with different detector geometries. A spatially integrating detector provides an incoherent hologram of the object's intensity distribution (absorption and/or fluorescence, for example, while a point detector in a conjugate plane of the pupil provides a coherent hologram of the object's complex amplitude, from which a quantitative measure of its phase distribution can be extracted. The possibility of capturing simultaneously holograms of three-dimensional specimens, leading to three-dimensional reconstructions with absorption contrast, reflectance contrast, fluorescence contrast, as was previously demonstrated, and quantitative phase contrast, as shown here for the first time, opens up new avenues for multimodal imaging in biological studies.

  10. Imaging Photon Lattice States by Scanning Defect Microscopy

    Directory of Open Access Journals (Sweden)

    D. L. Underwood

    2016-06-01

    Full Text Available Microwave photons inside lattices of coupled resonators and superconducting qubits can exhibit surprising matterlike behavior. Realizing such open-system quantum simulators presents an experimental challenge and requires new tools and measurement techniques. Here, we introduce scanning defect microscopy as one such tool and illustrate its use in mapping the normal-mode structure of microwave photons inside a 49-site kagome lattice of coplanar waveguide resonators. Scanning is accomplished by moving a probe equipped with a sapphire tip across the lattice. This locally perturbs resonator frequencies and induces shifts of the lattice resonance frequencies, which we determine by measuring the transmission spectrum. From the magnitude of mode shifts, we can reconstruct photon field amplitudes at each lattice site and thus create spatial images of the photon-lattice normal modes.

  11. Microstructure and properties of laser clad coatings studied by orientation imaging microscopy

    NARCIS (Netherlands)

    Ocelik, V.; Furar, I.; De Hosson, J. Th. M.

    2010-01-01

    In this work orientation imaging microscopy (OIM), based on electron backscatter diffraction in scanning electron microscopy, was employed to examine in detail the relationship between laser cladding processing parameters and he properties and the microstructure of single and overlapping laser

  12. Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging.

    NARCIS (Netherlands)

    Hoebe, R.A.; van Oven, C.H.; Gadella, Th.W.J.; Dhonukshe, P.B.; van Noorden, C.J.F.; Manders, E.M.M.

    2007-01-01

    Fluorescence microscopy of living cells enables visualization of the dynamics and interactions of intracellular molecules. However, fluorescence live-cell imaging is limited by photobleaching and phototoxicity induced by the excitation light. Here we describe controlled light-exposure microscopy

  13. Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging

    NARCIS (Netherlands)

    Hoebe, R. A.; van Oven, C. H.; Gadella, T. W. J.; Dhonukshe, P. B.; van Noorden, C. J. F.; Manders, E. M. M.

    2007-01-01

    Fluorescence microscopy of living cells enables visualization of the dynamics and interactions of intracellular molecules. However, fluorescence live-cell imaging is limited by photobleaching and phototoxicity induced by the excitation light. Here we describe controlled light-exposure microscopy (

  14. Transmission electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1984-01-01

    The aim of this book is to outline the physics of image formation, electron­ specimen interactions and image interpretation in transmission electron mic­ roscopy. The book evolved from lectures delivered at the University of Munster and is a revised version of the first part of my earlier book Elek­ tronenmikroskopische Untersuchungs- und Priiparationsmethoden, omitting the part which describes specimen-preparation methods. In the introductory chapter, the different types of electron microscope are compared, the various electron-specimen interactions and their applications are summarized and the most important aspects of high-resolution, analytical and high-voltage electron microscopy are discussed. The optics of electron lenses is discussed in Chapter 2 in order to bring out electron-lens properties that are important for an understanding of the function of an electron microscope. In Chapter 3, the wave optics of elec­ trons and the phase shifts by electrostatic and magnetic fields are introduced; Fresne...

  15. Imaging leukocytes in vivo with third harmonic generation microscopy

    Science.gov (United States)

    Tsai, Cheng-Kun; Chen, Chien-Kuo; Chen, Yu-Shing; Wu, Pei-Chun; Hsieh, Tsung-Yuan; Liu, Han-Wen; Yeh, Chiou-Yueh; Lin, Win-Li; Chia, Jean-San; Liu, Tzu-Ming

    2013-02-01

    Without a labeling, we demonstrated that lipid granules in leukocytes have distinctive third harmonic generation (THG) contrast. Excited by a 1230nm femtosecond laser, THG signals were generated at a significantly higher level in neutrophils than other mononuclear cells, whereas signals in agranular lymphocytes were one order smaller. These characteristic THG features can also be observed in vivo to trace the newly recruited leukocytes following lipopolysaccharide (LPS) challenge. Furthermore, using video-rate THG microscopy, we also captured images of blood cells in human capillaries. Quite different from red-blood-cells, every now and then, round and granule rich blood cells with strong THG contrast appeared in circulation. The corresponding volume densities in blood, evaluated from their frequencies of appearance and the velocity of circulation, fall within the physiological range of human white blood cell counts. These results suggested that labeling-free THG imaging may provide timely tracing of leukocyte movement and hematology inspection without disturbing the normal cellular or physiological status.

  16. Using FLIM-FRET to measure conformational changes of transglutaminase type 2 in live cells.

    Directory of Open Access Journals (Sweden)

    Nicholas S Caron

    Full Text Available Transglutaminase type 2 (TG2 is a ubiquitously expressed member of the transglutaminase family, capable of mediating a transamidation reaction between a variety of protein substrates. TG2 also has a unique role as a G-protein with GTPase activity. In response to GDP/GTP binding and increases in intracellular calcium levels, TG2 can undergo a large conformational change that reciprocally modulates the enzymatic activities of TG2. We have generated a TG2 biosensor that allows for quantitative assessment of TG2 conformational changes in live cells using Förster resonance energy transfer (FRET, as measured by fluorescence lifetime imaging microscopy (FLIM. Quantifying FRET efficiency with this biosensor provides a robust assay to quickly measure the effects of cell stress, changes in calcium levels, point mutations and chemical inhibitors on the conformation and localization of TG2 in living cells. The TG2 FRET biosensor was validated using established TG2 conformational point mutants, as well as cell stress events known to elevate intracellular calcium levels. We demonstrate in live cells that inhibitors of TG2 transamidation activity can differentially influence the conformation of the enzyme. The irreversible inhibitor of TG2, NC9, forces the enzyme into an open conformation, whereas the reversible inhibitor CP4d traps TG2 in the closed conformation. Thus, this biosensor provides new mechanistic insights into the action of two TG2 inhibitors and defines two new classes based on ability to alter TG2 conformation in addition to inhibiting transamidation activity. Future applications of this biosensor could be to discover small molecules that specifically alter TG2 conformation to affect GDP/GTP or calcium binding.

  17. Extracting quantitative parameters from images in multiphoton microscopy

    Science.gov (United States)

    Zimmerley, Maxwell Stuart

    Coherent anti-Stokes Raman scattering (CARS) microscopy allows for fast, three-dimensionally resolved detection of molecules based on vibrational contrast. In CARS, the generated signal is nonlinearly dependent upon the concentration of the vibrational mode of interest. This makes it challenging to extract quantitative parameters (such as the concentration or orientation) from CARS images of biological and synthetic samples. Because of this, many investigations which employ CARS microscopy generally only report qualitative information extracted from these images. In this thesis, three methods have been developed to extract the quantitative concentration information from CARS images. In the first, the ratio of the forward-propagating and back-reflected CARS signal generated in tissue is used to monitor the percolation of DMSO into excised human cadaver skin. Through this, we find that the maximum clearing of skin with DMSO occurs at 40% v/v. We also combine CARS with second harmonic generation (SHG) to investigate the effects of DMSO on collagen. Up to a 20% v/v concentration of DMSO in the skin, the collagen becomes disrupted, resulting in a significant drop in the generated SHG. In the second method, the ratio between the CARS resonance peak and dip is correlated with the concentration to measure the concentration of water and deuterated glycine in hair. Both molecules are found to distribute throughout the hair fiber homogenously, water at a 34% v/v concentration, and d-glycine with a 0.22 M concentration. In the final method, CARS spectra over one vibrational mode are used to extract the imaginary part of the third-order nonlinear susceptibility. This quantity is linearly dependent upon the concentration of the vibrational mode of interest. This procedure is used to determine the degree of conversion of two-photon polymerized microstructures synthesized with varying writing powers. A sigmoidal relationship is observed between the applied intensity and the degree

  18. Microscopy Techniques for Topography Image Acquisition of Marks on Cartridge Cases

    Directory of Open Access Journals (Sweden)

    Milan Navrátil

    2016-11-01

    Full Text Available Despite very high importance of tool mark analysis in criminalistics domain, the image acquisition and comparison of tool marks remains a difficult and time-consuming effort. This work deals with description of selected microscopy techniques applied to examination of marks on the surface of fired cartridge cases, specifically on marks after firing pin. They are represented by 3-D topography images (scanning probe microscopy and laser scanning confocal microscopy and 2-D images (scanning electron microscopy and light microscopy in bright field.

  19. Characterization of gold nanoparticle films: Rutherford backscattering spectroscopy, scanning electron microscopy with image analysis, and atomic force microscopy

    Directory of Open Access Journals (Sweden)

    Pia C. Lansåker

    2014-10-01

    Full Text Available Gold nanoparticle films are of interest in several branches of science and technology, and accurate sample characterization is needed but technically demanding. We prepared such films by DC magnetron sputtering and recorded their mass thickness by Rutherford backscattering spectroscopy. The geometric thickness dg—from the substrate to the tops of the nanoparticles—was obtained by scanning electron microscopy (SEM combined with image analysis as well as by atomic force microscopy (AFM. The various techniques yielded an internally consistent characterization of the films. In particular, very similar results for dg were obtained by SEM with image analysis and by AFM.

  20. Robust image alignment for cryogenic transmission electron microscopy.

    Science.gov (United States)

    McLeod, Robert A; Kowal, Julia; Ringler, Philippe; Stahlberg, Henning

    2017-03-01

    Cryo-electron microscopy recently experienced great improvements in structure resolution due to direct electron detectors with improved contrast and fast read-out leading to single electron counting. High frames rates enabled dose fractionation, where a long exposure is broken into a movie, permitting specimen drift to be registered and corrected. The typical approach for image registration, with high shot noise and low contrast, is multi-reference (MR) cross-correlation. Here we present the software package Zorro, which provides robust drift correction for dose fractionation by use of an intensity-normalized cross-correlation and logistic noise model to weight each cross-correlation in the MR model and filter each cross-correlation optimally. Frames are reliably registered by Zorro with low dose and defocus. Methods to evaluate performance are presented, by use of independently-evaluated even- and odd-frame stacks by trajectory comparison and Fourier ring correlation. Alignment of tiled sub-frames is also introduced, and demonstrated on an example dataset. Zorro source code is available at github.com/CINA/zorro. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Segmentation of phase contrast microscopy images based on multi-scale local Basic Image Features histograms.

    Science.gov (United States)

    Jaccard, N; Szita, N; Griffin, L D

    2017-09-03

    Phase contrast microscopy (PCM) is routinely used for the inspection of adherent cell cultures in all fields of biology and biomedicine. Key decisions for experimental protocols are often taken by an operator based on typically qualitative observations. However, automated processing and analysis of PCM images remain challenging due to the low contrast between foreground objects (cells) and background as well as various imaging artefacts. We propose a trainable pixel-wise segmentation approach whereby image structures and symmetries are encoded in the form of multi-scale Basic Image Features local histograms, and classification of them is learned by random decision trees. This approach was validated for segmentation of cell versus background, and discrimination between two different cell types. Performance close to that of state-of-the-art specialised algorithms was achieved despite the general nature of the method. The low processing time ( images) is suitable for batch processing of experimental data as well as for interactive segmentation applications.

  2. Understanding the phase contrast optics to restore artifact-free microscopy images for segmentation.

    Science.gov (United States)

    Yin, Zhaozheng; Kanade, Takeo; Chen, Mei

    2012-07-01

    Phase contrast, a noninvasive microscopy imaging technique, is widely used to capture time-lapse images to monitor the behavior of transparent cells without staining or altering them. Due to the optical principle, phase contrast microscopy images contain artifacts such as the halo and shade-off that hinder image segmentation, a critical step in automated microscopy image analysis. Rather than treating phase contrast microscopy images as general natural images and applying generic image processing techniques on them, we propose to study the optical properties of the phase contrast microscope to model its image formation process. The phase contrast imaging system can be approximated by a linear imaging model. Based on this model and input image properties, we formulate a regularized quadratic cost function to restore artifact-free phase contrast images that directly correspond to the specimen's optical path length. With artifacts removed, high quality segmentation can be achieved by simply thresholding the restored images. The imaging model and restoration method are quantitatively evaluated on microscopy image sequences with thousands of cells captured over several days. We also demonstrate that accurate restoration lays the foundation for high performance in cell detection and tracking. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Spot detection in microscopy images using Convolutional Neural Network with sliding-window approach

    CSIR Research Space (South Africa)

    Mabaso, Matsilele A

    2018-01-01

    Full Text Available Robust spot detection in microscopy image analysis serves as a critical prerequisite in many biomedical applications. Various approaches that automatically detect spots have been proposed to improve the analysis of biological images. In this paper...

  4. Algorithms for Reconstruction of Undersampled Atomic Force Microscopy Images Supplementary Material

    DEFF Research Database (Denmark)

    2017-01-01

    Two Jupyter Notebooks showcasing reconstructions of undersampled atomic force microscopy images. The reconstructions were obtained using a variety of interpolation and reconstruction methods.......Two Jupyter Notebooks showcasing reconstructions of undersampled atomic force microscopy images. The reconstructions were obtained using a variety of interpolation and reconstruction methods....

  5. Multiphoton fluorescence lifetime imaging of chemotherapy distribution in solid tumors

    Science.gov (United States)

    Carlson, Marjorie; Watson, Adrienne L.; Anderson, Leah; Largaespada, David A.; Provenzano, Paolo P.

    2017-11-01

    Doxorubicin is a commonly used chemotherapeutic employed to treat multiple human cancers, including numerous sarcomas and carcinomas. Furthermore, doxorubicin possesses strong fluorescent properties that make it an ideal reagent for modeling drug delivery by examining its distribution in cells and tissues. However, while doxorubicin fluorescence and lifetime have been imaged in live tissue, its behavior in archival samples that frequently result from drug and treatment studies in human and animal patients, and murine models of human cancer, has to date been largely unexplored. Here, we demonstrate imaging of doxorubicin intensity and lifetimes in archival formalin-fixed paraffin-embedded sections from mouse models of human cancer with multiphoton excitation and multiphoton fluorescence lifetime imaging microscopy (FLIM). Multiphoton excitation imaging reveals robust doxorubicin emission in tissue sections and captures spatial heterogeneity in cells and tissues. However, quantifying the amount of doxorubicin signal in distinct cell compartments, particularly the nucleus, often remains challenging due to strong signals in multiple compartments. The addition of FLIM analysis to display the spatial distribution of excited state lifetimes clearly distinguishes between signals in distinct compartments such as the cell nuclei versus cytoplasm and allows for quantification of doxorubicin signal in each compartment. Furthermore, we observed a shift in lifetime values in the nuclei of transformed cells versus nontransformed cells, suggesting a possible diagnostic role for doxorubicin lifetime imaging to distinguish normal versus transformed cells. Thus, data here demonstrate that multiphoton FLIM is a highly sensitive platform for imaging doxorubicin distribution in normal and diseased archival tissues.

  6. Optical biomarkers of serous and mucinous human ovarian tumor assessed with nonlinear optics microscopies.

    Science.gov (United States)

    Adur, Javier; Pelegati, Vitor B; de Thomaz, Andre A; Baratti, Mariana O; Almeida, Diogo B; Andrade, L A L A; Bottcher-Luiz, Fátima; Carvalho, Hernandes F; Cesar, Carlos L

    2012-01-01

    Nonlinear optical (NLO) microscopy techniques have potential to improve the early detection of epithelial ovarian cancer. In this study we showed that multimodal NLO microscopies, including two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third-harmonic generation (THG) and fluorescence lifetime imaging microscopy (FLIM) can detect morphological and metabolic changes associated with ovarian cancer progression. We obtained strong TPEF + SHG + THG signals from fixed samples stained with Hematoxylin & Eosin (H&E) and robust FLIM signal from fixed unstained samples. Particularly, we imaged 34 ovarian biopsies from different patients (median age, 49 years) including 5 normal ovarian tissue, 18 serous tumors and 11 mucinous tumors with the multimodal NLO platform developed in our laboratory. We have been able to distinguish adenomas, borderline, and adenocarcinomas specimens. Using a complete set of scoring methods we found significant differences in the content, distribution and organization of collagen fibrils in the stroma as well as in the morphology and fluorescence lifetime from epithelial ovarian cells. NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for serous and mucinous ovarian tumors. The results provide a basis to interpret future NLO images of ovarian tissue and lay the foundation for future in vivo optical evaluation of premature ovarian lesions.

  7. Optical biomarkers of serous and mucinous human ovarian tumor assessed with nonlinear optics microscopies.

    Directory of Open Access Journals (Sweden)

    Javier Adur

    Full Text Available Nonlinear optical (NLO microscopy techniques have potential to improve the early detection of epithelial ovarian cancer. In this study we showed that multimodal NLO microscopies, including two-photon excitation fluorescence (TPEF, second-harmonic generation (SHG, third-harmonic generation (THG and fluorescence lifetime imaging microscopy (FLIM can detect morphological and metabolic changes associated with ovarian cancer progression.We obtained strong TPEF + SHG + THG signals from fixed samples stained with Hematoxylin & Eosin (H&E and robust FLIM signal from fixed unstained samples. Particularly, we imaged 34 ovarian biopsies from different patients (median age, 49 years including 5 normal ovarian tissue, 18 serous tumors and 11 mucinous tumors with the multimodal NLO platform developed in our laboratory. We have been able to distinguish adenomas, borderline, and adenocarcinomas specimens. Using a complete set of scoring methods we found significant differences in the content, distribution and organization of collagen fibrils in the stroma as well as in the morphology and fluorescence lifetime from epithelial ovarian cells.NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for serous and mucinous ovarian tumors. The results provide a basis to interpret future NLO images of ovarian tissue and lay the foundation for future in vivo optical evaluation of premature ovarian lesions.

  8. Structural and Functional Characterization of Human Stem-Cell-Derived Retinal Organoids by Live Imaging.

    Science.gov (United States)

    Browne, Andrew W; Arnesano, Cosimo; Harutyunyan, Narine; Khuu, Thien; Martinez, Juan Carlos; Pollack, Harvey A; Koos, David S; Lee, Thomas C; Fraser, Scott E; Moats, Rex A; Aparicio, Jennifer G; Cobrinik, David

    2017-07-01

    Human pluripotent stem cell (hPSC)-derived retinal organoids are a platform for investigating retinal development, pathophysiology, and cellular therapies. In contrast to histologic analysis in which multiple specimens fixed at different times are used to reconstruct developmental processes, repeated analysis of the same living organoids provides a more direct means to characterize changes. New live imaging modalities can provide insights into retinal organoid structure and metabolic function during in vitro growth. This study employed live tissue imaging to characterize retinal organoid development, including metabolic changes accompanying photoreceptor differentiation. Live hPSC-derived retinal organoids at different developmental stages were examined for microanatomic organization and metabolic function by phase contrast microscopy, optical coherence tomography (OCT), fluorescence lifetime imaging microscopy (FLIM), and hyperspectral imaging (HSpec). Features were compared to those revealed by histologic staining, immunostaining, and microcomputed tomography (micro-CT) of fixed organoid tissue. We used FLIM and HSpec to detect changes in metabolic activity as organoids differentiated into organized lamellae. FLIM detected increased glycolytic activity and HSpec detected retinol and retinoic acid accumulation in the organoid outer layer, coinciding with photoreceptor genesis. OCT enabled imaging of lamellae formed during organoid maturation. Micro-CT revealed three-dimensional structure, but failed to detect lamellae. Live imaging modalities facilitate real-time and nondestructive imaging of retinal organoids as they organize into lamellar structures. FLIM and HSpec enable rapid detection of lamellar structure and photoreceptor metabolism. Live imaging techniques may aid in the continuous evaluation of retinal organoid development in diverse experimental and cell therapy settings.

  9. Multimodal nonlinear optical microscopy used to discriminate human colon cancer

    Science.gov (United States)

    Adur, Javier; Pelegati, Vitor B.; Bianchi, Mariana; de Thomaz, André A.; Baratti, Mariana O.; Carvalho, Hernandes F.; Casco, Víctor H.; Cesar, Carlos L.

    2013-02-01

    Colon cancer is one of the most diffused cancers in the Western World, ranking third worldwide in frequency of incidence after lung and breast cancers. Even if it is curable when detected and treated early, a more accurate premature diagnosis would be a suitable aim for both cancer prognostic and treatment. Combined multimodal nonlinear optical (NLO) microscopies, such as two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third harmonic generation (THG), and fluorescence lifetime imaging microscopy (FLIM) can be used to detect morphological and metabolic changes associated with stroma and epithelial transformation in colon cancer disease. NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns between normal and malignant human colonic mucosa. Using a set of scoring methods significant differences both in the content, distribution and organization of stroma collagen fibrils, and lifetime components of NADH and FAD cofactors of human colon mucosa biopsies were found. Our results provide a framework for using NLO techniques as a clinical diagnostic tool for human colon cancer, and also suggest that the SHG and FLIM metrics could be applied to other intestinal disorders, which are characterized by abnormal cell proliferation and collagen assembly.

  10. Diverse Protocols for Correlative Super-Resolution Fluorescence Imaging and Electron Microscopy of Cells and Tissue

    Science.gov (United States)

    2016-05-25

    super - resolution fluorescence imaging and electron microscopy of cells and tissue Benjamin G. Kopek1, Maria G...have recently developed related approaches for super - resolution imaging within endogenous cellular environments using correlative light and electron...low as ~10 nm under ideal conditions), collectively dubbed “ super - resolution imaging ”5-10. A major super - resolution imaging modality is

  11. SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking

    NARCIS (Netherlands)

    Antolovic, Ivan Michel; Burri, Samuel; Bruschini, Claudio; Hoebe, Ron A.; Charbon, Edoardo

    2017-01-01

    sCMOS imagers are currently utilized (replacing EMCCD imagers) to increase the acquisition speed in super resolution localization microscopy. Single-photon avalanche diode (SPAD) imagers feature frame rates per bit depth comparable to or higher than sCMOS imagers, while generating microsecond

  12. B-Spline potential function for maximum a-posteriori image reconstruction in fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Shilpa Dilipkumar

    2015-03-01

    Full Text Available An iterative image reconstruction technique employing B-Spline potential function in a Bayesian framework is proposed for fluorescence microscopy images. B-splines are piecewise polynomials with smooth transition, compact support and are the shortest polynomial splines. Incorporation of the B-spline potential function in the maximum-a-posteriori reconstruction technique resulted in improved contrast, enhanced resolution and substantial background reduction. The proposed technique is validated on simulated data as well as on the images acquired from fluorescence microscopes (widefield, confocal laser scanning fluorescence and super-resolution 4Pi microscopy. A comparative study of the proposed technique with the state-of-art maximum likelihood (ML and maximum-a-posteriori (MAP with quadratic potential function shows its superiority over the others. B-Spline MAP technique can find applications in several imaging modalities of fluorescence microscopy like selective plane illumination microscopy, localization microscopy and STED.

  13. Dental caries imaging using hyperspectral stimulated Raman scattering microscopy

    Science.gov (United States)

    Wang, Zi; Zheng, Wei; Jian, Lin; Huang, Zhiwei

    2016-03-01

    We report the development of a polarization-resolved hyperspectral stimulated Raman scattering (SRS) imaging technique based on a picosecond (ps) laser-pumped optical parametric oscillator system for label-free imaging of dental caries. In our imaging system, hyperspectral SRS images (512×512 pixels) in both fingerprint region (800-1800 cm-1) and high-wavenumber region (2800-3600 cm-1) are acquired in minutes by scanning the wavelength of OPO output, which is a thousand times faster than conventional confocal micro Raman imaging. SRS spectra variations from normal enamel to caries obtained from the hyperspectral SRS images show the loss of phosphate and carbonate in the carious region. While polarization-resolved SRS images at 959 cm-1 demonstrate that the caries has higher depolarization ratio. Our results demonstrate that the polarization resolved-hyperspectral SRS imaging technique developed allows for rapid identification of the biochemical and structural changes of dental caries.

  14. Technical Review: Microscopy and Image Processing Tools to Analyze Plant Chromatin: Practical Considerations.

    Science.gov (United States)

    Baroux, Célia; Schubert, Veit

    2018-01-01

    In situ nucleus and chromatin analyses rely on microscopy imaging that benefits from versatile, efficient fluorescent probes and proteins for static or live imaging. Yet the broad choice in imaging instruments offered to the user poses orientation problems. Which imaging instrument should be used for which purpose? What are the main caveats and what are the considerations to best exploit each instrument's ability to obtain informative and high-quality images? How to infer quantitative information on chromatin or nuclear organization from microscopy images? In this review, we present an overview of common, fluorescence-based microscopy systems and discuss recently developed super-resolution microscopy systems, which are able to bridge the resolution gap between common fluorescence microscopy and electron microscopy. We briefly present their basic principles and discuss their possible applications in the field, while providing experience-based recommendations to guide the user toward best-possible imaging. In addition to raw data acquisition methods, we discuss commercial and noncommercial processing tools required for optimal image presentation and signal evaluation in two and three dimensions.

  15. Extremely sensitive dual imaging system in solid phantoms

    Science.gov (United States)

    Barnoy, Eran A.; Fixler, Dror; Popovtzer, Rachela; Nayhoz, Tsviya; Ray, Krishanu

    2016-03-01

    Herein we describe promising results from the combination of fluorescent lifetime imaging microscopy (FLIM) and diffusion reflection (DR) medical imaging techniques. Three different geometries of gold nanoparticles (GNPs) were prepared: spheres of 20nm diameter, rods (GNRs) of aspect ratio (AR) 2.5, and GNRs of AR 3.3. Each GNP geometry was then conjugated using PEG linkers estimated to be 10nm in length to each of 3 different fluorescent dyes: Fluorescein, Rhodamine B, and Sulforhodamine B. DR provided deep-volume measurements (up to 1cm) from within solid, tissue-imitating phantoms, indicating GNR presence corresponding to the light used by recording light scattered from the GNPs with increasing distance to a photodetector. FLIM imaged solutions as well as phantom surfaces, recording both the fluorescence lifetimes as well as the fluorescence intensities. Fluorescence quenching was observed for Fluorescein, while metal-enhanced fluorescence (MEF) was observed in Rhodamine B and Sulforhodamine B - the dyes with an absorption peak at a slightly longer wavelength than the GNP plasmon resonance peak. Our system is highly sensitive due to the increased intensity provided by MEF, and also because of the inherent sensitivity of both FLIM and DR. Together, these two modalities and MEF can provide a lot of meaningful information for molecular and functional imaging of biological samples.

  16. Multi-photon excitation microscopy for advanced biomedical imaging

    NARCIS (Netherlands)

    Gadella, B.M.; Haeften, T.W. van; Bavel, Kees van; Valentijn, Jack A.

    Fluorescence microscopy (FM) is a technique traditionally used for determining biological structures [33]; its basic concept is summarised in Figure 1a. The biological specimen under examination is labelled with one or more fluorescent probes before being placed in the microscope. A single photon

  17. Post-processing for statistical image analysis in light microscopy.

    Science.gov (United States)

    Cardullo, Richard A; Hinchcliffe, Edward H

    2013-01-01

    Image processing of images serves a number of important functions including noise reduction, contrast enhancement, and feature extraction. Whatever the final goal, an understanding of the nature of image acquisition and digitization and subsequent mathematical manipulations of that digitized image is essential. Here we discuss the basic mathematical and statistical processes that are routinely used by microscopists to routinely produce high quality digital images and to extract key features of interest using a variety of extraction and thresholding tools. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Improving spatial resolution of confocal Raman microscopy by super-resolution image restoration.

    Science.gov (United States)

    Cui, Han; Zhao, Weiqian; Wang, Yun; Fan, Ying; Qiu, Lirong; Zhu, Ke

    2016-05-16

    A new super-resolution image restoration confocal Raman microscopy method (SRIR-RAMAN) is proposed for improving the spatial resolution of confocal Raman microscopy. This method can recover the lost high spatial frequency of the confocal Raman microscopy by using Poisson-MAP super-resolution imaging restoration, thereby improving the spatial resolution of confocal Raman microscopy and realizing its super-resolution imaging. Simulation analyses and experimental results indicate that the spatial resolution of SRIR-RAMAN can be improved by 65% to achieve 200 nm with the same confocal Raman microscopy system. This method can provide a new tool for high spatial resolution micro-probe structure detection in physical chemistry, materials science, biomedical science and other areas.

  19. Magni: A Python Package for Compressive Sampling and Reconstruction of Atomic Force Microscopy Images

    DEFF Research Database (Denmark)

    Oxvig, Christian Schou; Pedersen, Patrick Steffen; Arildsen, Thomas

    2014-01-01

    Magni is an open source Python package that embraces compressed sensing and Atomic Force Microscopy (AFM) imaging techniques. It provides AFM-specific functionality for undersampling and reconstructing images from AFM equipment and thereby accelerating the acquisition of AFM images. Magni also...

  20. Superresolution upgrade for confocal spinning disk systems using image scanning microscopy (Conference Presentation)

    Science.gov (United States)

    Isbaner, Sebastian; Hähnel, Dirk; Gregor, Ingo; Enderlein, Jörg

    2017-02-01

    Confocal Spinning Disk Systems are widely used for 3D cell imaging because they offer the advantage of optical sectioning at high framerates and are easy to use. However, as in confocal microscopy, the imaging resolution is diffraction limited, which can be theoretically improved by a factor of 2 using the principle of Image Scanning Microscopy (ISM) [1]. ISM with a Confocal Spinning Disk setup (CSDISM) has been shown to improve contrast as well as lateral resolution (FWHM) from 201 +/- 20 nm to 130 +/- 10 nm at 488 nm excitation. A minimum total acquisition time of one second per ISM image makes this method highly suitable for 3D live cell imaging [2]. Here, we present a multicolor implementation of CSDISM for the popular Micro-Manager Open Source Microscopy platform. Since changes in the optical path are not necessary, this will allow any researcher to easily upgrade their standard Confocal Spinning Disk system at remarkable low cost ( 5000 USD) with an ISM superresolution option. [1]. Müller, C.B. and Enderlein, J. Image Scanning Microscopy. Physical Review Letters 104, (2010). [2]. Schulz, O. et al. Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy. Proceedings of the National Academy of Sciences of the United States of America 110, 21000-5 (2013).

  1. Making Microscopy Motivating, Memorable, & Manageable for Undergraduate Students with Digital Imaging Laboratories

    Science.gov (United States)

    Weeks, Andrea; Bachman. Beverly; Josway, Sarah; North, Brittany; Tsuchiya, Mirian T.N.

    2013-01-01

    Microscopy and precise observation are essential skills that are challenging to teach effectively to large numbers of undergraduate biology students. We implemented student-driven digital imaging assignments for microscopy in a large enrollment laboratory for organismal biology. We detail how we promoted student engagement with the material and…

  2. Multiphoton microscopy for skin wound healing study in terms of cellular metabolism and collagen regeneration

    Science.gov (United States)

    Deka, Gitanjal; Okano, Kazunori; Wu, Wei-Wen; Kao, Fu-Jen

    2014-02-01

    Multiphoton microscopy was employed to study normal skin wound healing in live rats noninvasively. Wound healing is a process involving series of biochemical events. This study evaluates the regeneration of collagen and change in cellular metabolic activity during wound healing in rats, with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM), respectively. In eukaryotic cells ATP is the molecule that holds the energy for cellular functioning. Whereas NADH is an electron donor in the metabolic pathways, required to generate ATP. Fluorescence lifetime of NADH free to protein bound ratio was evaluated to determine the relative metabolic activity. The FLIM data were acquired by a TCSPC system using SPCM software and analyzed by SPCImage software. Additionally, polarization resolved SHG signals were also collected to observe the changes in optical birefringence and hence the anisotropy of regenerated collagens from rat wound biopsy samples. Mat lab programming was used to process the data to construct the anisotropy images. Results indicated that, cells involved in healing had higher metabolic activity during the first week of healing, which decreases gradually and become equivalent to normal skin upon healing completes. A net degradation of collagen during the inflammatory phase and net regeneration starting from day 5 were observed in terms of SHG signal intensity change. Polarization resolved SHG imaging of the wound biopsy sample indicates higher value of anisotropy in proliferative phase, from day 4th to 8th, of wound formation; however the anisotropy decreases upon healing.

  3. Characterization of human arterial tissue affected by atherosclerosis using multimodal nonlinear optical microscopy

    Science.gov (United States)

    Baria, Enrico; Cicchi, Riccardo; Rotellini, Matteo; Nesi, Gabriella; Massi, Daniela; Pavone, Francesco S.

    2016-03-01

    Atherosclerosis is a widespread cardiovascular disease caused by the deposition of lipids (such as cholesterol and triglycerides) on the inner arterial wall. The rupture of an atherosclerotic plaque, resulting in a thrombus, is one of the leading causes of death in the Western World. Preventive assessment of plaque vulnerability is therefore extremely important and can be performed by studying collagen organization and lipid composition in atherosclerotic arterial tissues. Routinely used diagnostic methods, such as histopathological examination, are limited to morphological analysis of the examined tissues, whereas an exhaustive characterization requires immune-histochemical examination and a morpho-functional approach. Instead, a label-free and non-invasive alternative is provided by nonlinear microscopy. In this study, we combined SHG and FLIM microscopy in order to characterize collagen organization and lipids in human carotid ex vivo tissues affected by atherosclerosis. SHG and TPF images, acquired from different regions within atherosclerotic plaques, were processed through image pattern analysis methods (FFT, GLCM). The resulting information on collagen and cholesterol distribution and anisotropy, combined with collagen and lipids fluorescence lifetime measured from FLIM images, allowed characterization of carotid samples and discrimination of different tissue regions. The presented method can be applied for automated classification of atherosclerotic lesions and plaque vulnerability. Moreover, it lays the foundation for a potential in vivo diagnostic tool to be used in clinical setting.

  4. Three dimensional refractive index imaging with differential interference contrast microscopy

    Science.gov (United States)

    Aung, Htet; Buckley, Jared; Kostyk, Piotr; Rodriguez, Braulio; Phelan, Shelley; Xu, M.

    2012-03-01

    We report here a new approach based on an extension of the transport of the intensity equation for three dimensional refractive index imaging of a weak phase object from a series of images recorded by a differential interference contrast microscope at different focus (z-stack). Our method is first validated by imaging polystyrene spheres. We then apply this method to monitor in vivo apoptosis of human breast MCF7 epithelial cells. The potential applications are discussed at the end.

  5. Image-based adaptive optics for two-photon microscopy

    OpenAIRE

    Débarre, Delphine; Botcherby, Edward J.; Watanabe, Tomoko; Srinivas, Shankar; Booth, Martin J.; Wilson, Tony

    2009-01-01

    We demonstrate wavefront sensorless aberration correction in a two-photon excited fluorescence microscope. Using analysis of the image-formation process, we have developed an optimized correction scheme permitting image-quality improvement with minimal additional exposure of the sample. We show that, as a result, our correction process induces little photobleaching and significantly improves the quality of images of biological samples. In particular, increased visibility of small structures i...

  6. Analysis of the Transition in Deformation Mechanisms in Superplastic 5083 Aluminum Alloys by Orientation Imaging Microscopy

    National Research Council Canada - National Science Library

    Harrell, James

    2001-01-01

    Recently developed Orientation Imaging Microscopy (OIM) methods have been applied to the analysis of microstructure and microtexture of 5083 aluminum alloy materials that have been processed to enable superplasticity...

  7. Characterization of human carotid atherosclerotic tissues imaged by combining multiple multiphoton microscopy techniques

    Science.gov (United States)

    Baria, E.; Cicchi, R.; Nesi, G.; Massi, D.; Pavone, F. S.

    2017-07-01

    We combined Second Harmonic Generation, Two-Photon Fluorescence and Fluorescence Lifetime Imaging Microscopy for studying human carotid ex vivo tissue sections affected by atherosclerosis, resulting in the discrimination of different arterial regions within the plaques.

  8. Evaluation of Yogurt Microstructure Using Confocal Laser Scanning Microscopy and Image Analysis

    DEFF Research Database (Denmark)

    Skytte, Jacob Lercke; Ghita, Ovidiu; Whelan, Paul F.

    2015-01-01

    The microstructure of protein networks in yogurts defines important physical properties of the yogurt and hereby partly its quality. Imaging this protein network using confocal scanning laser microscopy (CSLM) has shown good results, and CSLM has become a standard measuring technique for fermented...... to image texture description. Here, CSLM images from a yogurt fermentation study are investigated, where production factors including fat content, protein content, heat treatment, and incubation temperature are varied. The descriptors are evaluated through nearest neighbor classification, variance analysis...... scanning microscopy images can be used to provide information on the protein microstructure in yogurt products. For large numbers of microscopy images, subjective evaluation becomes a difficult or even impossible approach, if the images should be incorporated in any form of statistical analysis alongside...

  9. Fusion of lens-free microscopy and mobile-phone microscopy images for high-color-accuracy and high-resolution pathology imaging

    Science.gov (United States)

    Zhang, Yibo; Wu, Yichen; Zhang, Yun; Ozcan, Aydogan

    2017-03-01

    Digital pathology and telepathology require imaging tools with high-throughput, high-resolution and accurate color reproduction. Lens-free on-chip microscopy based on digital in-line holography is a promising technique towards these needs, as it offers a wide field of view (FOV >20 mm2) and high resolution with a compact, low-cost and portable setup. Color imaging has been previously demonstrated by combining reconstructed images at three discrete wavelengths in the red, green and blue parts of the visible spectrum, i.e., the RGB combination method. However, this RGB combination method is subject to color distortions. To improve the color performance of lens-free microscopy for pathology imaging, here we present a wavelet-based color fusion imaging framework, termed "digital color fusion microscopy" (DCFM), which digitally fuses together a grayscale lens-free microscope image taken at a single wavelength and a low-resolution and low-magnification color-calibrated image taken by a lens-based microscope, which can simply be a mobile phone based cost-effective microscope. We show that the imaging results of an H&E stained breast cancer tissue slide with the DCFM technique come very close to a color-calibrated microscope using a 40x objective lens with 0.75 NA. Quantitative comparison showed 2-fold reduction in the mean color distance using the DCFM method compared to the RGB combination method, while also preserving the high-resolution features of the lens-free microscope. Due to the cost-effective and field-portable nature of both lens-free and mobile-phone microscopy techniques, their combination through the DCFM framework could be useful for digital pathology and telepathology applications, in low-resource and point-of-care settings.

  10. Fluorescence lifetime imaging using light emitting diodes

    International Nuclear Information System (INIS)

    Kennedy, Gordon T; Munro, Ian; Poher, Vincent; French, Paul M W; Neil, Mark A A; Elson, Daniel S; Hares, Jonathan D

    2008-01-01

    We demonstrate flexible use of low cost, high-power light emitting diodes as illumination sources for fluorescence lifetime imaging (FLIM). Both time-domain and frequency-domain techniques have been implemented at wavelengths spanning the range 450-640 nm. Additionally, we demonstrate optically sectioned fluorescence lifetime imaging by combining structured illumination with frequency-domain FLIM

  11. Tomographic diffractive microscopy with agile illuminations for imaging targets in a noisy background.

    Science.gov (United States)

    Zhang, T; Godavarthi, C; Chaumet, P C; Maire, G; Giovannini, H; Talneau, A; Prada, C; Sentenac, A; Belkebir, K

    2015-02-15

    Tomographic diffractive microscopy is a marker-free optical digital imaging technique in which three-dimensional samples are reconstructed from a set of holograms recorded under different angles of incidence. We show experimentally that, by processing the holograms with singular value decomposition, it is possible to image objects in a noisy background that are invisible with classical wide-field microscopy and conventional tomographic reconstruction procedure. The targets can be further characterized with a selective quantitative inversion.

  12. Image processing of small protein-crystals in electron microscopy

    International Nuclear Information System (INIS)

    Feinberg, D.A.

    1978-11-01

    This electron microscope study was undertaken to determine whether high resolution reconstructed images could be obtained from statistically noisy micrographs by the super-position of several small areas of images of well-ordered crystals of biological macromolecules. Methods of rotational and translational alignment which use Fourier space data were demonstrated to be superior to methods which use Real space image data. After alignment, the addition of the diffraction patterns of four small areas did not produce higher resolution because of unexpected image distortion effects. A method was developed to determine the location of the distortion origin and the coefficients of spiral distortion and pincushion/barrel distortion in order to make future correction of distortions in electron microscope images of large area crystals

  13. Image processing of small protein-crystals in electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Feinberg, D.A.

    1978-11-01

    This electron microscope study was undertaken to determine whether high resolution reconstructed images could be obtained from statistically noisy micrographs by the super-position of several small areas of images of well-ordered crystals of biological macromolecules. Methods of rotational and translational alignment which use Fourier space data were demonstrated to be superior to methods which use Real space image data. After alignment, the addition of the diffraction patterns of four small areas did not produce higher resolution because of unexpected image distortion effects. A method was developed to determine the location of the distortion origin and the coefficients of spiral distortion and pincushion/barrel distortion in order to make future correction of distortions in electron microscope images of large area crystals.

  14. Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms.

    Science.gov (United States)

    Bouchard, Matthew B; Voleti, Venkatakaushik; Mendes, César S; Lacefield, Clay; Grueber, Wesley B; Mann, Richard S; Bruno, Randy M; Hillman, Elizabeth M C

    2015-02-01

    We report a new 3D microscopy technique that allows volumetric imaging of living samples at ultra-high speeds: Swept, confocally-aligned planar excitation (SCAPE) microscopy. While confocal and two-photon microscopy have revolutionized biomedical research, current implementations are costly, complex and limited in their ability to image 3D volumes at high speeds. Light-sheet microscopy techniques using two-objective, orthogonal illumination and detection require a highly constrained sample geometry, and either physical sample translation or complex synchronization of illumination and detection planes. In contrast, SCAPE microscopy acquires images using an angled, swept light-sheet in a single-objective, en-face geometry. Unique confocal descanning and image rotation optics map this moving plane onto a stationary high-speed camera, permitting completely translationless 3D imaging of intact samples at rates exceeding 20 volumes per second. We demonstrate SCAPE microscopy by imaging spontaneous neuronal firing in the intact brain of awake behaving mice, as well as freely moving transgenic Drosophila larvae.

  15. Three-dimensional super-resolution structured illumination microscopy with maximum a posteriori probability image estimation.

    Science.gov (United States)

    Lukeš, Tomáš; Křížek, Pavel; Švindrych, Zdeněk; Benda, Jakub; Ovesný, Martin; Fliegel, Karel; Klíma, Miloš; Hagen, Guy M

    2014-12-01

    We introduce and demonstrate a new high performance image reconstruction method for super-resolution structured illumination microscopy based on maximum a posteriori probability estimation (MAP-SIM). Imaging performance is demonstrated on a variety of fluorescent samples of different thickness, labeling density and noise levels. The method provides good suppression of out of focus light, improves spatial resolution, and allows reconstruction of both 2D and 3D images of cells even in the case of weak signals. The method can be used to process both optical sectioning and super-resolution structured illumination microscopy data to create high quality super-resolution images.

  16. Application of oblique plane microscopy to high speed live cell imaging

    Science.gov (United States)

    Kumar, Sunil; Wilding, Dean; Sikkel, Markus B.; Lyon, Alexander R.; MacLeod, Ken T.; Dunsby, Chris

    2011-07-01

    Oblique Plane Microscopy (OPM) is a light sheet microscopy technique that combines oblique illumination with correction optics that tilt the focal plane of the collection system. OPM can be used to image conventionally mounted specimens on coverslips or tissue culture dishes and has low out-of-plane photobleaching and phototoxicity. No moving parts are required to achieve an optically sectioned image and so high speed optically sectioned imaging is possible. We present high speed 2D and 3D optically sectioned OPM imaging of live cells using a high NA water immersion lens.

  17. Electron Microscopy and Image Analysis for Selected Materials

    Science.gov (United States)

    Williams, George

    1999-01-01

    This particular project was completed in collaboration with the metallurgical diagnostics facility. The objective of this research had four major components. First, we required training in the operation of the environmental scanning electron microscope (ESEM) for imaging of selected materials including biological specimens. The types of materials range from cyanobacteria and diatoms to cloth, metals, sand, composites and other materials. Second, to obtain training in surface elemental analysis technology using energy dispersive x-ray (EDX) analysis, and in the preparation of x-ray maps of these same materials. Third, to provide training for the staff of the metallurgical diagnostics and failure analysis team in the area of image processing and image analysis technology using NIH Image software. Finally, we were to assist in the sample preparation, observing, imaging, and elemental analysis for Mr. Richard Hoover, one of NASA MSFC's solar physicists and Marshall's principal scientist for the agency-wide virtual Astrobiology Institute. These materials have been collected from various places around the world including the Fox Tunnel in Alaska, Siberia, Antarctica, ice core samples from near Lake Vostoc, thermal vents in the ocean floor, hot springs and many others. We were successful in our efforts to obtain high quality, high resolution images of various materials including selected biological ones. Surface analyses (EDX) and x-ray maps were easily prepared with this technology. We also discovered and used some applications for NIH Image software in the metallurgical diagnostics facility.

  18. Development of a new device for ultrasensitive electrochemiluminescence microscopy imaging.

    Science.gov (United States)

    Dolci, Luisa S; Zanarini, Simone; Della Ciana, Leopoldo; Paolucci, Francesco; Roda, Aldo

    2009-08-01

    Electrochemiluminescence (ECL) is widely used in biosensors and immunoassays thanks to the high sensitivity and specificity of the electrochemically triggered luminescence signal. So far, no applications have been reported on the use of ECL as a probe for ultrasensitive low-light microscope imaging. This work reports the development of a new transparent electrochemical cell for ECL imaging suitable for single cell analysis. The system is based on the use of a microscope placed in a dark box equipped with a CCD camera and a potentiostat. Transparent conducting glass coated with fluorine-doped tin oxide (FTO) has been used, and a three electrode configuration has been designed. The electrochemical cell was optimized using 8 microm diameter polystyrene beads coated with a Ru(bpy)(3)(2+) complex in order to simulate living cells. The Ru(bpy)(3)(2+) immobilized on the microbeads can be imaged and quantified at a concentration as low as 1 x 10(-19) mol/microm(2). Microscope imaging showed that the ECL signal was detected only in correspondence to the beads present on the electrode surface, and the probe could be accurately localized with a spatial resolution of 0.4 microm. The new ECL imaging device can be used in conjunction with other chemiluminescence-based imaging methods for ultrasensitive multiplex imaging on cells and tissues.

  19. Image-based adaptive optics for two-photon microscopy.

    Science.gov (United States)

    Débarre, Delphine; Botcherby, Edward J; Watanabe, Tomoko; Srinivas, Shankar; Booth, Martin J; Wilson, Tony

    2009-08-15

    We demonstrate wavefront sensorless aberration correction in a two-photon excited fluorescence microscope. Using analysis of the image-formation process, we have developed an optimized correction scheme permitting image-quality improvement with minimal additional exposure of the sample. We show that, as a result, our correction process induces little photobleaching and significantly improves the quality of images of biological samples. In particular, increased visibility of small structures is demonstrated. Finally, we illustrate the use of this technique on various fresh and fixed biological tissues.

  20. Imaging of single retinal ganglion cell with differential interference contrast microscopy (Conference Presentation)

    Science.gov (United States)

    Oh, Juyeong; Kim, Yu Jeong; Kim, Chul-Ki; Lee, Taik Jin; Seo, Mina; Lee, Seok; Woo, Deok Ha; Jun, Seong Chan; Park, Ki-Ho; Kim, Seok Hwan; Kim, Jae Hun

    2017-02-01

    Glaucoma is a progressive optic neuropathy, characterized by the selective loss of retinal ganglion cells (RGCs). Therefore, monitoring the change of number or morphology of RGC is essential for the early detection as well as investigation of pathophysiology of glaucoma. Since RGC layer is transparent and hyporeflective, the direct optical visualization of RGCs has not been successful so far. Therefore, glaucoma evaluation mostly depends on indirect diagnostic methods such as the evaluation of optic disc morphology or retinal nerve fiber layer thickness measurement by optical coherence tomography. We have previously demonstrated single photoreceptor cell imaging with differential interference contrast (DIC) microscopy. Herein, we successfully visualized single RGC using DIC microscopy. Since RGC layer is much less reflective than photoreceptor layer, various techniques including the control of light wavelength and bandwidth using a tunable band pass filter were adopted to reduce the chromatic aberration in z-axis for higher and clearer resolution. To verify that the imaged cells were the RGCs, the flat-mounted retina of Sprague-Dawley rat, in which the RGCs were retrogradely labeled with fluorescence, was observed by both fluorescence and DIC microscopies for direct comparison. We have confirmed that the cell images obtained by fluorescence microscopy were perfectly matched with cell images by DIC microscopy. As conclusion, we have visualized single RGC with DIC microscopy, and confirmed with fluorescence microscopy.

  1. Resolution and contrast enhancement in laser scanning microscopy using dark beam imaging.

    Science.gov (United States)

    Dehez, Harold; Piché, Michel; De Koninck, Yves

    2013-07-01

    Laser scanning microscopy allows for three-dimensional imaging of cells with molecular specific labeling. However the spatial resolution of optical microscopy is fundamentally limited by the diffraction of light. In the last two decades many techniques have been introduced to enhance the resolution of laser scanning microscopes. However most of these techniques impose strong constraints on the specimen or rely on complex optical systems. These constraints limit the applicability of resolution improvement to various imaging modalities and sample types. To overcome these limitations, we introduce here a novel approach, which we called Switching LAser Mode (SLAM) microscopy, to enhance resolution and contrast in laser scanning microscopy. SLAM microscopy relies on subtracting images obtained with dark and bright modes, and exploits the smaller dimensions of the dark spot of the azimuthally polarized TE 01 mode. With this approach, resolution is improved by a factor of two in confocal microscopy. The technique is not based on complex nonlinear processes and thus requires laser power similar to that used in conventional imaging, minimizing photo-damage. The flexibility of the approach enables retrofitting in commercial confocal and two-photon microscopes and opens avenues for resolution enhancement in fluorescence-independent microscopy.

  2. Research and application on imaging technology of line structure light based on confocal microscopy

    Science.gov (United States)

    Han, Wenfeng; Xiao, Zexin; Wang, Xiaofen

    2009-11-01

    In 2005, the theory of line structure light confocal microscopy was put forward firstly in China by Xingyu Gao and Zexin Xiao in the Institute of Opt-mechatronics of Guilin University of Electronic Technology. Though the lateral resolution of line confocal microscopy can only reach or approach the level of the traditional dot confocal microscopy. But compared with traditional dot confocal microscopy, it has two advantages: first, by substituting line scanning for dot scanning, plane imaging only performs one-dimensional scanning, with imaging velocity greatly improved and scanning mechanism simplified, second, transfer quantity of light is greatly improved by substituting detection hairline for detection pinhole, and low illumination CCD is used directly to collect images instead of photoelectric intensifier. In order to apply the line confocal microscopy to practical system, based on the further research on the theory of the line confocal microscopy, imaging technology of line structure light is put forward on condition of implementation of confocal microscopy. Its validity and reliability are also verified by experiments.

  3. Automatic detection of NIL defects using microscopy and image processing

    KAUST Repository

    Pietroy, David

    2013-12-01

    Nanoimprint Lithography (NIL) is a promising technology for low cost and large scale nanostructure fabrication. This technique is based on a contact molding-demolding process, that can produce number of defects such as incomplete filling, negative patterns, sticking. In this paper, microscopic imaging combined to a specific processing algorithm is used to detect numerically defects in printed patterns. Results obtained for 1D and 2D imprinted gratings with different microscopic image magnifications are presented. Results are independent on the device which captures the image (optical, confocal or electron microscope). The use of numerical images allows the possibility to automate the detection and to compute a statistical analysis of defects. This method provides a fast analysis of printed gratings and could be used to monitor the production of such structures. © 2013 Elsevier B.V. All rights reserved.

  4. Dielectric and fluorescent samples imaged by scanning near-field optical microscopy in reflection

    OpenAIRE

    Jalocha, A.; Jalocha, A.; van Hulst, N.F.

    1995-01-01

    Dielectric fluorescent samples are imaged by scanning near- field optical microscopy in reflection. A non-metallized tapered fibre tip is used both as an emitter and a detector. Shear force feedback controls the distance between the tip and the sample and gives simultaneously a topographic image of the surface. A direct correlation with the optical image is obtained. We demonstrate that this reflection setup is suitable for dielectric samples. Images in fluorescence have been obtained o­n Lan...

  5. Quantitative characterization of articular cartilage using Mueller matrix imaging and multiphoton microscopy.

    Science.gov (United States)

    Ellingsen, Pål Gunnar; Lilledahl, Magnus Borstad; Aas, Lars Martin Sandvik; Davies, Catharina de Lange; Kildemo, Morten

    2011-11-01

    The collagen meshwork in articular cartilage of chicken knee is characterized using Mueller matrix imaging and multiphoton microscopy. Direction and degree of dispersion of the collagen fibers in the superficial layer are found using a Fourier transform image-analysis technique of the second-harmonic generated image. Mueller matrix images are used to acquire structural data from the intermediate layer of articular cartilage where the collagen fibers are too small to be resolved by optical microscopy, providing a powerful multimodal measurement technique. Furthermore, we show that Mueller matrix imaging provides more information about the tissue compared to standard polarization microscopy. The combination of these techniques can find use in improved diagnosis of diseases in articular cartilage, improved histopathology, and additional information for accurate biomechanical modeling of cartilage.

  6. New developments in electron microscopy for serial image acquisition of neuronal profiles.

    Science.gov (United States)

    Kubota, Yoshiyuki

    2015-02-01

    Recent developments in electron microscopy largely automate the continuous acquisition of serial electron micrographs (EMGs), previously achieved by laborious manual serial ultrathin sectioning using an ultramicrotome and ultrastructural image capture process with transmission electron microscopy. The new systems cut thin sections and capture serial EMGs automatically, allowing for acquisition of large data sets in a reasonably short time. The new methods are focused ion beam/scanning electron microscopy, ultramicrotome/serial block-face scanning electron microscopy, automated tape-collection ultramicrotome/scanning electron microscopy and transmission electron microscope camera array. In this review, their positive and negative aspects are discussed. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Microsphere-aided optical microscopy and its applications for super-resolution imaging

    Science.gov (United States)

    Upputuri, Paul Kumar; Pramanik, Manojit

    2017-12-01

    The spatial resolution of a standard optical microscope (SOM) is limited by diffraction. In visible spectrum, SOM can provide ∼ 200 nm resolution. To break the diffraction limit several approaches were developed including scanning near field microscopy, metamaterial super-lenses, nanoscale solid immersion lenses, super-oscillatory lenses, confocal fluorescence microscopy, techniques that exploit non-linear response of fluorophores like stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, etc. Recently, photonic nanojet generated by a dielectric microsphere was used to break the diffraction limit. The microsphere-approach is simple, cost-effective and can be implemented under a standard microscope, hence it has gained enormous attention for super-resolution imaging. In this article, we briefly review the microsphere approach and its applications for super-resolution imaging in various optical imaging modalities.

  8. Whole slide imaging of unstained tissue using lensfree microscopy

    Science.gov (United States)

    Morel, Sophie Nhu An; Hervé, Lionel; Bordy, Thomas; Cioni, Olivier; Delon, Antoine; Fromentin, Catherine; Dinten, Jean-Marc; Allier, Cédric

    2016-04-01

    Pathologist examination of tissue slides provides insightful information about a patient's disease. Traditional analysis of tissue slides is performed under a binocular microscope, which requires staining of the sample and delays the examination. We present a simple cost-effective lensfree imaging method to record 2-4μm resolution wide-field (10 mm2 to 6 cm2) images of unstained tissue slides. The sample processing time is reduced as there is no need for staining. A wide field of view (10 mm2) lensfree hologram is recorded in a single shot and the image is reconstructed in 2s providing a very fast acquisition chain. The acquisition is multispectral, i.e. multiple holograms are recorded simultaneously at three different wavelengths, and a dedicated holographic reconstruction algorithm is used to retrieve both amplitude and phase. Whole tissue slides imaging is obtained by recording 130 holograms with X-Y translation stages and by computing the mosaic of a 25 x 25 mm2 reconstructed image. The reconstructed phase provides a phase-contrast-like image of the unstained specimen, revealing structures of healthy and diseased tissue. Slides from various organs can be reconstructed, e.g. lung, colon, ganglion, etc. To our knowledge, our method is the first technique that enables fast wide-field lensfree imaging of such unlabeled dense samples. This technique is much cheaper and compact than a conventional phase contrast microscope and could be made portable. In sum, we present a new methodology that could quickly provide useful information when a rapid diagnosis is needed, such as tumor margin identification on frozen section biopsies during surgery.

  9. Visualizing liver anatomy, physiology and pharmacology using multiphoton microscopy.

    Science.gov (United States)

    Wang, Haolu; Liang, Xiaowen; Gravot, Germain; Thorling, Camilla A; Crawford, Darrell H G; Xu, Zhi Ping; Liu, Xin; Roberts, Michael S

    2017-01-01

    Multiphoton microscopy (MPM) has become increasingly popular and widely used in both basic and clinical liver studies over the past few years. This technology provides insights into deep live tissues with less photobleaching and phototoxicity, which helps us to better understand the cellular morphology, microenvironment, immune responses and spatiotemporal dynamics of drugs and therapeutic cells in the healthy and diseased liver. This review summarizes the principles, opportunities, applications and limitations of MPM in hepatology. A key emphasis is on the use of fluorescence lifetime imaging (FLIM) to add additional quantification and specificity to the detection of endogenous fluorescent species in the liver as well as exogenous molecules and nanoparticles that are applied to the liver in vivo. We anticipate that in the near future MPM-FLIM will advance our understanding of the cellular and molecular mechanisms of liver diseases, and will be evaluated from bench to bedside, leading to real-time histology of human liver diseases. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Towards simultaneous single emission microscopy and magnetic resonance imaging

    Science.gov (United States)

    Cai, Liang

    In recent years, the combined nuclear imaging and magnetic resonance imaging (MRI) has drawn extensive research effort. They can provide simultaneously acquired anatomical and functional information inside the human/small animal body in vivo. In this dissertation, the development of an ultrahigh resolution MR-compatible SPECT (Single Photon Emission Computed Tomography) system that can be operated inside a pre-existing clinical MR scanner for simultaneous dual-modality imaging of small animals will be discussed. This system is constructed with 40 small pixel CdTe detector modules assembled in a fully stationary ring SPECT geometry. Series of experiments have demonstrated that this system is capable of providing an imaging resolution of CdTe detector module that we recently developed. Each module consists of CdTe detectors having an overall size of 2.2 cm x 1.1 cm, divided into 64 x 32 pixels of 350 mum in size. A novel hybrid pixel-waveform (HPWF) readout system is also designed to alleviate several challenges for using small-pixel CdTe detectors in ultrahigh-resolution SPECT imaging applications. The HPWF system utilizes a modified version of a 2048-channel 2-D CMOS ASIC to readout the anode pixel, and a digitizing circuitry to sample the signal waveform induced on the cathode. The cathode waveform acquired with the HPWF circuitry offers excellent spatial resolution, energy resolution and depth of interaction (DOI) information, even with the presence of excessive charge-sharing/charge-loss between the small anode pixels. The HPWF CdTe detector is designed and constructed with a minimum amount of ferromagnetic materials, to ensure the MR-compatibility. To achieve sub-500?m imaging resolution, two special designed SPECT apertures have been constructed with different pinhole sizes of 300?m and 500?m respectively. It has 40 pinhole inserts that are made of cast platinum (90%)-iridium (10%) alloy, which provides the maximum stopping power and are compatible with MR

  11. A minimal optical trapping and imaging microscopy system.

    Directory of Open Access Journals (Sweden)

    Carmen Noemí Hernández Candia

    Full Text Available We report the construction and testing of a simple and versatile optical trapping apparatus, suitable for visualizing individual microtubules (∼25 nm in diameter and performing single-molecule studies, using a minimal set of components. This design is based on a conventional, inverted microscope, operating under plain bright field illumination. A single laser beam enables standard optical trapping and the measurement of molecular displacements and forces, whereas digital image processing affords real-time sample visualization with reduced noise and enhanced contrast. We have tested our trapping and imaging instrument by measuring the persistence length of individual double-stranded DNA molecules, and by following the stepping of single kinesin motor proteins along clearly imaged microtubules. The approach presented here provides a straightforward alternative for studies of biomaterials and individual biomolecules.

  12. Neural imaging in songbirds using fiber optic fluorescence microscopy

    Science.gov (United States)

    Nooshabadi, Fatemeh; Hearn, Gentry; Lints, Thierry; Maitland, Kristen C.

    2012-02-01

    The song control system of juvenile songbirds is an important model for studying the developmental acquisition and generation of complex learned vocal motor sequences, two processes that are fundamental to human speech and language. To understand the neural mechanisms underlying song production, it is critical to characterize the activity of identified neurons in the song control system when the bird is singing. Neural imaging in unrestrained singing birds, although technically challenging, will advance our understanding of neural ensemble coding mechanisms in this system. We are exploring the use of a fiber optic microscope for functional imaging in the brain of behaving and singing birds in order to better understand the contribution of a key brain nucleus (high vocal center nucleus; HVC) to temporal aspects of song motor control. We have constructed a fluorescence microscope with LED illumination, a fiber bundle for transmission of fluorescence excitation and emission light, a ~2x GRIN lens, and a CCD for image acquisition. The system has 2 μm resolution, 375 μm field of view, 200 μm working distance, and 1 mm outer diameter. As an initial characterization of this setup, neurons in HVC were imaged using the fiber optic microscope after injection of quantum dots or fluorescent retrograde tracers into different song nuclei. A Lucid Vivascope confocal microscope was used to confirm the imaging results. Long-term imaging of the activity of these neurons in juvenile birds during singing may lead us to a better understanding of the central motor codes for song and the central mechanism by which auditory experience modifies song motor commands to enable vocal learning and imitation.

  13. Optical Imaging and Microscopy Techniques and Advanced Systems

    CERN Document Server

    Török, Peter

    2007-01-01

    This text on contemporary optical systems is intended for optical researchers and engineers, graduate students and optical microscopists in the biological and biomedical sciences. This second edition contains two completely new chapters. In addition most of the chapters from the first edition have been revised and updated. The book consists of three parts: The first discusses high-aperture optical systems, which form the backbone of optical microscopes. An example is a chapter new in the second edition on the emerging field of high numerical aperture diffractive lenses which seems to have particular promise in improving the correction of lenses. In this part particular attention is paid to optical data storage. The second part is on the use of non-linear optical techniques, including nonlinear optical excitation (total internal reflection fluorescence, second and third harmonic generation and two photon microscopy) and non-linear spectroscopy (CARS). The final part of the book presents miscellaneous technique...

  14. Fluorescence cell imaging and manipulation using conventional halogen lamp microscopy.

    Directory of Open Access Journals (Sweden)

    Kazuo Yamagata

    Full Text Available Technologies for vitally labeling cells with fluorescent dyes have advanced remarkably. However, to excite fluorescent dyes currently requires powerful illumination, which can cause phototoxic damage to the cells and increases the cost of microscopy. We have developed a filter system to excite fluorescent dyes using a conventional transmission microscope equipped with a halogen lamp. This method allows us to observe previously invisible cell organelles, such as the metaphase spindle of oocytes, without causing phototoxicity. Cells remain healthy even after intensive manipulation under fluorescence observation, such as during bovine, porcine and mouse somatic cell cloning using nuclear transfer. This method does not require expensive epifluorescence equipment and so could help to reduce the science gap between developed and developing countries.

  15. Nanoscale imaging of Bacillus thuringiensis flagella using atomic force microscopy

    Science.gov (United States)

    Gillis, Annika; Dupres, Vincent; Delestrait, Guillaume; Mahillon, Jacques; Dufrêne, Yves F.

    2012-02-01

    Because bacterial flagella play essential roles in various processes (motility, adhesion, host interactions, secretion), studying their expression in relation to function is an important challenge. Here, we use atomic force microscopy (AFM) to gain insight into the nanoscale surface properties of two wild-type and four mutant strains of Bacillus thuringiensis exhibiting various levels of flagellation. We show that, unlike AFM in liquid, AFM in air is a simple and reliable approach to observe the morphological details of the bacteria, and to quantify the density and dimensions of their flagella. We found that the amount of flagella expressed by the six strains, as observed at the nanoscale, correlates with their microscopic swarming motility. These observations provide novel information on flagella expression in Gram-positive bacteria and demonstrate the power of AFM in genetic studies for the fast assessment of the phenotypic characteristics of bacterial strains altered in cell surface appendages.Because bacterial flagella play essential roles in various processes (motility, adhesion, host interactions, secretion), studying their expression in relation to function is an important challenge. Here, we use atomic force microscopy (AFM) to gain insight into the nanoscale surface properties of two wild-type and four mutant strains of Bacillus thuringiensis exhibiting various levels of flagellation. We show that, unlike AFM in liquid, AFM in air is a simple and reliable approach to observe the morphological details of the bacteria, and to quantify the density and dimensions of their flagella. We found that the amount of flagella expressed by the six strains, as observed at the nanoscale, correlates with their microscopic swarming motility. These observations provide novel information on flagella expression in Gram-positive bacteria and demonstrate the power of AFM in genetic studies for the fast assessment of the phenotypic characteristics of bacterial strains altered in

  16. Simulation of bright-field microscopy images depicting pap-smear specimen.

    Science.gov (United States)

    Malm, Patrik; Brun, Anders; Bengtsson, Ewert

    2015-03-01

    As digital imaging is becoming a fundamental part of medical and biomedical research, the demand for computer-based evaluation using advanced image analysis is becoming an integral part of many research projects. A common problem when developing new image analysis algorithms is the need of large datasets with ground truth on which the algorithms can be tested and optimized. Generating such datasets is often tedious and introduces subjectivity and interindividual and intraindividual variations. An alternative to manually created ground-truth data is to generate synthetic images where the ground truth is known. The challenge then is to make the images sufficiently similar to the real ones to be useful in algorithm development. One of the first and most widely studied medical image analysis tasks is to automate screening for cervical cancer through Pap-smear analysis. As part of an effort to develop a new generation cervical cancer screening system, we have developed a framework for the creation of realistic synthetic bright-field microscopy images that can be used for algorithm development and benchmarking. The resulting framework has been assessed through a visual evaluation by experts with extensive experience of Pap-smear images. The results show that images produced using our described methods are realistic enough to be mistaken for real microscopy images. The developed simulation framework is very flexible and can be modified to mimic many other types of bright-field microscopy images. © 2015 The Authors. Published by Wiley Periodicals, Inc. on behalf of ISAC.

  17. Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging.

    Science.gov (United States)

    Iijima, Hirofumi; Fukuda, Yoshiyuki; Arai, Yoshihiro; Terakawa, Susumu; Yamamoto, Naoki; Nagayama, Kuniaki

    2014-01-01

    Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. General Purpose Segmentation for Microorganisms in Microscopy Images

    DEFF Research Database (Denmark)

    Jensen, Sebastian H. Nesgaard; Moeslund, Thomas B.; Rankl, Christian

    2014-01-01

    In this paper, we propose an approach for achieving generalized segmentation of microorganisms in mi- croscopy images. It employs a pixel-wise classification strategy based on local features. Multilayer percep- trons are utilized for classification of the local features and is trained for each...

  19. A robust method for processing scanning probe microscopy images and determining nanoobject position and dimensions

    NARCIS (Netherlands)

    Silly, F.

    2009-01-01

    P>Processing of scanning probe microscopy (SPM) images is essential to explore nanoscale phenomena. Image processing and pattern recognition techniques are developed to improve the accuracy and consistency of nanoobject and surface characterization. We present a robust and versatile method to

  20. Cellular features of psoriatic skin: imaging and quantification using in vivo reflectance confocal microscopy

    NARCIS (Netherlands)

    Wolberink, E.A.W.; Erp, P.E.J. van; Teussink, M.M.; Kerkhof, P.C.M. van de; Gerritsen, M.J.P.

    2011-01-01

    BACKGROUND: In vivo reflectance confocal microscopy (RCM) is a novel, exciting imaging technique. It provides images of cell-and tissue structures and dynamics in situ, in real time, without the need for ex vivo tissue samples. RCM visualizes the superficial part of human skin up to a depth of 250

  1. A simple methodology for obtaining X-ray color images in scanning electron microscopy

    International Nuclear Information System (INIS)

    Veiga, M.M. da; Pietroluongo, L.R.V.

    1985-01-01

    A simple methodology for obtaining at least 3 elements X-ray images in only one photography is described. The fluorescent X-ray image is obtained from scanning electron microscopy with energy dispersion analysis system. The change of detector analytic channels, color cellophane foils and color films are used sequentially. (M.C.K.) [pt

  2. Dielectric and fluorescent samples imaged by scanning near-field optical microscopy in reflection

    NARCIS (Netherlands)

    Jalocha, A.; Jalocha, A.; van Hulst, N.F.

    1995-01-01

    Dielectric fluorescent samples are imaged by scanning near- field optical microscopy in reflection. A non-metallized tapered fibre tip is used both as an emitter and a detector. Shear force feedback controls the distance between the tip and the sample and gives simultaneously a topographic image of

  3. Imaging of phase change materials below a capping layer using correlative infrared near-field microscopy and electron microscopy

    Science.gov (United States)

    Lewin, M.; Hauer, B.; Bornhöfft, M.; Jung, L.; Benke, J.; Michel, A.-K. U.; Mayer, J.; Wuttig, M.; Taubner, T.

    2015-10-01

    Phase Change Materials (PCM) show two stable states in the solid phase with significantly different optical and electronic properties. They can be switched reversibly between those two states and are promising candidates for future non-volatile memory applications. The development of phase change devices demands characterization tools, yielding information about the switching process at high spatial resolution. Scattering-type Scanning Near-field Optical Microscopy (s-SNOM) allows for spectroscopic analyses of the different optical properties of the PCMs on the nm-scale. By correlating the optical s-SNOM images with transmission electron microscopy images of the same sample, we unambiguously demonstrate the correlation of the infrared optical contrast with the structural state of the phase change material. The investigated sample consists of sandwiched amorphous and crystalline regions of Ag 4 In 3 Sb 67 Te 26 below a 100 nm thick ( ZnS ) 80 - ( SiO2 ) 20 capping layer. Our results demonstrate the sensitivity of s-SNOM to small dielectric near-field contrasts even below a comparably thick capping layer ( 100 nm ).

  4. Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

    Science.gov (United States)

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei

    2014-09-01

    We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

  5. Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei, E-mail: biehzw@nus.edu.sg [Optical Bioimaging Laboratory, Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore 117576 (Singapore)

    2014-09-08

    We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

  6. Quantitative 3D imaging of whole, unstained cells by using X-ray diffraction microscopy.

    Science.gov (United States)

    Jiang, Huaidong; Song, Changyong; Chen, Chien-Chun; Xu, Rui; Raines, Kevin S; Fahimian, Benjamin P; Lu, Chien-Hung; Lee, Ting-Kuo; Nakashima, Akio; Urano, Jun; Ishikawa, Tetsuya; Tamanoi, Fuyuhiko; Miao, Jianwei

    2010-06-22

    Microscopy has greatly advanced our understanding of biology. Although significant progress has recently been made in optical microscopy to break the diffraction-limit barrier, reliance of such techniques on fluorescent labeling technologies prohibits quantitative 3D imaging of the entire contents of cells. Cryoelectron microscopy can image pleomorphic structures at a resolution of 3-5 nm, but is only applicable to thin or sectioned specimens. Here, we report quantitative 3D imaging of a whole, unstained cell at a resolution of 50-60 nm by X-ray diffraction microscopy. We identified the 3D morphology and structure of cellular organelles including cell wall, vacuole, endoplasmic reticulum, mitochondria, granules, nucleus, and nucleolus inside a yeast spore cell. Furthermore, we observed a 3D structure protruding from the reconstructed yeast spore, suggesting the spore germination process. Using cryogenic technologies, a 3D resolution of 5-10 nm should be achievable by X-ray diffraction microscopy. This work hence paves a way for quantitative 3D imaging of a wide range of biological specimens at nanometer-scale resolutions that are too thick for electron microscopy.

  7. Simulation study of secondary electron images in scanning ion microscopy

    CERN Document Server

    Ohya, K

    2003-01-01

    The target atomic number, Z sub 2 , dependence of secondary electron yield is simulated by applying a Monte Carlo code for 17 species of metals bombarded by Ga ions and electrons in order to study the contrast difference between scanning ion microscopes (SIM) and scanning electron microscopes (SEM). In addition to the remarkable reversal of the Z sub 2 dependence between the Ga ion and electron bombardment, a fine structure, which is correlated to the density of the conduction band electrons in the metal, is calculated for both. The brightness changes of the secondary electron images in SIM and SEM are simulated using Au and Al surfaces adjacent to each other. The results indicate that the image contrast in SIM is much more sensitive to the material species and is clearer than that for SEM. The origin of the difference between SIM and SEM comes from the difference in the lateral distribution of secondary electrons excited within the escape depth.

  8. Comparing Fourier optics and contrast transfer function modeling of image formation in low energy electron microscopy.

    Science.gov (United States)

    Yu, K M; Locatelli, A; Altman, M S

    2017-12-01

    A theoretical understanding of image formation in cathode lens microscopy can facilitate image interpretation. We compare Fourier Optics (FO) and Contrast Transfer Function (CTF) approaches that were recently adapted from other realms of microscopy to model image formation in low energy electron microscopy (LEEM). Although these two approaches incorporate imaging errors from several sources similarly, they differ in the way that the image intensity is calculated. The simplification that is used in the CTF calculation advantageously leads to its computational efficiency. However, we find that lens aberrations, and spatial and temporal coherence may affect the validity of the CTF approach to model LEEM image formation under certain conditions. In particular, these effects depend strongly on the nature of the object being imaged and also become more pronounced with increasing defocus. While the use of the CTF approach appears to be justified for objects that are routinely imaged with LEEM, comparison of theory to experimental observations of a focal image series for rippled, suspended graphene reveals one example where FO works, but CTF does not. This work alerts us to potential pitfalls and guides the effective use of FO and CTF approaches. It also lays the foundation for quantitative image evaluation using these methods. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Imaging by Electrochemical Scanning Tunneling Microscopy and Deconvolution Resolving More Details of Surfaces Nanomorphology

    DEFF Research Database (Denmark)

    Andersen, Jens Enevold Thaulov

    Upon imaging, electrochemical scanning tunneling microscopy (ESTM), scanning electrochemical micro-scopy (SECM) and in situ STM resolve information on electronic structures and on surface topography. At very high resolution, imaging processing is required, as to obtain information that relates...... to crystallographic-surface structures. Within the wide range of new technologies, those images surface features, the electrochemical scanning tunneling microscope (ESTM) provides means of atomic resolution where the tip participates actively in the process of imaging. Two metallic surfaces influence ions trapped...... of the characteristic details of the images. A large proportion of the observed noise may be explained by the scanning actions of the feedback circuitry while a minor fraction of the image details may be explained by surface drift phenomena. As opposed to the method of deconvolution, conventional methods of filtering...

  10. Spatiotemporal Rank Filtering Improves Image Quality Compared to Frame Averaging in 2-Photon Laser Scanning Microscopy.

    Directory of Open Access Journals (Sweden)

    Henry Pinkard

    Full Text Available Live imaging of biological specimens using optical microscopy is limited by tradeoffs between spatial and temporal resolution, depth into intact samples, and phototoxicity. Two-photon laser scanning microscopy (2P-LSM, the gold standard for imaging turbid samples in vivo, has conventionally constructed images with sufficient signal-to-noise ratio (SNR generated by sequential raster scans of the focal plane and temporal integration of the collected signals. Here, we describe spatiotemporal rank filtering, a nonlinear alternative to temporal integration, which makes more efficient use of collected photons by selectively reducing noise in 2P-LSM images during acquisition. This results in much higher SNR while preserving image edges and fine details. Practically, this allows for at least a four fold decrease in collection times, a substantial improvement for time-course imaging in biological systems.

  11. Spatiotemporal Rank Filtering Improves Image Quality Compared to Frame Averaging in 2-Photon Laser Scanning Microscopy.

    Science.gov (United States)

    Pinkard, Henry; Corbin, Kaitlin; Krummel, Matthew F

    2016-01-01

    Live imaging of biological specimens using optical microscopy is limited by tradeoffs between spatial and temporal resolution, depth into intact samples, and phototoxicity. Two-photon laser scanning microscopy (2P-LSM), the gold standard for imaging turbid samples in vivo, has conventionally constructed images with sufficient signal-to-noise ratio (SNR) generated by sequential raster scans of the focal plane and temporal integration of the collected signals. Here, we describe spatiotemporal rank filtering, a nonlinear alternative to temporal integration, which makes more efficient use of collected photons by selectively reducing noise in 2P-LSM images during acquisition. This results in much higher SNR while preserving image edges and fine details. Practically, this allows for at least a four fold decrease in collection times, a substantial improvement for time-course imaging in biological systems.

  12. Spatiotemporal Rank Filtering Improves Image Quality Compared to Frame Averaging in 2-Photon Laser Scanning Microscopy

    Science.gov (United States)

    Pinkard, Henry; Corbin, Kaitlin; Krummel, Matthew F.

    2016-01-01

    Live imaging of biological specimens using optical microscopy is limited by tradeoffs between spatial and temporal resolution, depth into intact samples, and phototoxicity. Two-photon laser scanning microscopy (2P-LSM), the gold standard for imaging turbid samples in vivo, has conventionally constructed images with sufficient signal-to-noise ratio (SNR) generated by sequential raster scans of the focal plane and temporal integration of the collected signals. Here, we describe spatiotemporal rank filtering, a nonlinear alternative to temporal integration, which makes more efficient use of collected photons by selectively reducing noise in 2P-LSM images during acquisition. This results in much higher SNR while preserving image edges and fine details. Practically, this allows for at least a four fold decrease in collection times, a substantial improvement for time-course imaging in biological systems. PMID:26938064

  13. Imaging nanoscale lattice variations by machine learning of x-ray diffraction microscopy data

    Science.gov (United States)

    Laanait, Nouamane; Zhang, Zhan; Schlepütz, Christian M.

    2016-09-01

    We present a novel methodology based on machine learning to extract lattice variations in crystalline materials, at the nanoscale, from an x-ray Bragg diffraction-based imaging technique. By employing a full-field microscopy setup, we capture real space images of materials, with imaging contrast determined solely by the x-ray diffracted signal. The data sets that emanate from this imaging technique are a hybrid of real space information (image spatial support) and reciprocal lattice space information (image contrast), and are intrinsically multidimensional (5D). By a judicious application of established unsupervised machine learning techniques and multivariate analysis to this multidimensional data cube, we show how to extract features that can be ascribed physical interpretations in terms of common structural distortions, such as lattice tilts and dislocation arrays. We demonstrate this ‘big data’ approach to x-ray diffraction microscopy by identifying structural defects present in an epitaxial ferroelectric thin-film of lead zirconate titanate.

  14. Three-dimensional tooth imaging using multiphoton and second harmonic generation microscopy

    Science.gov (United States)

    Chen, Min-Huey; Chen, Wei-Liang; Sun, Yen; Fwu, Peter Tramyeon; Lin, Ming-Gu; Dong, Chen-Yuan

    2007-02-01

    Detailed morphological and cellular information relating to the human tooth have traditionally been obtained through histological studies that required decalcification, staining, and fixation. With the recent invention of multiphoton microscopy, it has become possible to acquire high resolution images without histological procedures. Using an epiilluminated multiphoton microscope, we obtained two-photon excited autofluorescence and second harmonic generation (SHG) images of ex vivo human tooth. By combining these two imaging modalities we obtained submicron resolution images of the enamel, dentin, and the periodontal ligaments. The enamel emits endogenous two-photon autofluorescence. The structure of the dentin is visible from both the autofluorescence and second harmonic generation signals. The periodontal ligament composed mostly of collagen can be visualized by SHG imaging. We also constructed three dimensional images of the enamel, dentin, and periodontal ligament. The effectiveness of using multiphoton and second harmonic generation microscopy to obtain structural information of teeth suggest its potential use in dental diagnostics.

  15. Deep Learning Segmentation of Optical Microscopy Images Improves 3-D Neuron Reconstruction.

    Science.gov (United States)

    Li, Rongjian; Zeng, Tao; Peng, Hanchuan; Ji, Shuiwang

    2017-07-01

    Digital reconstruction, or tracing, of 3-D neuron structure from microscopy images is a critical step toward reversing engineering the wiring and anatomy of a brain. Despite a number of prior attempts, this task remains very challenging, especially when images are contaminated by noises or have discontinued segments of neurite patterns. An approach for addressing such problems is to identify the locations of neuronal voxels using image segmentation methods, prior to applying tracing or reconstruction techniques. This preprocessing step is expected to remove noises in the data, thereby leading to improved reconstruction results. In this paper, we proposed to use 3-D convolutional neural networks (CNNs) for segmenting the neuronal microscopy images. Specifically, we designed a novel CNN architecture, that takes volumetric images as the inputs and their voxel-wise segmentation maps as the outputs. The developed architecture allows us to train and predict using large microscopy images in an end-to-end manner. We evaluated the performance of our model on a variety of challenging 3-D microscopy images from different organisms. Results showed that the proposed methods improved the tracing performance significantly when combined with different reconstruction algorithms.

  16. Simulation of specular microscopy images of corneal endothelium, a tool for control of measurement errors.

    Science.gov (United States)

    Bucht, Curry; Söderberg, Per; Manneberg, Göran

    2011-05-01

    We aimed at developing simulation software capable of producing images of corneal endothelium close to identical to images captured by clinical specular microscopy with defined morphometrical characteristics. It was further planned to demonstrate the usefulness of the simulator by analysing measurement errors associated with a trained operator using a commercially available semi-automatic algorithm for analysis of simulated images. Software was developed that allows creation of unique images of the corneal endothelium expressing morphology close to identical with that seen in images of corneal specular microscopy. Several hundred unique images of the corneal endothelium were generated with randomization, spanning a physiological range of endothelial cell density. As an example of the usefulness of the simulator for analysis of measurement errors in corneal specular microscopy, a total of 12 of all the images generated were randomly selected such that the endothelial cell density expressed was evenly distributed over the physiological range of endothelial cell density. The images were transferred to a personal computer. The imagenet-640 software was used to analyse endothelial cell size variation, percentage of hexagonal endothelial cells, and endothelial cell density. The simulator developed allows randomized generation of corneal specular microscopy images with a preset expected average and variation of cell structure. Calculated morphometric information of each cell is stored in the simulator. The image quality can secondarily be varied with a toolbox of filters to approximate a large spectrum of clinically captured images. As an example of the use of the simulator, measurement errors associated with one trained operator using the imagenet-640 software, and focusing on endothelial cell density, were examined. The functional dependence between morphometric information estimated with the imagenet-640 software algorithm and real morphometric information as provided

  17. Image simulations of kinked vortices for transmission electron microscopy

    DEFF Research Database (Denmark)

    Beleggia, Marco; Pozzi, G.; Tonomura, A.

    2010-01-01

    We present an improved model of kinked vortices in high-Tc superconductors suitable for the interpretation of Fresnel or holographic observations carried out with a transmission electron microscope. A kinked vortex is composed of two displaced half-vortices, perpendicular to the film plane...... observations of high-Tc superconducting films, where the Fresnel contrast associated with some vortices showed a dumbbell like appearance. Here, we show that under suitable conditions the JV segment may reveal itself in Fresnel imaging or holographic phase mapping in a transmission electron microscope....

  18. Membrane lipid domains and rafts: current applications of fluorescence lifetime spectroscopy and imaging.

    Science.gov (United States)

    de Almeida, Rodrigo F M; Loura, Luís M S; Prieto, Manuel

    2009-02-01

    Membrane microdomains and their involvement in cellular processes are part of the current paradigm of biomembranes. However, a better characterization of domains, namely lipid rafts, is needed. In this review, it is shown how the use of time-resolved fluorescence, with the adequate parameters and probes, helps elucidating the type, number, fraction, composition and size of lipid phases and domains in multicomponent model systems. The determination of phase diagrams for lipid mixtures containing sphingolipids and/or cholesterol is exemplified. The use of fluorescence quenching and Förster resonance energy transfer (FRET) are also illustrated. Strategies for studying protein-induced domains are presented. The advantages of using single point microscopic decays and fluorescence lifetime imaging microscopy (FLIM) in systems with three-phase coexistence are explained. Finally, the introduction of FLIM allows studies in live cell membranes, and the nature of the microdomains observed is readily elucidated due to the information retrieved from fluorescence lifetimes.

  19. Note: Fast imaging of DNA in atomic force microscopy enabled by a local raster scan algorithm.

    Science.gov (United States)

    Huang, Peng; Andersson, Sean B

    2014-06-01

    Approaches to high-speed atomic force microscopy typically involve some combination of novel mechanical design to increase the physical bandwidth and advanced controllers to take maximum advantage of the physical capabilities. For certain classes of samples, however, imaging time can be reduced on standard instruments by reducing the amount of measurement that is performed to image the sample. One such technique is the local raster scan algorithm, developed for imaging of string-like samples. Here we provide experimental results on the use of this technique to image DNA samples, demonstrating the efficacy of the scheme and illustrating the order-of-magnitude improvement in imaging time that it provides.

  20. Live cardiomyocyte imaging via hybrid TPEF-SHG microscopy

    Science.gov (United States)

    Liu, Honghai; Qin, Wan; Shao, Yonghong; Liu, Qiuying; Ma, Zhen; Borg, Thomas K.; Gao, Bruce Z.

    2012-03-01

    Utilizing a custom-built, on-stage incubator-combined, two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) imaging system, we observed new-sarcomere addition in rat neonatal cardiomyocytes during 10 hours of on-stage incubation. This addition occurred at one end of an existing myofibril, the sides of existing myofibrils, and at the interstice of several separated myofibrils; in the cases of the latter two, we observed mature myofibrils acting as templates. We found that during sarcomeric addition, myosin filaments are assembled onto the premyofibril laterally. This lateral addition, which proceeds stepwise along the axial direction, plays an important role in the accumulation of Z-bodies to form mature Z-disks and in the regulation of sarcomeric length during maturation.

  1. Video Object Tracking in Neural Axons with Fluorescence Microscopy Images

    Directory of Open Access Journals (Sweden)

    Liang Yuan

    2014-01-01

    tracking. In this paper, we describe two automated tracking methods for analyzing neurofilament movement based on two different techniques: constrained particle filtering and tracking-by-detection. First, we introduce the constrained particle filtering approach. In this approach, the orientation and position of a particle are constrained by the axon’s shape such that fewer particles are necessary for tracking neurofilament movement than object tracking techniques based on generic particle filtering. Secondly, a tracking-by-detection approach to neurofilament tracking is presented. For this approach, the axon is decomposed into blocks, and the blocks encompassing the moving neurofilaments are detected by graph labeling using Markov random field. Finally, we compare two tracking methods by performing tracking experiments on real time-lapse image sequences of neurofilament movement, and the experimental results show that both methods demonstrate good performance in comparison with the existing approaches, and the tracking accuracy of the tracing-by-detection approach is slightly better between the two.

  2. High-sensitivity chemical imaging for biomedicine by SRS microscopy (Conference Presentation)

    Science.gov (United States)

    Min, Wei

    2017-02-01

    Innovations in spectroscopy principles and microscopy technology have significantly impacted modern biology and medicine. While most of the contemporary bio-imaging modalities harness electronic transition, nuclear spin or radioactivity, vibrational spectroscopy has not been widely used yet. Here we will discuss an emerging chemical imaging platform, stimulated Raman scattering (SRS) microscopy, which can enhance the otherwise feeble spontaneous Raman eight orders of magnitude by virtue of stimulated emission. When coupled with stable isotopes (e.g., deuterium and 13C) or bioorthogonal chemical moieties (e.g., alkynes), SRS microscopy is well suited for probing in vivo metabolic dynamics of small bio-molecules which cannot be labeled by bulky fluorophores. Physical principle of the underlying optical spectroscopy and exciting biomedical applications such as imaging lipid metabolism, protein synthesis, DNA replication, protein degradation, RNA synthesis, glucose uptake, drug trafficking and tumor metabolism will be presented.

  3. Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy

    Directory of Open Access Journals (Sweden)

    Segall Jeffrey E

    2005-05-01

    Full Text Available Abstract Background The development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues. However, the use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy. Results Here we describe a method for the simultaneous imaging, by multiphoton microscopy, of Green Fluorescent Protein, Cyan Fluorescent Protein and collagen in vivo in living tumors. This novel method enables: 1 the simultaneous visualization of overall cell shape and sub-cellular structures such as the plasma membrane or proteins of interest in cells inside living animals, 2 direct comparison of the behavior of single cells from different cell lines in the same microenvironment in vivo. Conclusion Using this multi-fluor, multiphoton technique, we demonstrate that motility and metastatic differences between carcinoma cells of differing metastatic potential can be imaged in the same animal simultaneously at sub-cellular resolution.

  4. Correlation of live-cell imaging with volume scanning electron microscopy.

    Science.gov (United States)

    Lucas, Miriam S; Günthert, Maja; Bittermann, Anne Greet; de Marco, Alex; Wepf, Roger

    2017-01-01

    Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach is a flow-chamber setup based on Ibidi μ-slides. Both live-cell imaging strategies can be followed up with serial blockface- or focused ion beam-scanning electron microscopy. Two types of resin embedding after heavy metal staining and dehydration are presented making best use of the particular advantages of each imaging modality: classical en-bloc embedding and thin-layer plastification. The latter can be used only for focused ion beam-scanning electron microscopy, but is advantageous for studying cell-interactions with specific substrates, or when the substrate cannot be removed. En-bloc embedding has diverse applications and can be applied for both described volume scanning electron microscopy techniques. Finally, strategies for relocating the cell of interest are discussed for both embedding approaches and in respect to the applied light and scanning electron microscopy methods. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. The virtual microscopy database-sharing digital microscope images for research and education.

    Science.gov (United States)

    Lee, Lisa M J; Goldman, Haviva M; Hortsch, Michael

    2018-02-14

    Over the last 20 years, virtual microscopy has become the predominant modus of teaching the structural organization of cells, tissues, and organs, replacing the use of optical microscopes and glass slides in a traditional histology or pathology laboratory setting. Although virtual microscopy image files can easily be duplicated, creating them requires not only quality histological glass slides but also an expensive whole slide microscopic scanner and massive data storage devices. These resources are not available to all educators and researchers, especially at new institutions in developing countries. This leaves many schools without access to virtual microscopy resources. The Virtual Microscopy Database (VMD) is a new resource established to address this problem. It is a virtual image file-sharing website that allows researchers and educators easy access to a large repository of virtual histology and pathology image files. With the support from the American Association of Anatomists (Bethesda, MD) and MBF Bioscience Inc. (Williston, VT), registration and use of the VMD are currently free of charge. However, the VMD site is restricted to faculty and staff of research and educational institutions. Virtual Microscopy Database users can upload their own collection of virtual slide files, as well as view and download image files for their own non-profit educational and research purposes that have been deposited by other VMD clients. Anat Sci Educ. © 2018 American Association of Anatomists. © 2018 American Association of Anatomists.

  6. Hybrid Microscopy: Enabling Inexpensive High-Performance Imaging through Combined Physical and Optical Magnifications.

    Science.gov (United States)

    Zhang, Yu Shrike; Chang, Jae-Byum; Alvarez, Mario Moisés; Trujillo-de Santiago, Grissel; Aleman, Julio; Batzaya, Byambaa; Krishnadoss, Vaishali; Ramanujam, Aishwarya Aravamudhan; Kazemzadeh-Narbat, Mehdi; Chen, Fei; Tillberg, Paul W; Dokmeci, Mehmet Remzi; Boyden, Edward S; Khademhosseini, Ali

    2016-03-15

    To date, much effort has been expended on making high-performance microscopes through better instrumentation. Recently, it was discovered that physical magnification of specimens was possible, through a technique called expansion microscopy (ExM), raising the question of whether physical magnification, coupled to inexpensive optics, could together match the performance of high-end optical equipment, at a tiny fraction of the price. Here we show that such "hybrid microscopy" methods--combining physical and optical magnifications--can indeed achieve high performance at low cost. By physically magnifying objects, then imaging them on cheap miniature fluorescence microscopes ("mini-microscopes"), it is possible to image at a resolution comparable to that previously attainable only with benchtop microscopes that present costs orders of magnitude higher. We believe that this unprecedented hybrid technology that combines expansion microscopy, based on physical magnification, and mini-microscopy, relying on conventional optics--a process we refer to as Expansion Mini-Microscopy (ExMM)--is a highly promising alternative method for performing cost-effective, high-resolution imaging of biological samples. With further advancement of the technology, we believe that ExMM will find widespread applications for high-resolution imaging particularly in research and healthcare scenarios in undeveloped countries or remote places.

  7. Cryogenic-temperature electron microscopy direct imaging of carbon nanotubes and graphene solutions in superacids.

    Science.gov (United States)

    Kleinerman, O; Parra-Vasquez, A Nicholas G; Green, M J; Behabtu, N; Schmidt, J; Kesselman, E; Young, C C; Cohen, Y; Pasquali, M; Talmon, Y

    2015-07-01

    Cryogenic electron microscopy (cryo-EM) is a powerful tool for imaging liquid and semiliquid systems. While cryogenic transmission electron microscopy (cryo-TEM) is a standard technique in many fields, cryogenic scanning electron microscopy (cryo-SEM) is still not that widely used and is far less developed. The vast majority of systems under investigation by cryo-EM involve either water or organic components. In this paper, we introduce the use of novel cryo-TEM and cryo-SEM specimen preparation and imaging methodologies, suitable for highly acidic and very reactive systems. Both preserve the native nanostructure in the system, while not harming the expensive equipment or the user. We present examples of direct imaging of single-walled, multiwalled carbon nanotubes and graphene, dissolved in chlorosulfonic acid and oleum. Moreover, we demonstrate the ability of these new cryo-TEM and cryo-SEM methodologies to follow phase transitions in carbon nanotube (CNT)/superacid systems, starting from dilute solutions up to the concentrated nematic liquid-crystalline CNT phases, used as the 'dope' for all-carbon-fibre spinning. Originally developed for direct imaging of CNTs and graphene dissolution and self-assembly in superacids, these methodologies can be implemented for a variety of highly acidic systems, paving a way for a new field of nonaqueous cryogenic electron microscopy. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

  8. Magnetic imaging of unconventional superconductors by scanning SQUID microscopy

    International Nuclear Information System (INIS)

    Hykel, D.

    2011-01-01

    We present the development of a scanning SQUID/AFM microscope and measurements performed on different samples. The microscope can take topographic and magnetic images simultaneously. The magnetic resolution is of the order of 10 -4 Φ 0 √Hz and the spatial resolution of the SQUIDs used in this thesis goes up to 600 nm. The scanning range is 70 μm * 85 μm. The temperature range accessible is between 200 mK and 10 K at the time of writing. Measurements on a thin rhenium film (80 nm) give an estimate of the minimal pinning force of a vortex of about 3.9 * 10 -16 N. Furthermore, the penetration depth λ on this sample was determined as a function of temperature. For T → 0, λ →79 nm. We have for the first time shown local measurements of the domain structure of the superconducting ferromagnet UCoGe and determined the average domain size in the virgin state (10 μm). By magnetic imaging we were capable of determining the magnetic field difference above opposite domains along the c-axis to be 45 G and 16 G along the b-axis. Due to these magnetic field measurements we were able to give an upper limit for the domain wall width (∼ 1μm) and domain reconstruction depth (100 nm). This is supported by simple calculations leading to a domain wall width of several angstroms. Thus UCoGe can be considered an ideal Ising ferromagnet. Different possible domain structures for an Ising ferromagnet have been discussed. The complicated domain structure found in the zero field cooled virgin state corresponds to up domains embedded in larger down domains and vice versa. We have shown evidence for coexistence of superconductivity and ferromagnetism. The weak Meissner effect can be explained by a spontaneous vortex state, put forward by other groups. Numerical simulations suggest that the strong magnetic background signal and the limited spatial and magnetic resolution of the used SQUID made it difficult to resolve the expected spontaneous vortex state. The relaxation of the

  9. Forming intermediate spatial resolution of microscopy images for continuous zooming on multi-resolution processing system

    Science.gov (United States)

    Putranto, Evan H. E.; Suzuki, Tomohiro; Usuki, Shin; Miura, Kenjiro T.

    2017-09-01

    Digital zooming especially on microscopy image has attempted to improve their quality of measurement into a better assessment. However, since the field of view of high-resolution image are not wide despite of the fact that high-resolution image has more information detail and low-resolution image has their merits which is bring a big picture of the whole structure, we need to observe the sample in any scale. This problem was been solved by developing dual-view of high and low images resolution1 but in a single interpolated images. The goal of this research is utilize multi-resolution images to develop smooth zooming magnification of microscopy image. In order to achieve smooth zooming magnification on different condition of the images, scheme process will be needed. First, we took a several spatial images of the same sample based on the different objective lens, author was used 4 objective lens which are 10×, 20×, 50× and 150× magnification. In this synthesize phase, we interpolate lower resolution image for synthesize purpose with the next higher resolution image of the sample. Second, continue to looking for the feature point of both images with SIFT feature point method until we synthesize both images. Third, author treat this synthesized image with discrete fourier transform (DFT) with low-pass filter as the same size with numerical aperture (NA) that was input on the first phase. Then the fourth phase is looping this processes until intermediate images are generated enough to be blend with pyramid blend method. In this article we also try to make a system that can arbitrarily generate intermediate image with hierarchical system.

  10. Quantitative changes in human epithelial cancers and osteogenesis imperfecta disease detected using nonlinear multicontrast microscopy

    Science.gov (United States)

    Adur, Javier; Pelegati, Vitor B.; de Thomaz, Andre A.; D'Souza-Li, Lilia; Assunção, Maria do Carmo; Bottcher-Luiz, Fátima; Andrade, Liliana A. L. A.; Cesar, Carlos L.

    2012-08-01

    We show that combined multimodal nonlinear optical (NLO) microscopies, including two-photon excitation fluorescence, second-harmonic generation (SHG), third harmonic generation, and fluorescence lifetime imaging microscopy (FLIM) can be used to detect morphological and metabolic changes associated with stroma and epithelial transformation during the progression of cancer and osteogenesis imperfecta (OI) disease. NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for different types of human breast cancer, mucinous ovarian tumors, and skin dermis of patients with OI. Using a set of scoring methods (anisotropy, correlation, uniformity, entropy, and lifetime components), we found significant differences in the content, distribution and organization of collagen fibrils in the stroma of breast and ovary as well as in the dermis of skin. We suggest that our results provide a framework for using NLO techniques as a clinical diagnostic tool for human cancer and OI. We further suggest that the SHG and FLIM metrics described could be applied to other connective or epithelial tissue disorders that are characterized by abnormal cells proliferation and collagen assembly.

  11. Advances in cellular, subcellular, and nanoscale imaging in vitro and in vivo.

    Science.gov (United States)

    Wessels, Johannes T; Yamauchi, Kensuke; Hoffman, Robert M; Wouters, Fred S

    2010-07-01

    This review focuses on technical advances in fluorescence microscopy techniques including laser scanning techniques, fluorescence-resonance energy transfer (FRET) microscopy, fluorescence lifetime imaging (FLIM), stimulated emission depletion (STED)-based super-resolution microscopy, scanning confocal endomicroscopes, thin-sheet laser imaging microscopy (TSLIM), and tomographic techniques such as early photon tomography (EPT) as well as on clinical laser-based endoscopic and microscopic techniques. We will also discuss the new developments in the field of fluorescent dyes and fluorescent genetic reporters that enable new possibilities in high-resolution and molecular imaging both in in vitro and in vivo. Small animal and tissue imaging benefit from the development of new fluorescent proteins, dyes, and sensing constructs that operate in the far red and near-infrared spectrum.

  12. High resolution multiplexed functional imaging in live embryos (Conference Presentation)

    Science.gov (United States)

    Xu, Dongli; Zhou, Weibin; Peng, Leilei

    2017-02-01

    Fourier multiplexed fluorescence lifetime imaging (FmFLIM) scanning laser optical tomography (FmFLIM-SLOT) combines FmFLIM and Scanning laser optical tomography (SLOT) to perform multiplexed 3D FLIM imaging of live embryos. The system had demonstrate multiplexed functional imaging of zebrafish embryos genetically express Foster Resonant Energy Transfer (FRET) sensors. However, previous system has a 20 micron resolution because the focused Gaussian beam diverges quickly from the focused plane, makes it difficult to achieve high resolution imaging over a long projection depth. Here, we present a high-resolution FmFLIM-SLOT system with achromatic Bessel beam, which achieves 3 micron resolution in 3D deep tissue imaging. In Bessel-FmFLIM-SLOT, multiple laser excitation lines are firstly intensity modulated by a Michelson interferometer with a spinning polygon mirror optical delay line, which enables Fourier multiplexed multi-channel lifetime measurements. Then, a spatial light modulator and a prism are used to transform the modulated Gaussian laser beam to an achromatic Bessel beam. The achromatic Bessel beam scans across the whole specimen with equal angular intervals as sample rotated. After tomography reconstruction and the frequency domain lifetime analysis method, both the 3D intensity and lifetime image of multiple excitation-emission can be obtained. Using Bessel-FmFLIM-SLOT system, we performed cellular-resolution FLIM tomography imaging of live zebrafish embryo. Genetically expressed FRET sensors in these embryo will allow non-invasive observation of multiple biochemical processes in vivo.

  13. On the Progress of Scanning Transmission Electron Microscopy (STEM) Imaging in a Scanning Electron Microscope.

    Science.gov (United States)

    Sun, Cheng; Müller, Erich; Meffert, Matthias; Gerthsen, Dagmar

    2018-03-28

    Transmission electron microscopy (TEM) with low-energy electrons has been recognized as an important addition to the family of electron microscopies as it may avoid knock-on damage and increase the contrast of weakly scattering objects. Scanning electron microscopes (SEMs) are well suited for low-energy electron microscopy with maximum electron energies of 30 keV, but they are mainly used for topography imaging of bulk samples. Implementation of a scanning transmission electron microscopy (STEM) detector and a charge-coupled-device camera for the acquisition of on-axis transmission electron diffraction (TED) patterns, in combination with recent resolution improvements, make SEMs highly interesting for structure analysis of some electron-transparent specimens which are traditionally investigated by TEM. A new aspect is correlative SEM, STEM, and TED imaging from the same specimen region in a SEM which leads to a wealth of information. Simultaneous image acquisition gives information on surface topography, inner structure including crystal defects and qualitative material contrast. Lattice-fringe resolution is obtained in bright-field STEM imaging. The benefits of correlative SEM/STEM/TED imaging in a SEM are exemplified by structure analyses from representative sample classes such as nanoparticulates and bulk materials.

  14. Confocal scanning microscopy with multiple optical probes for high speed measurements and better imaging

    Science.gov (United States)

    Chun, Wanhee; Lee, SeungWoo; Gweon, Dae-Gab

    2008-02-01

    Confocal scanning microscopy (CSM) needs a scanning mechanism because only one point information of specimen can be obtained. Therefore the speed of the confocal scanning microscopy is limited by the speed of the scanning tool. To overcome this limitation from scanning tool we propose another scanning mechanism. We make three optical probes in the specimen under confocal condition of each point. Three optical probes are moved by beam scanning mechanism with shared resonant scanning mirror (RM) and galvanometer driven mirror (GM). As each optical probe scan allocated region of the specimen, information from three points is obtained simultaneously and image acquisition time is reduced. Therefore confocal scanning microscopy with multiple optical probes is expected to have three times faster speed of the image acquisition than conventional one. And as another use, multiple optical probes to which different light wavelength is applied can scan whole same region respectively. It helps to obtain better contrast image in case of specimens having different optical characteristics for specific light wavelength. In conclusion confocal scanning microscopy with multiple optical probes is useful technique for views of image acquisition speed and image quality.

  15. Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

    Energy Technology Data Exchange (ETDEWEB)

    Duman, M; Pfleger, M; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Ebner, A; Schuetz, G J; Hinterdorfer, P [Institute for Biophysics, University of Linz, Altenbergerstrasse 69, A-4040 Linz (Austria); Zhu, R; Mayer, B [Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Institute for Biophysics, University of Linz, Altenbergerstrasse 69, A-4040 Linz (Austria); Rankl, C; Moertelmaier, M; Kada, G; Kienberger, F [Agilent Technologies Austria GmbH, Aubrunnerweg 11, A-4040 Linz (Austria); Salio, M; Shepherd, D; Polzella, P; Cerundolo, V [Cancer Research UK Tumor Immunology Group, Weatherall Institute of Molecular Medicine, Nuffield Department of Medicine, University of Oxford, Oxford OX3 9DS (United Kingdom); Dieudonne, M, E-mail: ferry_kienberger@agilent.com [Agilent Technologies Belgium, Wingepark 51, Rotselaar, AN B-3110 (Belgium)

    2010-03-19

    The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on {alpha}-galactosylceramide ({alpha}GalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from {approx} 25 to {approx} 160 nm, with the smaller domains corresponding to a single CD1d molecule.

  16. Improved localization accuracy in stochastic super-resolution fluorescence microscopy by K-factor image deshadowing.

    Science.gov (United States)

    Ilovitsh, Tali; Meiri, Amihai; Ebeling, Carl G; Menon, Rajesh; Gerton, Jordan M; Jorgensen, Erik M; Zalevsky, Zeev

    2013-12-16

    Localization of a single fluorescent particle with sub-diffraction-limit accuracy is a key merit in localization microscopy. Existing methods such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) achieve localization accuracies of single emitters that can reach an order of magnitude lower than the conventional resolving capabilities of optical microscopy. However, these techniques require a sparse distribution of simultaneously activated fluorophores in the field of view, resulting in larger time needed for the construction of the full image. In this paper we present the use of a nonlinear image decomposition algorithm termed K-factor, which reduces an image into a nonlinear set of contrast-ordered decompositions whose joint product reassembles the original image. The K-factor technique, when implemented on raw data prior to localization, can improve the localization accuracy of standard existing methods, and also enable the localization of overlapping particles, allowing the use of increased fluorophore activation density, and thereby increased data collection speed. Numerical simulations of fluorescence data with random probe positions, and especially at high densities of activated fluorophores, demonstrate an improvement of up to 85% in the localization precision compared to single fitting techniques. Implementing the proposed concept on experimental data of cellular structures yielded a 37% improvement in resolution for the same super-resolution image acquisition time, and a decrease of 42% in the collection time of super-resolution data with the same resolution.

  17. Denoising time-resolved microscopy image sequences with singular value thresholding

    Energy Technology Data Exchange (ETDEWEB)

    Furnival, Tom, E-mail: tjof2@cam.ac.uk; Leary, Rowan K., E-mail: rkl26@cam.ac.uk; Midgley, Paul A., E-mail: pam33@cam.ac.uk

    2017-07-15

    Time-resolved imaging in microscopy is important for the direct observation of a range of dynamic processes in both the physical and life sciences. However, the image sequences are often corrupted by noise, either as a result of high frame rates or a need to limit the radiation dose received by the sample. Here we exploit both spatial and temporal correlations using low-rank matrix recovery methods to denoise microscopy image sequences. We also make use of an unbiased risk estimator to address the issue of how much thresholding to apply in a robust and automated manner. The performance of the technique is demonstrated using simulated image sequences, as well as experimental scanning transmission electron microscopy data, where surface adatom motion and nanoparticle structural dynamics are recovered at rates of up to 32 frames per second. - Highlights: • Correlations in space and time are harnessed to denoise microscopy image sequences. • A robust estimator provides automated selection of the denoising parameter. • Motion tracking and automated noise estimation provides a versatile algorithm. • Application to time-resolved STEM enables study of atomic and nanoparticle dynamics.

  18. Rapid volumetric imaging with Bessel-Beam three-photon microscopy

    Science.gov (United States)

    Chen, Bingying; Huang, Xiaoshuai; Gou, Dongzhou; Zeng, Jianzhi; Chen, Guoqing; Pang, Meijun; Hu, Yanhui; Zhao, Zhe; Zhang, Yunfeng; Zhou, Zhuan; Wu, Haitao; Cheng, Heping; Zhang, Zhigang; Xu, Chris; Li, Yulong; Chen, Liangyi; Wang, Aimin

    2018-01-01

    Owing to its tissue-penetration ability, multi-photon fluorescence microscopy allows for the high-resolution, non-invasive imaging of deep tissue in vivo; the recently developed three-photon microscopy (3PM) has extended the depth of high-resolution, non-invasive functional imaging of mouse brains to beyond 1.0 mm. However, the low repetition rate of femtosecond lasers that are normally used in 3PM limits the temporal resolution of point-scanning three-photon microscopy. To increase the volumetric imaging speed of 3PM, we propose a combination of an axially elongated needle-like Bessel-beam with three-photon excitation (3PE) to image biological samples with an extended depth of focus. We demonstrate the higher signal-to-background ratio (SBR) of the Bessel-beam 3PM compared to the two-photon version both theoretically and experimentally. Finally, we perform simultaneous calcium imaging of brain regions at different axial locations in live fruit flies and rapid volumetric imaging of neuronal structures in live mouse brains. These results highlight the unique advantage of conducting rapid volumetric imaging with a high SBR in the deep brain in vivo using scanning Bessel-3PM.

  19. Imaging by Electrochemical Scanning Tunneling Microscopy and Deconvolution Resolving More Details of Surfaces Nanomorphology

    DEFF Research Database (Denmark)

    Andersen, Jens Enevold Thaulov

    Upon imaging, electrochemical scanning tunneling microscopy (ESTM), scanning electrochemical micro-scopy (SECM) and in situ STM resolve information on electronic structures and on surface topography. At very high resolution, imaging processing is required, as to obtain information that relates...... to crystallographic-surface structures. Within the wide range of new technologies, those images surface features, the electrochemical scanning tunneling microscope (ESTM) provides means of atomic resolution where the tip participates actively in the process of imaging. Two metallic surfaces influence ions trapped.......g., nanoelectronics and single-molecule probing. In principle, the ESTM is capable of sub-atomic resolution but many details at this level of magnification need further treatment of recorded data before real information is obtained. Deconvolution of the data according to the instrument response may explain some...

  20. Parallel detecting super-resolution microscopy using correlation based image restoration

    Science.gov (United States)

    Yu, Zhongzhi; Liu, Shaocong; Zhu, Dazhao; Kuang, Cuifang; Liu, Xu

    2017-12-01

    A novel approach to achieve the image restoration is proposed in which each detector's relative position in the detector array is no longer a necessity. We can identify each detector's relative location by extracting a certain area from one of the detector's image and scanning it on other detectors' images. According to this location, we can generate the point spread functions (PSF) for each detector and perform deconvolution for image restoration. Equipped with this method, the microscope with discretionally designed detector array can be easily constructed without the concern of exact relative locations of detectors. The simulated results and experimental results show the total improvement in resolution with a factor of 1.7 compared to conventional confocal fluorescence microscopy. With the significant enhancement in resolution and easiness for application of this method, this novel method should have potential for a wide range of application in fluorescence microscopy based on parallel detecting.

  1. Multiparametric atomic force microscopy imaging of single bacteriophages extruding from living bacteria

    Science.gov (United States)

    Alsteens, David; Trabelsi, Heykel; Soumillion, Patrice; Dufrêne, Yves F.

    2013-12-01

    Force-distance (FD) curve-based atomic force microscopy is a valuable tool to simultaneously image the structure and map the biophysical properties of biological samples at the nanoscale. Traditionally, FD-based atomic force microscopy has been severely limited by its poor temporal and lateral resolutions. Here we report the use of advanced FD-based technology combined with biochemically sensitive tips to image filamentous bacteriophages extruding from living bacteria at unprecedented speed and resolution. Directly correlated multiparametric images of the structure, adhesion and elasticity of infected bacteria demonstrate that the sites of assembly and extrusion localize at the bacterial septum in the form of soft nanodomains surrounded by stiff cell wall material. The quantitative nano-bio-imaging method presented here offers a wealth of opportunities for mapping the physical properties and molecular interactions of complex biosystems, from viruses to tissues.

  2. Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis.

    Science.gov (United States)

    Supatto, Willy; McMahon, Amy; Fraser, Scott E; Stathopoulos, Angelike

    2009-01-01

    This protocol describes imaging and computational tools to collect and analyze live imaging data of embryonic cell migration. Our five-step protocol requires a few weeks to move through embryo preparation and four-dimensional (4D) live imaging using multi-photon microscopy, to 3D cell tracking using image processing, registration of tracking data and their quantitative analysis using computational tools. It uses commercially available equipment and requires expertise in microscopy and programming that is appropriate for a biology laboratory. Custom-made scripts are provided, as well as sample datasets to permit readers without experimental data to carry out the analysis. The protocol has offered new insights into the genetic control of cell migration during Drosophila gastrulation. With simple modifications, this systematic analysis could be applied to any developing system to define cell positions in accordance with the body plan, to decompose complex 3D movements and to quantify the collective nature of cell migration.

  3. Wavelength-Dependent Differential Interference Contrast Microscopy: Selectively Imaging Nanoparticle Probes in Live Cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Wei; Wang, Gufeng; Fang, Ning; and Yeung, Edward S.

    2009-11-15

    Gold and silver nanoparticles display extraordinarily large apparent refractive indices near their plasmon resonance (PR) wavelengths. These nanoparticles show good contrast in a narrow spectral band but are poorly resolved at other wavelengths in differential interference contrast (DIC) microscopy. The wavelength dependence of DIC contrast of gold/silver nanoparticles is interpreted in terms of Mie's theory and DIC working principles. We further exploit this wavelength dependence by modifying a DIC microscope to enable simultaneous imaging at two wavelengths. We demonstrate that gold/silver nanoparticles immobilized on the same glass slides through hybridization can be differentiated and imaged separately. High-contrast, video-rate images of living cells can be recorded both with and without illuminating the gold nanoparticle probes, providing definitive probe identification. Dual-wavelength DIC microscopy thus presents a new approach to the simultaneous detection of multiple probes of interest for high-speed live-cell imaging.

  4. Microscopy mineral image enhancement based on improved adaptive threshold in nonsubsampled shearlet transform domain

    Science.gov (United States)

    Li, Liangliang; Si, Yujuan; Jia, Zhenhong

    2018-03-01

    In this paper, a novel microscopy mineral image enhancement method based on adaptive threshold in non-subsampled shearlet transform (NSST) domain is proposed. First, the image is decomposed into one low-frequency sub-band and several high-frequency sub-bands. Second, the gamma correction is applied to process the low-frequency sub-band coefficients, and the improved adaptive threshold is adopted to suppress the noise of the high-frequency sub-bands coefficients. Third, the processed coefficients are reconstructed with the inverse NSST. Finally, the unsharp filter is used to enhance the details of the reconstructed image. Experimental results on various microscopy mineral images demonstrated that the proposed approach has a better enhancement effect in terms of objective metric and subjective metric.

  5. Noninvasive multiphoton imaging of cardiovascular structures using NIR femtosecond laser scanning microscopy

    Science.gov (United States)

    Schenke-Layland, Katja; Riemann, Iris; Stock, Ulrich A.; Konig, Karsten

    2004-07-01

    Near infrared (NIR) femtosecond laser scanning microscopy represents a novel and very promising medical diagnostic imaging technology for non-invasive cross-sectional analysis of living biological tissues. In this study multiphoton imaging has been performed to analyze the structural features of extracellular matrix (ECM) components, e.g. collagen and elastin, of living pulmonary and aortic heart valves. High-resolution autofluorescence and second harmonic generation (SHG) images of collagenous and elastic fibers were demonstrated using multifluorophore, multiphoton excitation at two different wavelengths and non-invasive optical sectioning, without the need of embedding or staining. The quality of the resulting three-dimensional images allowed exact differentiation of the ECM components. These experimental results indicated that NIR femtosecond laser scanning microscopy may prove to be a useful tool for the non-destructive monitoring and characterization of cardiovascular structures.

  6. High-contrast imaging of mycobacterium tuberculosis using third-harmonic generation microscopy

    Science.gov (United States)

    Kim, Bo Ram; Lee, Eungjang; Park, Seung-Han

    2015-07-01

    Nonlinear optical microcopy has become an important tool in investigating biomaterials due to its various advantages such as label-free imaging capabilities. In particular, it has been shown that third-harmonic generation (THG) signals can be produced at interfaces between an aqueous medium (e.g. cytoplasm, interstitial fluid) and a mineralized lipidic surface. In this work, we have demonstrated that label-free high-contrast THG images of the mycobacterium tuberculosis can be obtained using THG microscopy.

  7. In vivo corneal neovascularization imaging by optical-resolution photoacoustic microscopy

    OpenAIRE

    Liu, Wenzhong; Schultz, Kathryn M.; Zhang, Kevin; Sasman, Amy; Gao, Fengli; Kume, Tsutomu; Zhang, Hao F.

    2014-01-01

    Corneal neovascularization leads to blurred vision, thus in vivo visualization is essential for pathological studies in animal models. Photoacoustic (PA) imaging can delineate microvasculature and hemodynamics noninvasively, which is suitable for investigating corneal neovascularization. In this study, we demonstrate in vivo imaging of corneal neovascularization in the mouse eye by optical-resolution photoacoustic microscopy (OR-PAM), where corneal neovascularization is induced by deliberate ...

  8. Segmentation of Drosophila Heart in Optical Coherence Microscopy Images Using Convolutional Neural Networks

    OpenAIRE

    Duan, Lian; Qin, Xi; He, Yuanhao; Sang, Xialin; Pan, Jinda; Xu, Tao; Men, Jing; Tanzi, Rudolph E.; Li, Airong; Ma, Yutao; Zhou, Chao

    2018-01-01

    Convolutional neural networks are powerful tools for image segmentation and classification. Here, we use this method to identify and mark the heart region of Drosophila at different developmental stages in the cross-sectional images acquired by a custom optical coherence microscopy (OCM) system. With our well-trained convolutional neural network model, the heart regions through multiple heartbeat cycles can be marked with an intersection over union (IOU) of ~86%. Various morphological and dyn...

  9. Combination of widefield fluorescence imaging and nonlinear optical microscopy of oral epithelial neoplasia

    Science.gov (United States)

    Pal, Rahul; Edward, Kert; Brown, Tyra; Ma, Liang; Yang, Jinping; McCammon, Susan; Motamedi, Massoud; Vargas, Gracie

    2013-03-01

    Multiphoton Autofluorescence Microscopy (MPAM) and Second Harmonic Generation Microscopy (SHGM) have shown the potential for noninvasive assessment of oral precancers and cancers. We have explored a combination of these nonlinear optical microscopic imaging techniques with widefield fluorescence imaging to assess morphometry similar to that of pathologic evaluation as well as information from endogenous fluorophores, which are altered with neoplastic transformation. Widefield fluorescence revealed areas of interest corresponding to sites with precancers or early tumors, generally resulting in a decrease in green emission or increase in red emission. Subsequent microscopy revealed significant differences in morphology between normal, dysplastic/neoplastic mucosa for all layers. Combination of a widefield and a microscopic technique provides a novel approach for tissue morphometric analysis along with large area assessment of tissue autofluorescence properties.

  10. Multimodal imaging of human cerebellum - merging X-ray phase microtomography, magnetic resonance microscopy and histology

    Science.gov (United States)

    Schulz, Georg; Waschkies, Conny; Pfeiffer, Franz; Zanette, Irene; Weitkamp, Timm; David, Christian; Müller, Bert

    2012-11-01

    Imaging modalities including magnetic resonance imaging and X-ray computed tomography are established methods in daily clinical diagnosis of human brain. Clinical equipment does not provide sufficient spatial resolution to obtain morphological information on the cellular level, essential for applying minimally or non-invasive surgical interventions. Therefore, generic data with lateral sub-micrometer resolution have been generated from histological slices post mortem. Sub-cellular spatial resolution, lost in the third dimension as a result of sectioning, is obtained using magnetic resonance microscopy and micro computed tomography. We demonstrate that for human cerebellum grating-based X-ray phase tomography shows complementary contrast to magnetic resonance microscopy and histology. In this study, the contrast-to-noise values of magnetic resonance microscopy and phase tomography were comparable whereas the spatial resolution in phase tomography is an order of magnitude better. The registered data with their complementary information permit the distinct segmentation of tissues within the human cerebellum.

  11. Adaptive and robust statistical methods for processing near-field scanning microwave microscopy images.

    Science.gov (United States)

    Coakley, K J; Imtiaz, A; Wallis, T M; Weber, J C; Berweger, S; Kabos, P

    2015-03-01

    Near-field scanning microwave microscopy offers great potential to facilitate characterization, development and modeling of materials. By acquiring microwave images at multiple frequencies and amplitudes (along with the other modalities) one can study material and device physics at different lateral and depth scales. Images are typically noisy and contaminated by artifacts that can vary from scan line to scan line and planar-like trends due to sample tilt errors. Here, we level images based on an estimate of a smooth 2-d trend determined with a robust implementation of a local regression method. In this robust approach, features and outliers which are not due to the trend are automatically downweighted. We denoise images with the Adaptive Weights Smoothing method. This method smooths out additive noise while preserving edge-like features in images. We demonstrate the feasibility of our methods on topography images and microwave |S11| images. For one challenging test case, we demonstrate that our method outperforms alternative methods from the scanning probe microscopy data analysis software package Gwyddion. Our methods should be useful for massive image data sets where manual selection of landmarks or image subsets by a user is impractical. Published by Elsevier B.V.

  12. Integrated optical coherence tomography and optical coherence microscopy imaging of human pathology

    Science.gov (United States)

    Lee, Hsiang-Chieh; Zhou, Chao; Wang, Yihong; Aquirre, Aaron D.; Tsai, Tsung-Han; Cohen, David W.; Connolly, James L.; Fujimoto, James G.

    2010-02-01

    Excisional biopsy is the current gold standard for disease diagnosis; however, it requires a relatively long processing time and it may also suffer from unacceptable false negative rates due to sampling errors. Optical coherence tomography (OCT) is a promising imaging technique that provide real-time, high resolution and three-dimensional (3D) images of tissue morphology. Optical coherence microscopy (OCM) is an extension of OCT, combining both the coherence gating and the confocal gating techniques. OCM imaging achieves cellular resolution with deeper imaging depth compared to confocal microscopy. An integrated OCT/OCM imaging system can provide co-registered multiscale imaging of tissue morphology. 3D-OCT provides architectural information with a large field of view and can be used to find regions of interest; while OCM provides high magnification to enable cellular imaging. The integrated OCT/OCM system has an axial resolution of pathologic specimens, including colon (58), thyroid (43), breast (34), and kidney (19), were imaged with OCT and OCM within 2 to 6 hours after excision. The images were compared with H & E histology to identify characteristic features useful for disease diagnosis. The feasibility of visualizing human pathology using integrated OCT/OCM was demonstrated in the pathology laboratory settings.

  13. Accuracy of surface strain measurements from transmission electron microscopy images of nanoparticles

    DEFF Research Database (Denmark)

    Madsen, Jacob; Liu, Pei; Wagner, Jakob Birkedal

    2017-01-01

    Strain analysis from high-resolution transmission electron microscopy (HRTEM) images offers a convenient tool for measuring strain in materials at the atomic scale. In this paper we present a theoretical study of the precision and accuracy of surface strain measurements directly from aberration...

  14. Diagnostic accuracy of confocal microscopy imaging vs. punch biopsy for diagnosing and subtyping basal cell carcinoma

    NARCIS (Netherlands)

    Kadouch, D. J.; Leeflang, M. M.; Elshot, Y. S.; Longo, C.; Ulrich, M.; van der Wal, A. C.; Wolkerstorfer, A.; Bekkenk, M. W.; de Rie, M. A.

    2017-01-01

    BackgroundIn vivo reflectance confocal microscopy (RCM) is a promising non-invasive skin imaging technique that could facilitate early diagnosis of basal cell carcinoma (BCC) instead of routine punch biopsies. However, the clinical value and utility of RCM vs. a punch biopsy in diagnosing and

  15. Covalently Immobilised Cytochrome C Imaged by In Situ Scanning Tunnelling Microscopy

    DEFF Research Database (Denmark)

    Andersen, Jens Enevold Thaulov; Olesen, Klaus G.; Danilov, Alexey I.

    1997-01-01

    In situ scanning tunnelling microscopy (STM) imaging of cytochrome c (cyt c) on polycrystalline Pt surfaces and on Au(lll) was achieved first by covalent immobilisation of 3-aminopropyltriethoxysilane (3-APTS) brought to react with oxide present on the Pt surfaces. Covalently bound 3-APTS forms...

  16. Optical Coherence Tomography and Biomolecular Imaging with Coherent Raman Scattering Microscopy

    DEFF Research Database (Denmark)

    Andersson-Engels, Stefan; Andersen, Peter E.

    2014-01-01

    The Special Section on Selected Topics in Biophotonics: Optical Coherence Tomography and Biomolecular Imaging with Coherent Raman Scattering Microscopy comprises two invited review papers and several contributed papers from the summer school Biophotonics ’13, as well as contributed papers within...

  17. Imaging of matrix-disorder in normal and pathological human dermis using nonlinear optical microscopy

    Science.gov (United States)

    Zhuo, Shuangmu; Chen, Jianxin; Xie, Shusen; Zheng, Liqin; Jiang, Xingshan

    2009-11-01

    In dermis, collagen and elastin are important structural proteins of extracellular maxtrix. The matrix-disorder is associated with various physiologic processes, such as localized scleroderma, anetoderma, photoaging. In this work, we demonstrate the capability of nonlinear optical microscopy in imaging structural proteins in normal and pathological human dermis.

  18. Optical imaging of non-fluorescent nanodiamonds in live cells using transient absorption microscopy.

    Science.gov (United States)

    Chen, Tao; Lu, Feng; Streets, Aaron M; Fei, Peng; Quan, Junmin; Huang, Yanyi

    2013-06-07

    We directly observe non-fluorescent nanodiamonds in living cells using transient absorption microscopy. This label-free technology provides a novel modality to study the dynamic behavior of nanodiamonds inside the cells with intrinsic three-dimensional imaging capability. We apply this method to capture the cellular uptake of nanodiamonds under various conditions, confirming the endocytosis mechanism.

  19. Imaging three-dimensional surface objects with submolecular resolution by atomic force microscopy

    Czech Academy of Sciences Publication Activity Database

    Moreno, C.; Stetsovych, Oleksandr; Shimizu, T.K.; Custance, O.

    2015-01-01

    Roč. 15, č. 4 (2015), s. 2257-2262 ISSN 1530-6984 Institutional support: RVO:68378271 Keywords : noncontact atomic force microscopy (NC- AFM ) * submolecular resolution * three-dimensional dynamic force spectroscopy * high-resolution imaging Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 13.779, year: 2015

  20. Determination of line edge roughness in low-dose top-down scanning electron microscopy images

    NARCIS (Netherlands)

    Verduin, T.; Kruit, P.; Hagen, C.W.

    2014-01-01

    We investigated the off-line metrology for line edge roughness (LER) determination by using the discrete power spectral density (PSD). The study specifically addresses low-dose scanning electron microscopy (SEM) images in order to reduce the acquisition time and the risk of resist shrinkage. The

  1. Self-interference fluorescence microscopy: three dimensional fluorescence imaging without depth scanning

    NARCIS (Netherlands)

    de Groot, M.; Evans, C.L.; de Boer, J.F.

    2012-01-01

    We present a new method for high-resolution, three-dimensional fluorescence imaging. In contrast to beam-scanning confocal microscopy, where the laser focus must be scanned both laterally and axially to collect a volume, we obtain depth information without the necessity of depth scanning. In this

  2. Batch fabrication of scanning microscopy probes for thermal and magnetic imaging using standard micromachining

    NARCIS (Netherlands)

    Sarajlic, Edin; Vermeer, Rolf; Delalande, M.Y.; Siekman, Martin Herman; Huijink, R.; Fujita, H.; Abelmann, Leon

    2010-01-01

    We present a process for batch fabrication of a novel scanning microscopy probe for thermal and magnetic imaging using standard micromachining and conventional optical contact lithography. The probe features an AFM-type cantilever with a sharp pyramidal tip composed of four freestanding silicon

  3. Polarization modulation laser scanning microscopy: A powerful tool to image molecular orientation and order

    Science.gov (United States)

    Gupta, Vinay K.; Kornfield, Julia A.

    1994-09-01

    To image the orientational order in a broad class of biological and manufactured materials, a new microscope has been developed that integrates laser scanning microscopy with polarization modulation polarimetry. Polarimetry allows quantitative characterization of the molecular orientation and the degree of order through characterization of optical anisotropy. Combined with laser scanning microscopy, it is used here to image the anisotropy with high spatial resolution, sensitivity, and speed. The design of the microscope is presented; and the vast improvement in sensitivity achieved using PM-LSM over conventional polarization microscopy is illustrated by imaging the linear dichroism of ultrathin Langmuir-Blodgett polymer films. PM-LSM allows imaging of the magnitude and orientation of linear dichroism in films as thin as three molecular layers (˜66 Å) at high resolution by rastering a diffraction limited spot of laser light across the sample. The rate of image acquisition is over 2000 pixels/s, two to three orders of magnitude faster than the previous methods of imaging optical anisotropy.

  4. Direct imaging of phase objects enables conventional deconvolution in bright field light microscopy.

    Directory of Open Access Journals (Sweden)

    Carmen Noemí Hernández Candia

    Full Text Available In transmitted optical microscopy, absorption structure and phase structure of the specimen determine the three-dimensional intensity distribution of the image. The elementary impulse responses of the bright field microscope therefore consist of separate absorptive and phase components, precluding general application of linear, conventional deconvolution processing methods to improve image contrast and resolution. However, conventional deconvolution can be applied in the case of pure phase (or pure absorptive objects if the corresponding phase (or absorptive impulse responses of the microscope are known. In this work, we present direct measurements of the phase point- and line-spread functions of a high-aperture microscope operating in transmitted bright field. Polystyrene nanoparticles and microtubules (biological polymer filaments serve as the pure phase point and line objects, respectively, that are imaged with high contrast and low noise using standard microscopy plus digital image processing. Our experimental results agree with a proposed model for the response functions, and confirm previous theoretical predictions. Finally, we use the measured phase point-spread function to apply conventional deconvolution on the bright field images of living, unstained bacteria, resulting in improved definition of cell boundaries and sub-cellular features. These developments demonstrate practical application of standard restoration methods to improve imaging of phase objects such as cells in transmitted light microscopy.

  5. Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging.

    Science.gov (United States)

    Errico, Claudia; Pierre, Juliette; Pezet, Sophie; Desailly, Yann; Lenkei, Zsolt; Couture, Olivier; Tanter, Mickael

    2015-11-26

    Non-invasive imaging deep into organs at microscopic scales remains an open quest in biomedical imaging. Although optical microscopy is still limited to surface imaging owing to optical wave diffusion and fast decorrelation in tissue, revolutionary approaches such as fluorescence photo-activated localization microscopy led to a striking increase in resolution by more than an order of magnitude in the last decade. In contrast with optics, ultrasonic waves propagate deep into organs without losing their coherence and are much less affected by in vivo decorrelation processes. However, their resolution is impeded by the fundamental limits of diffraction, which impose a long-standing trade-off between resolution and penetration. This limits clinical and preclinical ultrasound imaging to a sub-millimetre scale. Here we demonstrate in vivo that ultrasound imaging at ultrafast frame rates (more than 500 frames per second) provides an analogue to optical localization microscopy by capturing the transient signal decorrelation of contrast agents--inert gas microbubbles. Ultrafast ultrasound localization microscopy allowed both non-invasive sub-wavelength structural imaging and haemodynamic quantification of rodent cerebral microvessels (less than ten micrometres in diameter) more than ten millimetres below the tissue surface, leading to transcranial whole-brain imaging within short acquisition times (tens of seconds). After intravenous injection, single echoes from individual microbubbles were detected through ultrafast imaging. Their localization, not limited by diffraction, was accumulated over 75,000 images, yielding 1,000,000 events per coronal plane and statistically independent pixels of ten micrometres in size. Precise temporal tracking of microbubble positions allowed us to extract accurately in-plane velocities of the blood flow with a large dynamic range (from one millimetre per second to several centimetres per second). These results pave the way for deep non

  6. Imaging and quantitative data acquisition of biological cell walls with Atomic Force Microscopy and Scanning Acoustic Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tittmann, B. R. [Penn State; Xi, X. [Penn State

    2014-09-01

    This chapter demonstrates the feasibility of Atomic Force Microscopy (AFM) and High Frequency Scanning Acoustic Microscopy (HF-SAM) as tools to characterize biological tissues. Both the AFM and the SAM have shown to provide imaging (with different resolution) and quantitative elasticity measuring abilities. Plant cell walls with minimal disturbance and under conditions of their native state have been examined with these two kinds of microscopy. After descriptions of both the SAM and AFM, their special features and the typical sample preparation is discussed. The sample preparation is focused here on epidermal peels of onion scales and celery epidermis cells which were sectioned for the AFM to visualize the inner surface (closest to the plasma membrane) of the outer epidermal wall. The nm-wide cellulose microfibrils orientation and multilayer structure were clearly observed. The microfibril orientation and alignment tend to be more organized in older scales compared with younger scales. The onion epidermis cell wall was also used as a test analog to study cell wall elasticity by the AFM nanoindentation and the SAM V(z) feature. The novelty in this work was to demonstrate the capability of these two techniques to analyze isolated, single layered plant cell walls in their natural state. AFM nanoindentation was also used to probe the effects of Ethylenediaminetetraacetic acid (EDTA), and calcium ion treatment to modify pectin networks in cell walls. The results suggest a significant modulus increase in the calcium ion treatment and a slight decrease in EDTA treatment. To complement the AFM measurements, the HF-SAM was used to obtain the V(z) signatures of the onion epidermis. These measurements were focused on documenting the effect of pectinase enzyme treatment. The results indicate a significant change in the V(z) signature curves with time into the enzyme treatment. Thus AFM and HF-SAM open the door to a systematic nondestructive structure and mechanical property

  7. Whole Slide Imaging Versus Microscopy for Primary Diagnosis in Surgical Pathology

    Science.gov (United States)

    Mukhopadhyay, Sanjay; Feldman, Michael D.; Abels, Esther; Ashfaq, Raheela; Beltaifa, Senda; Cacciabeve, Nicolas G.; Cathro, Helen P.; Cheng, Liang; Cooper, Kumarasen; Dickey, Glenn E.; Gill, Ryan M.; Heaton, Robert P.; Kerstens, René; Lindberg, Guy M.; Malhotra, Reenu K.; Mandell, James W.; Manlucu, Ellen D.; Mills, Anne M.; Mills, Stacey E.; Moskaluk, Christopher A.; Nelis, Mischa; Patil, Deepa T.; Przybycin, Christopher G.; Reynolds, Jordan P.; Rubin, Brian P.; Saboorian, Mohammad H.; Salicru, Mauricio; Samols, Mark A.; Sturgis, Charles D.; Turner, Kevin O.; Wick, Mark R.; Yoon, Ji Y.; Zhao, Po

    2018-01-01

    Most prior studies of primary diagnosis in surgical pathology using whole slide imaging (WSI) versus microscopy have focused on specific organ systems or included relatively few cases. The objective of this study was to demonstrate that WSI is noninferior to microscopy for primary diagnosis in surgical pathology. A blinded randomized noninferiority study was conducted across the entire range of surgical pathology cases (biopsies and resections, including hematoxylin and eosin, immunohistochemistry, and special stains) from 4 institutions using the original sign-out diagnosis (baseline diagnosis) as the reference standard. Cases were scanned, converted to WSI and randomized. Sixteen pathologists interpreted cases by microscopy or WSI, followed by a wash-out period of ≥4 weeks, after which cases were read by the same observers using the other modality. Major discordances were identified by an adjudication panel, and the differences between major discordance rates for both microscopy (against the reference standard) and WSI (against the reference standard) were calculated. A total of 1992 cases were included, resulting in 15,925 reads. The major discordance rate with the reference standard diagnosis was 4.9% for WSI and 4.6% for microscopy. The difference between major discordance rates for microscopy and WSI was 0.4% (95% confidence interval, −0.30% to 1.01%). The difference in major discordance rates for WSI and microscopy was highest in endocrine pathology (1.8%), neoplastic kidney pathology (1.5%), urinary bladder pathology (1.3%), and gynecologic pathology (1.2%). Detailed analysis of these cases revealed no instances where interpretation by WSI was consistently inaccurate compared with microscopy for multiple observers. We conclude that WSI is noninferior to microscopy for primary diagnosis in surgical pathology, including biopsies and resections stained with hematoxylin and eosin, immunohistochemistry and special stains. This conclusion is valid across a

  8. Imaging of Au nanoparticles deeply buried in polymer matrix by various atomic force microscopy techniques

    International Nuclear Information System (INIS)

    Kimura, Kuniko; Kobayashi, Kei; Matsushige, Kazumi; Yamada, Hirofumi

    2013-01-01

    Recently, some papers reported successful imaging of subsurface features using atomic force microscopy (AFM). Some theoretical studies have also been presented, however the imaging mechanisms are not fully understood yet. In the preceeding papers, imaging of deeply buried nanometer-scale features has been successful only if they were buried in a soft matrix. In this paper, subsurface features (Au nanoparticles) buried in a soft polymer matrix were visualized. To elucidate the imaging mechanisms, various AFM techniques; heterodyne force microscopy, ultrasonic atomic force microscopy (UAFM), 2nd-harmonic UAFM and force modulation microscopy (FMM) were employed. The particles buried under 960 nm from the surface were successfully visualized which has never been achieved. The results elucidated that it is important for subsurface imaging to choose a cantilever with a suitable stiffness range for a matrix. In case of using the most suitable cantilever, the nanoparticles were visualized using every technique shown above except for FMM. The experimental results suggest that the subsurface features buried in a soft matrix with a depth of at least 1 µm can affect the local viscoelasticity (mainly viscosity) detected as the variation of the amplitude and phase of the tip oscillation on the surface. This phenomenon presumably makes it possible to visualize such deeply buried nanometer-scale features in a soft matrix. - Highlights: • We visualized subsurface features buried in soft matrix, and investigated its imaging mechanism. • AFM techniques; UAFM, FMM, HFM and 2nd-harmonic UAFM were applied to elucidate the mechanism. • Au nanoparticles buried under 960 nm from surface were visualized, which has never been achieved. • Imaging at contact resonance using a cantilever of suitable stiffness is important. • Subsurface features in a soft matrix affect surface viscoelasticity, which are detected by AFM

  9. Modifications for soft x-ray contact microscopy -- quantification of carbon density discrimination and stereo imaging

    Energy Technology Data Exchange (ETDEWEB)

    Stead, A.D.; Page, A.M.; Cotton, R.A. [Univ. of London, Egham (United Kingdom). Dept. of Biology] [and others

    1995-12-31

    Soft x-ray contact microscopy successfully images hydrated biological material with resolution superior to light microscopy. Nanosecond laser pulses record the image before movement or radiation damage can occur, eliminating concerns of fixation-induced artifacts (cf electron microscopy). X-rays make the recording polymethyl methacrylate (PMMA) photoresist more soluble, thus a contour map of x-ray absorbency is produced in which relative heights, measured by atomic force microscopy, reflect specimen carbon density. Until now quantification of the carbon-density differences was impossible, neither has the minimum carbon density difference which is detectable been determined. Since biological specimens are composed of structures differing only marginally in carbon density the discrimination between carbon densities is critical. Using Si{sub 3}N{sub 4} windows coated with differing carbon thicknesses the authors have followed the rate of PMMA dissolution in order to produce calibration curves from which specimen carbon density can be determined. These experiments have also attempted to determine the minimum detectable carbon density difference. When using relatively thick (>5{micro}m) specimens image interpretation can be difficult as spatially separated structures in the original specimen become superimposed in the x-ray image. To provide spatial resolution in three dimensions they are developing a soft x-ray stereo imaging system. Using two laser plasma x-ray sources and contoured photoresists they have obtained two simultaneous images of the same specimen from different angles. E-beam lithography, cutting and imprinting have been tested as means of producing contoured photoresists. The merits of each will be discussed and preliminary stereo images of hydrated biological specimens presented.

  10. Visualization of macro-immune complexes in the antiphospholipid syndrome by multi-modal microscopy imaging.

    Science.gov (United States)

    Taatjes, Douglas J; Bouffard, Nicole; von Turkovich, Michele; Quinn, Anthony S; Wu, Xiao-Xuan; Vasovic, Ljiljana V; Rand, Jacob H

    2017-09-01

    The antiphospholipid syndrome (APS) is an autoimmune thrombotic condition that is marked by autoantibodies against phospholipid-binding proteins. The mechanism(s) of thrombogenesis has (have) resisted elucidation since its description over thirty years ago. Nevertheless, a defining aspect of the disorder is positivity for clinical laboratory tests that confirm antibody binding to anionic phospholipids. It is remarkable that, to our knowledge, the binding of proteins from plasmas of APS patients to phospholipid has not been previously imaged. We therefore investigated this with high resolution microscopy-based imaging techniques that have not been previously used to address this question, namely atomic force microscopy and scanning electron microscopy. Atomic force microscopy imaging of APS plasmas incubated on an anionic planar phospholipid layer revealed the formation of distinct complex three-dimensional structures, which were morphologically dissimilar to structures formed from control plasmas from healthy patients. Likewise, scanning electron microscopy analysis of phospholipid vesicles incubated with APS plasmas in suspension showed formation of layered macro-immune complexes demonstrated by the significant agglomeration of a complex proteinaceous matrix from soluble plasma and aggregation of particles. In contrast, plasmas from healthy control samples bound to phospholipid vesicles in suspension generally displayed a more flattened, mat-like appearance by scanning electron microscopy. Scanning electron microscopy of plasma samples incubated on planar phospholipid layers and previously imaged by atomic force microscopy, corroborated the results obtained by mixing the plasmas with phospholipids in solution. Analysis of the incorporated proteins by silver stained SDS-polyacrylamide gel electrophoresis indicated considerable heterogeneity in the composition of the phospholipid vesicle-adsorbed proteins among APS patients. To our knowledge, these results provide

  11. Imaging Drosophila brain by combining cryo-soft X-ray microscopy of thick vitreous sections and cryo-electron microscopy of ultrathin vitreous sections.

    Science.gov (United States)

    Leforestier, Amélie; Levitz, Pierre; Preat, Thomas; Guttmann, Peter; Michot, Laurent J; Tchénio, Paul

    2014-11-01

    Cryo-soft X-ray microscopy is an emerging imaging tool complementary to cryo-electron microscopy, allowing to image frozen hydrated specimens ten to hundred times thicker, but at lower resolution. We describe how the method, so far restricted to isolated small cells or cell monolayers, can be extended to large cells and tissue. We image the synapses of the Kenyon cells in frozen hydrated Drosophila brains combining cryo-soft X-ray microscopy of thick vitreous sections, and cryo-electron microscopy of ultrathin vitreous sections. We show how to obtain frozen hydrated sections of thicknesses ranging from 40 nm up to 2.5 μm, by tuning the sectioning speed of the cryo-microtome. A fluorescent stereo-microscope mounted on the cryo-microtome allowed us to target the regions of interest after GFP-labeling of synapses. Thick cryo-sections were imaged by cryo-soft X-ray microscopy at a resolution better than 25 nm, while ultrathin cryo-sections of the same regions were explored in parallel at the nanometre level of resolution by cryo-electron microscopy. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering microscopy

    International Nuclear Information System (INIS)

    Volkmer, Andreas

    2005-01-01

    For noninvasive characterization of chemical species or biological components within a complex heterogeneous system, their intrinsic molecular vibrational properties can be used in contrast mechanisms in optical microscopy. A series of recent advances have made coherent anti-Stokes Raman scattering (CARS) microscopy a powerful technique that allows vibrational imaging with high sensitivity, high spectral resolution and three-dimensional sectioning capability. In this review, we discuss theoretical and experimental aspects of CARS microscopy in a collinear excitation beam geometry. Particular attention is given to the underlying physical principles behind the new features of CARS signal generation under tight focusing conditions. We provide a brief overview of the instrumentation of CARS microscopy and its experimental characterization by means of imaging of model systems and live unstained cells. CARS microscopy offers the possibility of spatially resolved vibrational spectroscopy, providing chemical and physical structure information of molecular specimens on the sub-micrometre length scale. We review multiplex CARS microspectroscopy allowing fast acquisition of frequency-resolved CARS spectra, time-resolved CARS microspectroscopy recording ultrafast Raman free induction decays and CARS correlation spectroscopy probing dynamical processes with chemical selectivity. (topical review)

  13. 65,000 shades of grey: use of digital image files in light microscopy.

    Science.gov (United States)

    Shaw, Sidney L; Hinchcliffe, Edward H

    2013-01-01

    Computers dominate image capture and analysis in modern light microscopy. The output of an imaging experiment is a binary coded file, called an image file, which contains the spatial, temporal and intensity information present in the sample. Understanding what comprises an image file, and how these files are generated is necessary in order to optimize the use of the digital light microscope. In this chapter, we discuss image file formats, and the various components of these files, such as bit-depth, sampling rate, color theory, and compression, from the perspective of the non-computer scientist. We also discuss the problem of proprietary file formats, and how these often are incompatible with certain types of imaging software. We present several solutions to this issue. Finally, we present the use of digital movie formats, compression routines, and provide some real world examples for optimizing the generation of digital movies. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Mathematical imaging methods for mitosis analysis in live-cell phase contrast microscopy.

    Science.gov (United States)

    Grah, Joana Sarah; Harrington, Jennifer Alison; Koh, Siang Boon; Pike, Jeremy Andrew; Schreiner, Alexander; Burger, Martin; Schönlieb, Carola-Bibiane; Reichelt, Stefanie

    2017-02-15

    In this paper we propose a workflow to detect and track mitotic cells in time-lapse microscopy image sequences. In order to avoid the requirement for cell lines expressing fluorescent markers and the associated phototoxicity, phase contrast microscopy is often preferred over fluorescence microscopy in live-cell imaging. However, common specific image characteristics complicate image processing and impede use of standard methods. Nevertheless, automated analysis is desirable due to manual analysis being subjective, biased and extremely time-consuming for large data sets. Here, we present the following workflow based on mathematical imaging methods. In the first step, mitosis detection is performed by means of the circular Hough transform. The obtained circular contour subsequently serves as an initialisation for the tracking algorithm based on variational methods. It is sub-divided into two parts: in order to determine the beginning of the whole mitosis cycle, a backwards tracking procedure is performed. After that, the cell is tracked forwards in time until the end of mitosis. As a result, the average of mitosis duration and ratios of different cell fates (cell death, no division, division into two or more daughter cells) can be measured and statistics on cell morphologies can be obtained. All of the tools are featured in the user-friendly MATLAB®Graphical User Interface MitosisAnalyser. Copyright © 2017. Published by Elsevier Inc.

  15. A comparison of reconstruction methods for undersampled atomic force microscopy images.

    Science.gov (United States)

    Luo, Yufan; Andersson, Sean B

    2015-12-18

    Non-raster scanning and undersampling of atomic force microscopy (AFM) images is a technique for improving imaging rate and reducing the amount of tip-sample interaction needed to produce an image. Generation of the final image can be done using a variety of image processing techniques based on interpolation or optimization. The choice of reconstruction method has a large impact on the quality of the recovered image and the proper choice depends on the sample under study. In this work we compare interpolation through the use of inpainting algorithms with reconstruction based on optimization through the use of the basis pursuit algorithm commonly used for signal recovery in compressive sensing. Using four different sampling patterns found in non-raster AFM, namely row subsampling, spiral scanning, Lissajous scanning, and random scanning, we subsample data from existing images and compare reconstruction performance against the original image. The results illustrate that inpainting generally produces superior results when the image contains primarily low frequency content while basis pursuit is better when the images have mixed, but sparse, frequency content. Using support vector machines, we then classify images based on their frequency content and sparsity and, from this classification, develop a fast decision strategy to select a reconstruction algorithm to be used on subsampled data. The performance of the classification and decision test are demonstrated on test AFM images.

  16. A comparison of reconstruction methods for undersampled atomic force microscopy images

    International Nuclear Information System (INIS)

    Luo, Yufan; Andersson, Sean B

    2015-01-01

    Non-raster scanning and undersampling of atomic force microscopy (AFM) images is a technique for improving imaging rate and reducing the amount of tip–sample interaction needed to produce an image. Generation of the final image can be done using a variety of image processing techniques based on interpolation or optimization. The choice of reconstruction method has a large impact on the quality of the recovered image and the proper choice depends on the sample under study. In this work we compare interpolation through the use of inpainting algorithms with reconstruction based on optimization through the use of the basis pursuit algorithm commonly used for signal recovery in compressive sensing. Using four different sampling patterns found in non-raster AFM, namely row subsampling, spiral scanning, Lissajous scanning, and random scanning, we subsample data from existing images and compare reconstruction performance against the original image. The results illustrate that inpainting generally produces superior results when the image contains primarily low frequency content while basis pursuit is better when the images have mixed, but sparse, frequency content. Using support vector machines, we then classify images based on their frequency content and sparsity and, from this classification, develop a fast decision strategy to select a reconstruction algorithm to be used on subsampled data. The performance of the classification and decision test are demonstrated on test AFM images. (paper)

  17. CHEMICAL MAPPING OF ELEMENTAL SULFUR ON PYRITE AND ARSENOPYRITE SURFACES USING NEAR-INFRARED RAMAN IMAGING MICROSCOPY. (R826189)

    Science.gov (United States)

    AbstractNear-infrared Raman imaging microscopy (NIRIM) was used to produce chemical images of the distribution of elemental sulfur on oxidized pyrite and arsenopyrite surfaces. Analysis using Savitsky¯Golay filtering permits an unambiguous identificati...

  18. Three-dimensional super-resolution imaging for fluorescence emission difference microscopy

    Directory of Open Access Journals (Sweden)

    Shangting You

    2015-08-01

    Full Text Available We propose a method theoretically to break the diffraction limit and to improve the resolution in all three dimensions for fluorescence emission difference microscopy. We produce two kinds of hollow focal spot by phase modulation. By incoherent superposition, these two kinds of focal spot yield a 3D hollow focal spot. The optimal proportion of these two kinds of spot is given in the paper. By employing 3D hollow focal spot, super-resolution image can be yielded by means of fluorescence emission difference microscopy, with resolution enhanced both laterally and axially. According to computation result, size of point spread function of three-dimensional super-resolution imaging is reduced by about 40% in all three spatial directions with respect to confocal imaging.

  19. Total three-dimensional imaging of phase objects using defocusing microscopy: Application to red blood cells

    Science.gov (United States)

    Roma, P. M. S.; Siman, L.; Amaral, F. T.; Agero, U.; Mesquita, O. N.

    2014-06-01

    We introduce Defocusing Microscopy (DM), a bright-field optical microscopy technique able to perform total three-dimensional (3D) imaging of transparent objects. By total 3D imaging, we mean the determination of the actual shapes of the upper and lower surfaces of a phase object. We propose a methodology using DM and apply it to red blood cells subject to different osmolality conditions: hypotonic, isotonic, and hypertonic solutions. For each situation, the shapes of the upper and lower cell surface-membranes (lipid bilayer/cytoskeleton) are completely recovered, displaying the deformation of red blood cell (RBC) surfaces due to adhesion on the glass-substrate. The axial resolution of our technique allowed us to image surface-membranes separated by distances as small as 300 nm. Finally, we determine the volume, surface area, sphericity index, and RBC refractive index for each osmotic condition.

  20. Multicomponent chemical imaging of pharmaceutical solid dosage forms with broadband CARS microscopy.

    Science.gov (United States)

    Hartshorn, Christopher M; Lee, Young Jong; Camp, Charles H; Liu, Zhen; Heddleston, John; Canfield, Nicole; Rhodes, Timothy A; Hight Walker, Angela R; Marsac, Patrick J; Cicerone, Marcus T

    2013-09-03

    We compare a coherent Raman imaging modality, broadband coherent anti-Stokes Raman scattering (BCARS) microscopy, with spontaneous Raman microscopy for quantitative and qualitative assessment of multicomponent pharmaceuticals. Indomethacin was used as a model active pharmaceutical ingredient (API) and was analyzed in a tabulated solid dosage form, embedded within commonly used excipients. In comparison with wide-field spontaneous Raman chemical imaging, BCARS acquired images 10× faster, at higher spatiochemical resolution and with spectra of much higher SNR, eliminating the need for multivariate methods to identify chemical components. The significant increase in spatiochemical resolution allowed identification of an unanticipated API phase that was missed by the spontaneous wide-field method and bulk Raman spectroscopy. We confirmed the presence of the unanticipated API phase using confocal spontaneous Raman, which provided spatiochemical resolution similar to BCARS but at 100× slower acquisition times.

  1. The reinvention of twentieth century microscopy for three-dimensional imaging.

    Science.gov (United States)

    Whitehead, Lachlan W; McArthur, Kate; Geoghegan, Niall D; Rogers, Kelly L

    2017-07-01

    In just over a decade, the field of biomedical research has witnessed a radical evolution in technologies for the 3- and 4-dimensional imaging of biological samples. Light sheet fluorescence microscopy is quickly developing into a powerful approach for fast, volumetric imaging of cells, tissues and living organisms. This review touches on the development of 3-dimensional imaging, from its foundations, namely from the invention of confocal microscopy in the twentieth century to more recent examples, notably the IsoView SPIM, the Lattice Light Sheet Microscope and swept confocally aligned planar excitation. These technologies overcome the limitations of conventional optical sectioning techniques and enable unprecedented levels of spatio-temporal resolution with low levels of phototoxicity. Developing in parallel with powerful computational approaches, light sheet based methods promise to completely transform cell biology as we know it today.

  2. Microscopy imaging system and method employing stimulated raman spectroscopy as a contrast mechanism

    Science.gov (United States)

    Xie, Xiaoliang Sunney [Lexington, MA; Freudiger, Christian [Boston, MA; Min, Wei [Cambridge, MA

    2011-09-27

    A microscopy imaging system includes a first light source for providing a first train of pulses at a first center optical frequency .omega..sub.1, a second light source for providing a second train of pulses at a second center optical frequency .omega..sub.2, a modulator system, an optical detector, and a processor. The modulator system is for modulating a beam property of the second train of pulses at a modulation frequency f of at least 100 kHz. The optical detector is for detecting an integrated intensity of substantially all optical frequency components of the first train of pulses from the common focal volume by blocking the second train of pulses being modulated. The processor is for detecting, a modulation at the modulation frequency f, of the integrated intensity of the optical frequency components of the first train of pulses to provide a pixel of an image for the microscopy imaging system.

  3. Imaging by in situ Scanning Tunnelling Microscopy and its Nanotechnological Perspectives

    DEFF Research Database (Denmark)

    Andersen, Jens Enevold Thaulov

    2002-01-01

    of the tip and working electrode. In collaboration with Danish Micro Engineering A/S, the instrument was constructed and tested in laboratory environments. The system was successfully developed, as to meet international-market requirements. Within the frame of the work, procedures of tip coating...... and bipotentiostat construction were evaluated. After the fulfilment of the instrument manufacturing process followed application of the system to scientific investigations. The generation of an image by in situ STM is founded on the principle of electron tunneling but the application of the instrument to aqueous...... in the interpretation of the imaging procedure. Other methods of in situ Scanning Probe Microscopy (in situ SPM), such as in situ Scanning Force Microscopy (in situ AFM) are considered for the sake of comparison and they are applied to imaging of non-conducting systems. Major results include demonstration of atomic...

  4. Imaging Single ZnO Vertical Nanowire Laser Cavities using UV-Laser Scanning Confocal Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Gargas, D.J.; Toimil-Molares, M.E.; Yang, P.

    2008-11-17

    We report the fabrication and optical characterization of individual ZnO vertical nanowire laser cavities. Dilute nanowire arrays with interwire spacing>10 ?m were produced by a modified chemical vapor transport (CVT) method yielding an ideal platform for single nanowire imaging and spectroscopy. Lasing characteristics of a single vertical nanowire are presented, as well as high-resolution photoluminescence imaging by UV-laser scanning confocal microscopy. In addition, three-dimensional (3D) mapping of the photoluminescence emission performed in both planar and vertical dimensions demonstrates height-selective imaging useful for vertical nanowires and heteronanostructures emerging in the field of optoelectronics and nanophotonics.

  5. Dynamics of annular bright field imaging in scanning transmission electron microscopy

    International Nuclear Information System (INIS)

    Findlay, S.D.; Shibata, N.; Sawada, H.; Okunishi, E.; Kondo, Y.; Ikuhara, Y.

    2010-01-01

    We explore the dynamics of image formation in the so-called annular bright field mode in scanning transmission electron microscopy, whereby an annular detector is used with detector collection range lying within the cone of illumination, i.e. the bright field region. We show that this imaging mode allows us to reliably image both light and heavy columns over a range of thickness and defocus values, and we explain the contrast mechanisms involved. The role of probe and detector aperture sizes is considered, as is the sensitivity of the method to intercolumn spacing and local disorder.

  6. Imaging of buried phosphorus nanostructures in silicon using scanning tunneling microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Oberbeck, Lars [Centre for Quantum Computation and Communication Technology, School of Physics, University of New South Wales, Sydney, New South Wales 2052 (Australia); TOTAL Marketing Services, New Energies, La Défense 10, 92069 Paris La Défense Cedex (France); Reusch, Thilo C. G.; Hallam, Toby; Simmons, Michelle Y., E-mail: n.curson@ucl.ac.uk, E-mail: michelle.simmons@unsw.edu.au [Centre for Quantum Computation and Communication Technology, School of Physics, University of New South Wales, Sydney, New South Wales 2052 (Australia); Schofield, Steven R. [Centre for Quantum Computation and Communication Technology, School of Physics, University of New South Wales, Sydney, New South Wales 2052 (Australia); London Centre for Nanotechnology, UCL, London WC1H 0AH (United Kingdom); Department of Physics and Astronomy, UCL, London WC1E 6BT (United Kingdom); Curson, Neil J., E-mail: n.curson@ucl.ac.uk, E-mail: michelle.simmons@unsw.edu.au [Centre for Quantum Computation and Communication Technology, School of Physics, University of New South Wales, Sydney, New South Wales 2052 (Australia); London Centre for Nanotechnology, UCL, London WC1H 0AH (United Kingdom); Department of Electronic and Electrical Engineering, UCL, London WC1E 7JE (United Kingdom)

    2014-06-23

    We demonstrate the locating and imaging of single phosphorus atoms and phosphorus dopant nanostructures, buried beneath the Si(001) surface using scanning tunneling microscopy. The buried dopant nanostructures have been fabricated in a bottom-up approach using scanning tunneling microscope lithography on Si(001). We find that current imaging tunneling spectroscopy is suited to locate and image buried nanostructures at room temperature and with residual surface roughness present. From these studies, we can place an upper limit on the lateral diffusion during encapsulation with low-temperature Si molecular beam epitaxy.

  7. Imaging of buried phosphorus nanostructures in silicon using scanning tunneling microscopy

    International Nuclear Information System (INIS)

    Oberbeck, Lars; Reusch, Thilo C. G.; Hallam, Toby; Simmons, Michelle Y.; Schofield, Steven R.; Curson, Neil J.

    2014-01-01

    We demonstrate the locating and imaging of single phosphorus atoms and phosphorus dopant nanostructures, buried beneath the Si(001) surface using scanning tunneling microscopy. The buried dopant nanostructures have been fabricated in a bottom-up approach using scanning tunneling microscope lithography on Si(001). We find that current imaging tunneling spectroscopy is suited to locate and image buried nanostructures at room temperature and with residual surface roughness present. From these studies, we can place an upper limit on the lateral diffusion during encapsulation with low-temperature Si molecular beam epitaxy.

  8. Advanced magneto-optical microscopy: Imaging from picoseconds to centimeters - imaging spin waves and temperature distributions (invited

    Directory of Open Access Journals (Sweden)

    Necdet Onur Urs

    2016-05-01

    Full Text Available Recent developments in the observation of magnetic domains and domain walls by wide-field optical microscopy based on the magneto-optical Kerr, Faraday, Voigt, and Gradient effect are reviewed. Emphasis is given to the existence of higher order magneto-optical effects for advanced magnetic imaging. Fundamental concepts and advances in methodology are discussed that allow for imaging of magnetic domains on various length and time scales. Time-resolved imaging of electric field induced domain wall rotation is shown. Visualization of magnetization dynamics down to picosecond temporal resolution for the imaging of spin-waves and magneto-optical multi-effect domain imaging techniques for obtaining vectorial information are demonstrated. Beyond conventional domain imaging, the use of a magneto-optical indicator technique for local temperature sensing is shown.

  9. Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ.

    Science.gov (United States)

    Müller, Marcel; Mönkemöller, Viola; Hennig, Simon; Hübner, Wolfgang; Huser, Thomas

    2016-03-21

    Super-resolved structured illumination microscopy (SR-SIM) is an important tool for fluorescence microscopy. SR-SIM microscopes perform multiple image acquisitions with varying illumination patterns, and reconstruct them to a super-resolved image. In its most frequent, linear implementation, SR-SIM doubles the spatial resolution. The reconstruction is performed numerically on the acquired wide-field image data, and thus relies on a software implementation of specific SR-SIM image reconstruction algorithms. We present fairSIM, an easy-to-use plugin that provides SR-SIM reconstructions for a wide range of SR-SIM platforms directly within ImageJ. For research groups developing their own implementations of super-resolution structured illumination microscopy, fairSIM takes away the hurdle of generating yet another implementation of the reconstruction algorithm. For users of commercial microscopes, it offers an additional, in-depth analysis option for their data independent of specific operating systems. As a modular, open-source solution, fairSIM can easily be adapted, automated and extended as the field of SR-SIM progresses.

  10. Imaging ferroelectric domains via charge gradient microscopy enhanced by principal component analysis

    Directory of Open Access Journals (Sweden)

    Ehsan Nasr Esfahani

    2017-12-01

    Full Text Available Local domain structures of ferroelectrics have been studied extensively using various modes of scanning probes at the nanoscale, including piezoresponse force microscopy (PFM and Kelvin probe force microscopy (KPFM, though none of these techniques measure the polarization directly, and the fast formation kinetics of domains and screening charges cannot be captured by these quasi-static measurements. In this study, we used charge gradient microscopy (CGM to image ferroelectric domains of lithium niobate based on current measured during fast scanning, and applied principal component analysis (PCA to enhance the signal-to-noise ratio of noisy raw data. We found that the CGM signal increases linearly with the scan speed while decreases with the temperature under power-law, consistent with proposed imaging mechanisms of scraping and refilling of surface charges within domains, and polarization change across domain wall. We then, based on CGM mappings, estimated the spontaneous polarization and the density of surface charges with order of magnitude agreement with literature data. The study demonstrates that PCA is a powerful method in imaging analysis of scanning probe microscopy (SPM, with which quantitative analysis of noisy raw data becomes possible.

  11. Wide-field imaging through scattering media by scattered light fluorescence microscopy

    Science.gov (United States)

    Zhou, Yulan; Li, Xun

    2017-08-01

    To obtain images through scattering media, scattered light fluorescence (SLF) microscopy that utilizes the optical memory effect has been developed. However, the small field of view (FOV) of SLF microscopy limits its application. In this paper, we have introduced a re-modulation method to achieve wide-field imaging through scattering media by SLF microscopy. In the re-modulation method, to raster scan the focus across the object plane, the incident wavefront is re-modulated via a spatial light modulator (SLM) in the updated phase compensation calculated using the optimized iterative algorithm. Compared with the conventional optical memory effect method, the re-modulation method can greatly increase the FOV of a SLF microscope. With the phase compensation theoretically calculated, the process of updating the phase compensation of a high speed SLM is fast. The re-modulation method does not increase the imaging time. The re-modulation method is, therefore, expected to make SLF microscopy have much wider applications in biology, medicine and physiology.

  12. Imaging transient blood vessel fusion events in zebrafish by correlative volume electron microscopy.

    Directory of Open Access Journals (Sweden)

    Hannah E J Armer

    Full Text Available The study of biological processes has become increasingly reliant on obtaining high-resolution spatial and temporal data through imaging techniques. As researchers demand molecular resolution of cellular events in the context of whole organisms, correlation of non-invasive live-organism imaging with electron microscopy in complex three-dimensional samples becomes critical. The developing blood vessels of vertebrates form a highly complex network which cannot be imaged at high resolution using traditional methods. Here we show that the point of fusion between growing blood vessels of transgenic zebrafish, identified in live confocal microscopy, can subsequently be traced through the structure of the organism using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM and Serial Block Face/Scanning Electron Microscopy (SBF/SEM. The resulting data give unprecedented microanatomical detail of the zebrafish and, for the first time, allow visualization of the ultrastructure of a time-limited biological event within the context of a whole organism.

  13. Non-invasive current and voltage imaging techniques for integrated circuits using scanning probe microscopy

    Science.gov (United States)

    Campbell, A. N.; Cole, E. I., Jr.; Tangyunyong, Paiboon

    1995-06-01

    This report describes the first practical, non-invasive technique for detecting and imaging currents internal to operating integrated circuits (IC's). This technique is based on magnetic force microscopy and was developed under Sandia National Laboratories' LDRD (Laboratory Directed Research and Development) program during FY 93 and FY 94. LDRD funds were also used to explore a related technique, charge force microscopy, for voltage probing of IC's. This report describes the technical work performed under this LDRD as well as the outcomes of the project in terms of publications and awards, intellectual property and licensing, synergistic work, potential future work, hiring of additional permanent staff, and benefits to DOE's defense programs (DP).

  14. Evaluation of autofocus measures for microscopy images of biopsy and cytology

    Science.gov (United States)

    Redondo, R.; Bueno, M. G.; Valdiviezo, J. C.; Nava, R.; Cristóbal, G.; García, M.; Déniz, O.; Escalante-Ramírez, B.

    2011-08-01

    An essential and indispensable component of automated microscopy is the automatic focusing system, which determines the in-focus position of a given field of view by searching for the maximal of an autofocus function over a range of z-axis positions. The autofocus function and its computation time are crucial to the accuracy and efficiency of the system. In this paper, we analyze and evaluate fifteen autofocus algorithms for biopsy and cytology microscopy images, ranging from the already well known methods to those proposed recently. Results have shown that there is a trade-off between computational cost and accuracy. Finally, the error committed by each of the algorithms is presented.

  15. Tracking multiple particles in fluorescence time-lapse microscopy images via probabilistic data association.

    Science.gov (United States)

    Godinez, William J; Rohr, Karl

    2015-02-01

    Tracking subcellular structures as well as viral structures displayed as 'particles' in fluorescence microscopy images yields quantitative information on the underlying dynamical processes. We have developed an approach for tracking multiple fluorescent particles based on probabilistic data association. The approach combines a localization scheme that uses a bottom-up strategy based on the spot-enhancing filter as well as a top-down strategy based on an ellipsoidal sampling scheme that uses the Gaussian probability distributions computed by a Kalman filter. The localization scheme yields multiple measurements that are incorporated into the Kalman filter via a combined innovation, where the association probabilities are interpreted as weights calculated using an image likelihood. To track objects in close proximity, we compute the support of each image position relative to the neighboring objects of a tracked object and use this support to recalculate the weights. To cope with multiple motion models, we integrated the interacting multiple model algorithm. The approach has been successfully applied to synthetic 2-D and 3-D images as well as to real 2-D and 3-D microscopy images, and the performance has been quantified. In addition, the approach was successfully applied to the 2-D and 3-D image data of the recent Particle Tracking Challenge at the IEEE International Symposium on Biomedical Imaging (ISBI) 2012.

  16. High-Throughput Light Sheet Microscopy for the Automated Live Imaging of Larval Zebrafish

    Science.gov (United States)

    Baker, Ryan; Logan, Savannah; Dudley, Christopher; Parthasarathy, Raghuveer

    The zebrafish is a model organism with a variety of useful properties; it is small and optically transparent, it reproduces quickly, it is a vertebrate, and there are a large variety of transgenic animals available. Because of these properties, the zebrafish is well suited to study using a variety of optical technologies including light sheet fluorescence microscopy (LSFM), which provides high-resolution three-dimensional imaging over large fields of view. Research progress, however, is often not limited by optical techniques but instead by the number of samples one can examine over the course of an experiment, which in the case of light sheet imaging has so far been severely limited. Here we present an integrated fluidic circuit and microscope which provides rapid, automated imaging of zebrafish using several imaging modes, including LSFM, Hyperspectral Imaging, and Differential Interference Contrast Microscopy. Using this system, we show that we can increase our imaging throughput by a factor of 10 compared to previous techniques. We also show preliminary results visualizing zebrafish immune response, which is sensitive to gut microbiota composition, and which shows a strong variability between individuals that highlights the utility of high throughput imaging. National Science Foundation, Award No. DBI-1427957.

  17. SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking

    Science.gov (United States)

    Antolovic, Ivan Michel; Burri, Samuel; Bruschini, Claudio; Hoebe, Ron A.; Charbon, Edoardo

    2017-01-01

    sCMOS imagers are currently utilized (replacing EMCCD imagers) to increase the acquisition speed in super resolution localization microscopy. Single-photon avalanche diode (SPAD) imagers feature frame rates per bit depth comparable to or higher than sCMOS imagers, while generating microsecond 1-bit-frames without readout noise, thus paving the way to in-depth time-resolved image analysis. High timing resolution can also be exploited to explore fluorescent dye blinking and other photophysical properties, which can be used for dye optimization. We present the methodology for the blinking analysis of fluorescent dyes on experimental data. Furthermore, the recent use of microlenses has enabled a substantial increase of SPAD imager overall sensitivity (12-fold in our case), reaching satisfactory values for sensitivity-critical applications. This has allowed us to record the first super resolution localization microscopy results obtained with a SPAD imager, with a localization uncertainty of 20 nm and a resolution of 80 nm. PMID:28287122

  18. Multiphoton imaging microscopy at deeper layers with adaptive optics control of spherical aberration.

    Science.gov (United States)

    Bueno, Juan M; Skorsetz, Martin; Palacios, Raquel; Gualda, Emilio J; Artal, Pablo

    2014-01-01

    Despite the inherent confocality and optical sectioning capabilities of multiphoton microscopy, three-dimensional (3-D) imaging of thick samples is limited by the specimen-induced aberrations. The combination of immersion objectives and sensorless adaptive optics (AO) techniques has been suggested to overcome this difficulty. However, a complex plane-by-plane correction of aberrations is required, and its performance depends on a set of image-based merit functions. We propose here an alternative approach to increase penetration depth in 3-D multiphoton microscopy imaging. It is based on the manipulation of the spherical aberration (SA) of the incident beam with an AO device while performing fast tomographic multiphoton imaging. When inducing SA, the image quality at best focus is reduced; however, better quality images are obtained from deeper planes within the sample. This is a compromise that enables registration of improved 3-D multiphoton images using nonimmersion objectives. Examples on ocular tissues and nonbiological samples providing different types of nonlinear signal are presented. The implementation of this technique in a future clinical instrument might provide a better visualization of corneal structures in living eyes.

  19. Measurement of capillary lenght from 3D images acquired by confocal microscopy using image analysis and stereology

    Czech Academy of Sciences Publication Activity Database

    Kubínová, Lucie; Janáček, Jiří; Eržen, I.; Mao, X. W.

    2010-01-01

    Roč. 16, Suppl.2 (2010), s. 736-737 ISSN 1431-9276. [Microscopy and Microanalysis 2010. Portland, 01.08.2010-05.08.2010] R&D Projects: GA MŠk(CZ) LC06063; GA MŠk(CZ) ME09010; GA MŠk(CZ) MEB090910; GA ČR(CZ) GA304/09/0733 Institutional research plan: CEZ:AV0Z50110509 Keywords : capillary length * confocal microscopy * image analysis Subject RIV: EA - Cell Biology Impact factor: 2.179, year: 2010

  20. Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy

    International Nuclear Information System (INIS)

    Pandžić, Elvis; Rossy, Jérémie; Gaus, Katharina

    2015-01-01

    Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins. (paper)

  1. Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy

    Science.gov (United States)

    Pandžić, Elvis; Rossy, Jérémie; Gaus, Katharina

    2015-03-01

    Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins.

  2. Activated sludge characterization through microscopy: A review on quantitative image analysis and chemometric techniques

    Energy Technology Data Exchange (ETDEWEB)

    Mesquita, Daniela P. [IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga (Portugal); Amaral, A. Luís [IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga (Portugal); Instituto Politécnico de Coimbra, ISEC, DEQB, Rua Pedro Nunes, Quinta da Nora, 3030-199 Coimbra (Portugal); Ferreira, Eugénio C., E-mail: ecferreira@deb.uminho.pt [IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga (Portugal)

    2013-11-13

    Graphical abstract: -- Highlights: •Quantitative image analysis shows potential to monitor activated sludge systems. •Staining techniques increase the potential for detection of operational problems. •Chemometrics combined with quantitative image analysis is valuable for process monitoring. -- Abstract: In wastewater treatment processes, and particularly in activated sludge systems, efficiency is quite dependent on the operating conditions, and a number of problems may arise due to sludge structure and proliferation of specific microorganisms. In fact, bacterial communities and protozoa identification by microscopy inspection is already routinely employed in a considerable number of cases. Furthermore, quantitative image analysis techniques have been increasingly used throughout the years for the assessment of aggregates and filamentous bacteria properties. These procedures are able to provide an ever growing amount of data for wastewater treatment processes in which chemometric techniques can be a valuable tool. However, the determination of microbial communities’ properties remains a current challenge in spite of the great diversity of microscopy techniques applied. In this review, activated sludge characterization is discussed highlighting the aggregates structure and filamentous bacteria determination by image analysis on bright-field, phase-contrast, and fluorescence microscopy. An in-depth analysis is performed to summarize the many new findings that have been obtained, and future developments for these biological processes are further discussed.

  3. Global error minimization in image mosaicing using graph connectivity and its applications in microscopy

    Directory of Open Access Journals (Sweden)

    Parmeshwar Khurd

    2011-01-01

    Full Text Available Several applications such as multiprojector displays and microscopy require the mosaicing of images (tiles acquired by a camera as it traverses an unknown trajectory in 3D space. A homography relates the image coordinates of a point in each tile to those of a reference tile provided the 3D scene is planar. Our approach in such applications is to first perform pairwise alignment of the tiles that have imaged common regions in order to recover a homography relating the tile pair. We then find the global set of homographies relating each individual tile to a reference tile such that the homographies relating all tile pairs are kept as consistent as possible. Using these global homographies, one can generate a mosaic of the entire scene. We derive a general analytical solution for the global homographies by representing the pair-wise homographies on a connectivity graph. Our solution can accommodate imprecise prior information regarding the global homographies whenever such information is available. We also derive equations for the special case of translation estimation of an X-Y microscopy stage used in histology imaging and present examples of stitched microscopy slices of specimens obtained after radical prostatectomy or prostate biopsy. In addition, we demonstrate the superiority of our approach over tree-structured approaches for global error minimization.

  4. Nanostructured imaging of biological specimens in vivo with laser plasma X-ray contact microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Cefalas, A.C.; Sarantopoulou, E.; Kollia, Z.; Argitis, P.; Tegou, E.; Ford, T.W.; Stead, A.D.; Danson, C.N.; Neely, D.; Kobe, S

    2003-01-15

    Soft X-ray contact microscopy (SXCM) enables the study of the ultrastructure of living hydrated specimens, without the need of dehydration or any other chemical pretreatment, by using suitable pulsed X-ray sources such as laser plasmas. The successful imaging of biological specimen requires the development of sensitive photoresist materials for image recording; these should have capabilities of high-resolution lithography and an extended grey scale. A very sensitive photoresist, used for the first time in SXCM, enabled the biological imaging with the specific source in single-pulse experiments in the water window spectral range. This photoresist is an epoxy novolac-based chemically amplified photoresist (EPR), which has been proven capable of resolving subtenth-micron features. The photoresist response was at least two orders of magnitude 'faster' than polymethyl methacrylate (PMMA), which is the standard resist used so far in SXCM. Atomic force microscopy (AFM) and scanning electron microscopy of the biological specimen images recorded in the resist clearly showed the flagella of the motile green alga, suggesting a lateral resolution better than 100 nm. The resist was also capable of providing height features, as small as 20 nm, in AFM depth profiles and discriminating the flagella intersection areas.

  5. Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy

    Science.gov (United States)

    Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Borri, Paola

    2016-01-01

    Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

  6. An introduction to sample preparation and imaging by cryo-electron microscopy for structural biology

    Science.gov (United States)

    Thompson, Rebecca F.; Walker, Matt; Siebert, C. Alistair; Muench, Stephen P.; Ranson, Neil A.

    2016-01-01

    Transmission electron microscopy (EM) is a versatile technique that can be used to image biological specimens ranging from intact eukaryotic cells to individual proteins >150 kDa. There are several strategies for preparing samples for imaging by EM, including negative staining and cryogenic freezing. In the last few years, cryo-EM has undergone a ‘resolution revolution’, owing to both advances in imaging hardware, image processing software, and improvements in sample preparation, leading to growing number of researchers using cryo-EM as a research tool. However, cryo-EM is still a rapidly growing field, with unique challenges. Here, we summarise considerations for imaging of a range of specimens from macromolecular complexes to cells using EM. PMID:26931652

  7. Nanohybrids Near-Field Optical Microscopy: From Image Shift to Biosensor Application

    Directory of Open Access Journals (Sweden)

    Nayla El-Kork

    2016-01-01

    Full Text Available Near-Field Optical Microscopy is a valuable tool for the optical and topographic study of objects at a nanometric scale. Nanoparticles constitute important candidates for such type of investigations, as they bear an important weight for medical, biomedical, and biosensing applications. One, however, has to be careful as artifacts can be easily reproduced. In this study, we examined hybrid nanoparticles (or nanohybrids in the near-field, while in solution and attached to gold nanoplots. We found out that they can be used for wavelength modulable near-field biosensors within conditions of artifact free imaging. In detail, we refer to the use of topographic/optical image shift and the imaging of Local Surface Plasmon hot spots to validate the genuineness of the obtained images. In summary, this study demonstrates a new way of using simple easily achievable comparative methods to prove the authenticity of near-field images and presents nanohybrid biosensors as an application.

  8. Phase sensitive optical coherence microscopy for photothermal imaging of gold nanorods

    Science.gov (United States)

    Hu, Yong; Podoleanu, Adrian G.; Dobre, George

    2018-03-01

    We describe a swept source based phase sensitive optical coherence microscopy (OCM) system for photothermal imaging of gold nanorods (GNR). The phase sensitive OCM system employed in the study has a displacement sensitivity of 0.17 nm to vibrations at single frequencies below 250 Hz. We demonstrate the generation of phase maps and confocal phase images. By displaying the difference between successive confocal phase images, we perform the confocal photothermal imaging of accumulated GNRs behind a glass coverslip and behind the scattering media separately. Compared with two-photon luminescence (TPL) detection techniques reported in literature, the technique in this study has the advantage of a simplified experimental setup and provides a more efficient method for imaging the aggregation of GNR. However, the repeatability performance of this technique suffers due to jitter noise from the swept laser source.

  9. Acoustic Imaging Frequency Dynamics of Ferroelectric Domains by Atomic Force Microscopy

    International Nuclear Information System (INIS)

    Kun-Yu, Zhao; Hua-Rong, Zeng; Hong-Zhang, Song; Sen-Xing, Hui; Guo-Rong, Li; Qing-Rui, Yin; Shimamura, Kiyoshi; Kannan, Chinna Venkadasamy; Villora, Encarnacion Antonia Garcia; Takekawa, Shunji; Kitamura, Kenji

    2008-01-01

    We report the acoustic imaging frequency dynamics of ferroelectric domains by low-frequency acoustic probe microscopy based on the commercial atomic force microscopy It is found that ferroelectric domain could be firstly visualized at lower frequency down to 0.5 kHz by AFM-based acoustic microscopy The frequency-dependent acoustic signal revealed a strong acoustic response in the frequency range from 7kHz to 10kHz, and reached maximum at 8.1kHz. The acoustic contrast mechanism can be ascribed to the different elastic response of ferroelectric microstructures to local elastic stress fields, which is induced by the acoustic wave transmitting in the sample when the piezoelectric transducer is vibrating and exciting acoustic wave under ac electric fields due to normal piezoelectric effects. (condensed matter: electronic structure, electrical, magnetic, and optical properties)

  10. SISGR: Room Temperature Single-Molecule Detection and Imaging by Stimulated Emission Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Xiaoliang Sunney [Harvard Univ., Cambridge, MA (United States). Dept. of Chemistry and Chemical Biology

    2017-03-13

    Single-molecule spectroscopy has made considerable impact on many disciplines including chemistry, physics, and biology. To date, most single-molecule spectroscopy work is accomplished by detecting fluorescence. On the other hand, many naturally occurring chromophores, such as retinal, hemoglobin and cytochromes, do not have detectable fluorescence. There is an emerging need for single-molecule spectroscopy techniques that do not require fluorescence. In the last proposal period, we have successfully demonstrated stimulated emission microscopy, single molecule absorption, and stimulated Raman microscopy based on a high-frequency modulation transfer technique. These first-of-a- kind new spectroscopy/microscopy methods tremendously improved our ability to observe molecules that fluorescence weakly, even to the limit of single molecule detection for absorption measurement. All of these methods employ two laser beams: one (pump beam) excites a single molecule to a real or virtual excited state, and the other (probe beam) monitors the absorption/emission property of the single. We extract the intensity change of the probe beam with high sensitivity by implementing a high-frequency phase-sensitive detection scheme, which offers orders of magnitude improvement in detection sensitivity over direct absorption/emission measurement. However, single molecule detection based on fluorescence or absorption is fundamentally limited due to their broad spectral response. It is important to explore other avenues in single molecule detection and imaging which provides higher molecular specificity for studying a wide variety of heterogeneous chemical and biological systems. This proposal aimed to achieve single-molecule detection sensitivity with near resonance stimulated Raman scattering (SRS) microscopy. SRS microscopy was developed in our lab as a powerful technique for imaging heterogeneous samples based on their intrinsic vibrational contrasts, which provides much higher molecular

  11. Objective for EUV microscopy, EUV lithography, and x-ray imaging

    Science.gov (United States)

    Bitter, Manfred; Hill, Kenneth W.; Efthimion, Philip

    2016-05-03

    Disclosed is an imaging apparatus for EUV spectroscopy, EUV microscopy, EUV lithography, and x-ray imaging. This new imaging apparatus could, in particular, make significant contributions to EUV lithography at wavelengths in the range from 10 to 15 nm, which is presently being developed for the manufacturing of the next-generation integrated circuits. The disclosure provides a novel adjustable imaging apparatus that allows for the production of stigmatic images in x-ray imaging, EUV imaging, and EUVL. The imaging apparatus of the present invention incorporates additional properties compared to previously described objectives. The use of a pair of spherical reflectors containing a concave and convex arrangement has been applied to a EUV imaging system to allow for the image and optics to all be placed on the same side of a vacuum chamber. Additionally, the two spherical reflector segments previously described have been replaced by two full spheres or, more precisely, two spherical annuli, so that the total photon throughput is largely increased. Finally, the range of permissible Bragg angles and possible magnifications of the objective has been largely increased.

  12. Image processing for cryogenic transmission electron microscopy of symmetry-mismatched complexes.

    Science.gov (United States)

    Huiskonen, Juha T

    2018-02-08

    Cryogenic transmission electron microscopy (cryo-TEM) is a high-resolution biological imaging method, whereby biological samples, such as purified proteins, macromolecular complexes, viral particles, organelles and cells, are embedded in vitreous ice preserving their native structures. Due to sensitivity of biological materials to the electron beam of the microscope, only relatively low electron doses can be applied during imaging. As a result, the signal arising from the structure of interest is overpowered by noise in the images. To increase the signal-to-noise ratio, different image processing-based strategies that aim at coherent averaging of signal have been devised. In such strategies, images are generally assumed to arise from multiple identical copies of the structure. Prior to averaging, the images must be grouped according to the view of the structure they represent and images representing the same view must be simultaneously aligned relatively to each other. For computational reconstruction of the three-dimensional structure, images must contain different views of the original structure. Structures with multiple symmetry-related substructures are advantageous in averaging approaches because each image provides multiple views of the substructures. However, the symmetry assumption may be valid for only parts of the structure, leading to incoherent averaging of the other parts. Several image processing approaches have been adapted to tackle symmetry-mismatched substructures with increasing success. Such structures are ubiquitous in nature and further computational method development is needed to understanding their biological functions. ©2018 The Author(s).

  13. Deblurring of class-averaged images in single-particle electron microscopy

    International Nuclear Information System (INIS)

    Park, Wooram; Chirikjian, Gregory S; Madden, Dean R; Rockmore, Daniel N

    2010-01-01

    This paper proposes a method for the deblurring of class-averaged images in single-particle electron microscopy (EM). Since EM images of biological samples are very noisy, the images which are nominally identical projection images are often grouped, aligned and averaged in order to cancel or reduce the background noise. However, the noise in the individual EM images generates errors in the alignment process, which creates an inherent limit on the accuracy of the resulting class averages. This inaccurate class average due to the alignment errors can be viewed as the result of a convolution of an underlying clear image with a blurring function. In this work, we develop a deconvolution method that gives an estimate for the underlying clear image from a blurred class-averaged image using precomputed statistics of misalignment. Since this convolution is over the group of rigid-body motions of the plane, SE(2), we use the Fourier transform for SE(2) in order to convert the convolution into a matrix multiplication in the corresponding Fourier space. For practical implementation we use a Hermite-function-based image modeling technique, because Hermite expansions enable lossless Cartesian-polar coordinate conversion using the Laguerre–Fourier expansions, and Hermite expansion and Laguerre–Fourier expansion retain their structures under the Fourier transform. Based on these mathematical properties, we can obtain the deconvolution of the blurred class average using simple matrix multiplication. Tests of the proposed deconvolution method using synthetic and experimental EM images confirm the performance of our method

  14. Quantitative image correction and calibration for confocal fluorescence microscopy using thin reference layers and SIPchart-based calibration procedures

    NARCIS (Netherlands)

    Zwier, J.M.; Oomen, L.; Brocks, L.; Jalink, K.; Brakenhoff, G.J.

    2008-01-01

    The fluorescence intensity image of an axially integrated through-focus series of a thin standardized uniform fluorescent layer can be used for image intensity correction and calibration in sectioning microscopy. This intensity image is in fact available from the earlier introduced Sectioned Imaging

  15. Imaging latex–carbon nanotube composites by subsurface electrostatic force microscopy

    International Nuclear Information System (INIS)

    Patel, Sajan; Petty, Clayton W.; Krafcik, Karen Lee

    2016-01-01

    Electrostatic modes of atomic force microscopy have shown to be non-destructive and relatively simple methods for imaging conductors embedded in insulating polymers. Here we use electrostatic force microscopy to image the dispersion of carbon nanotubes in a latex-based conductive composite, which brings forth features not observed in previously studied systems employing linear polymer films. A fixed-potential model of the probe-nanotube electrostatics is presented which in principle gives access to the conductive nanoparticle's depth and radius, and the polymer film dielectric constant. Comparing this model to the data results in nanotube depths that appear to be slightly above the film–air interface. Furthermore, this result suggests that water-mediated charge build-up at the film–air interface may be the source of electrostatic phase contrast in ambient conditions.

  16. Infrared microscopy imaging applied to obtain the index finger pad's thermoregulation curves

    Science.gov (United States)

    Viafora, Laura A.; Torres, Sergio N.; Ramírez, Wagner; Gutiérrez, Pablo A.; Machuca, Guillermo; Jara, Anselmo

    2017-03-01

    In this work, mid wavelength infrared microscopy imaging videos of several index finger pads, from voluntary people, are recorded to obtain their thermoregulation curves. The proposed non-invasive technique is able to capture spatial and temporal thermal information emitted from blood vessels under-skin, and the irrigation finger pad system, making possible to capture features that a visual-spectrum microscopy cannot detect. Using an infrared laboratory prepared method several voluntary patients exposed theirs fingers to thermal stress while the infrared data is recorded. Using standard infrared imaging and signal processing techniques the thermoregulation curves are estimated. The Cold/Hot Stress experiments have shown infrared data with exponential trend curves, with different recovering slopes for each voluntary person, and sometimes with two steps increasing slope in one person thermoregulation curve response.

  17. Accurate Classification of Protein Subcellular Localization from High-Throughput Microscopy Images Using Deep Learning

    Directory of Open Access Journals (Sweden)

    Tanel Pärnamaa

    2017-05-01

    Full Text Available High-throughput microscopy of many single cells generates high-dimensional data that are far from straightforward to analyze. One important problem is automatically detecting the cellular compartment where a fluorescently-tagged protein resides, a task relatively simple for an experienced human, but difficult to automate on a computer. Here, we train an 11-layer neural network on data from mapping thousands of yeast proteins, achieving per cell localization classification accuracy of 91%, and per protein accuracy of 99% on held-out images. We confirm that low-level network features correspond to basic image characteristics, while deeper layers separate localization classes. Using this network as a feature calculator, we train standard classifiers that assign proteins to previously unseen compartments after observing only a small number of training examples. Our results are the most accurate subcellular localization classifications to date, and demonstrate the usefulness of deep learning for high-throughput microscopy.

  18. Accurate Classification of Protein Subcellular Localization from High-Throughput Microscopy Images Using Deep Learning.

    Science.gov (United States)

    Pärnamaa, Tanel; Parts, Leopold

    2017-05-05

    High-throughput microscopy of many single cells generates high-dimensional data that are far from straightforward to analyze. One important problem is automatically detecting the cellular compartment where a fluorescently-tagged protein resides, a task relatively simple for an experienced human, but difficult to automate on a computer. Here, we train an 11-layer neural network on data from mapping thousands of yeast proteins, achieving per cell localization classification accuracy of 91%, and per protein accuracy of 99% on held-out images. We confirm that low-level network features correspond to basic image characteristics, while deeper layers separate localization classes. Using this network as a feature calculator, we train standard classifiers that assign proteins to previously unseen compartments after observing only a small number of training examples. Our results are the most accurate subcellular localization classifications to date, and demonstrate the usefulness of deep learning for high-throughput microscopy. Copyright © 2017 Parnamaa and Parts.

  19. Structured oblique illumination microscopy for enhanced resolution imaging of non-fluorescent, coherently scattering samples.

    Science.gov (United States)

    Chowdhury, Shwetadwip; Dhalla, Al-Hafeez; Izatt, Joseph

    2012-08-01

    Many biological structures of interest are beyond the diffraction limit of conventional microscopes and their visualization requires application of super-resolution techniques. Such techniques have found remarkable success in surpassing the diffraction limit to achieve sub-diffraction limited resolution; however, they are predominantly limited to fluorescent samples. Here, we introduce a non-fluorescent analogue to structured illumination microscopy, termed structured oblique illumination microscopy (SOIM), where we use simultaneous oblique illuminations of the sample to multiplex high spatial-frequency content into the frequency support of the system. We introduce a theoretical framework describing how to demodulate this multiplexed information to reconstruct an image with a spatial-frequency support exceeding that of the system's classical diffraction limit. This approach allows enhanced-resolution imaging of non-fluorescent samples. Experimental confirmation of the approach is obtained in a reflection test target with moderate numerical aperture.

  20. RGB color coded images in scanning electron microscopy of biological surfaces

    Czech Academy of Sciences Publication Activity Database

    Kofroňová, Olga; Benada, Oldřich

    2017-01-01

    Roč. 61, č. 3 (2017), s. 349-352 ISSN 0001-723X R&D Projects: GA MŠk(CZ) LO1509; GA ČR(CZ) GA16-20229S Institutional support: RVO:61388971 Keywords : Biological surfaces * Color image s * Scanning electron microscopy Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 0.673, year: 2016

  1. Microstructural orientation of isotactic polypropylene studied by computerized scanning eletron microscopy image analysis

    OpenAIRE

    Machado,Giovanna; Luca,Maria Augusta de; Samios,Dimitrios

    2001-01-01

    We evaluate the orientation of microstructural elements of isotatic polypropylene (i-PP) before and after deformation using computerized "Quantikov" software analysis of Scanning Electron Microscopy (SEM) images. We observed that before deformation through uniaxial compression, the polymeric material doesn’t exhibit any significant orientation. After deformation at 1349 MPa the material clearly showed preferential orientation that was attested by the orientation axis seen between two petals o...

  2. Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium-tin-oxide.

    Science.gov (United States)

    Rodighiero, Simona; Torre, Bruno; Sogne, Elisa; Ruffilli, Roberta; Cagnoli, Cinzia; Francolini, Maura; Di Fabrizio, Enzo; Falqui, Andrea

    2015-06-01

    Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field-of-view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold-labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium-tin-oxide was deposited by ion-sputtering on gold-decorated HeLa cells and neurons. Indium-tin-oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold-conjugated markers. © 2015 Wiley Periodicals, Inc.

  3. Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium-tin-oxide

    KAUST Repository

    Rodighiero, Simona

    2015-03-22

    Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field-of-view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold-labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium-tin-oxide was deposited by ion-sputtering on gold-decorated HeLa cells and neurons. Indium-tin-oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold-conjugated markers. © 2015 Wiley Periodicals, Inc.

  4. All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide

    Science.gov (United States)

    Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

    2017-01-01

    While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab. PMID:28186188

  5. All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide

    Science.gov (United States)

    Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

    2017-02-01

    While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab.

  6. Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM).

    Science.gov (United States)

    Tang, Anson H L; Lai, Queenie T K; Chung, Bob M F; Lee, Kelvin C M; Mok, Aaron T Y; Yip, G K; Shum, Anderson H C; Wong, Kenneth K Y; Tsia, Kevin K

    2017-06-28

    Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization

  7. A method for improved clustering and classification of microscopy images using quantitative co-localization coefficients

    LENUS (Irish Health Repository)

    Singan, Vasanth R

    2012-06-08

    AbstractBackgroundThe localization of proteins to specific subcellular structures in eukaryotic cells provides important information with respect to their function. Fluorescence microscopy approaches to determine localization distribution have proved to be an essential tool in the characterization of unknown proteins, and are now particularly pertinent as a result of the wide availability of fluorescently-tagged constructs and antibodies. However, there are currently very few image analysis options able to effectively discriminate proteins with apparently similar distributions in cells, despite this information being important for protein characterization.FindingsWe have developed a novel method for combining two existing image analysis approaches, which results in highly efficient and accurate discrimination of proteins with seemingly similar distributions. We have combined image texture-based analysis with quantitative co-localization coefficients, a method that has traditionally only been used to study the spatial overlap between two populations of molecules. Here we describe and present a novel application for quantitative co-localization, as applied to the study of Rab family small GTP binding proteins localizing to the endomembrane system of cultured cells.ConclusionsWe show how quantitative co-localization can be used alongside texture feature analysis, resulting in improved clustering of microscopy images. The use of co-localization as an additional clustering parameter is non-biased and highly applicable to high-throughput image data sets.

  8. Functional connectivity in the mouse brain imaged by B-mode photoacoustic microscopy

    Science.gov (United States)

    Nasiriavanaki, Mohammadreza; Xing, Wenxin; Xia, Jun; Wang, Lihong V.

    2014-03-01

    The increasing use of mouse models for human brain disease studies, coupled with the fact that existing functional imaging modalities cannot be easily applied to mice, presents an emerging need for a new functional imaging modality. Utilizing acoustic-resolution photoacoustic microscopy (AR-PAM), we imaged spontaneous cerebral hemodynamic fluctuations and their associated functional connections in the mouse brain. The images were acquired noninvasively in B-scan mode with a fast frame rate, a large field of view, and a high spatial resolution. At a location relative to the bregma 0, correlations were investigated inter-hemispherically between bilaterally homologous regions, as well as intra-hemispherically within the same functional regions. The functional connectivity in different functional regions was studied. The locations of these regions agreed well with the Paxinos mouse brain atlas. The functional connectivity map obtained in this study can then be used in the investigation of brain disorders such as stroke, Alzheimer's, schizophrenia, multiple sclerosis, autism, and epilepsy. Our experiments show that photoacoustic microscopy is capable to detect connectivities between different functional regions in B-scan mode, promising a powerful functional imaging modality for future brain research.

  9. Image formation mechanisms in scanning electron microscopy of carbon nanotubes, and retrieval of their intrinsic dimensions.

    Science.gov (United States)

    Jackman, H; Krakhmalev, P; Svensson, K

    2013-01-01

    We present a detailed analysis of the image formation mechanisms that are involved in the imaging of carbon nanotubes with scanning electron microscopy (SEM). We show how SEM images can be modelled by accounting for surface enhancement effects together with the absorption coefficient for secondary electrons, and the electron-probe shape. Images can then be deconvoluted, enabling retrieval of the intrinsic nanotube dimensions. Accurate estimates of their dimensions can thereby be obtained even for structures that are comparable to the electron-probe size (on the order of 2 nm). We also present a simple and robust model for obtaining the outer diameter of nanotubes without any detailed knowledge about the electron-probe shape. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. SMLocalizer, a GPU accelerated ImageJ plugin for single molecule localization microscopy.

    Science.gov (United States)

    Bernhem, Kristoffer; Brismar, Hjalmar

    2018-01-01

    SMLocalizer combines the availability of ImageJ with the power of GPU processing for fast and accurate analysis of single molecule localization microscopy data. Analysis of 2D and 3D data in multiple channels is supported. Plugin freely available for Fiji and ImageJ2.0 through https://sourceforge.net/projects/smlocalizer/. Plugin also available for continuous updates through ImageJ update system, add http://sites.imagej.net/Cellular-Biophysics-KTH/ as update site in ImageJ. Java and CUDA source code freely available on the web at https://github.com/KristofferBernhem/SMlocalizer. brismar@kth.se. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

  11. Imaging modes of atomic force microscopy for application in molecular and cell biology.

    Science.gov (United States)

    Dufrêne, Yves F; Ando, Toshio; Garcia, Ricardo; Alsteens, David; Martinez-Martin, David; Engel, Andreas; Gerber, Christoph; Müller, Daniel J

    2017-04-06

    Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today. Here, we review the basic principles, advantages and limitations of the most common AFM bioimaging modes, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging. For each of these modes, we discuss recent experiments that highlight their unique capabilities.

  12. Real-time histological imaging of kidneys stained with food dyes using multiphoton microscopy.

    Science.gov (United States)

    Nagao, Yasuaki; Kimura, Kazushi; Wang, Shujie; Fujiwara, Takeshi; Mizoguchi, Akira

    2015-10-01

    We have developed a real-time imaging technique for diagnosis of kidney diseases which is composed of two steps, staining renal cells safely with food dyes and optical sectioning of living renal tissue to obtain histological images by multiphoton microscopy (MPM). Here, we demonstrated that the MPM imaging with food dyes, including erythrosine and indigo carmine, could be used as fluorescent agents to visualize renal functions and structures such as glomerular bloodstreams, glomerular filtration, and morphology of glomeruli and renal tubules. We also showed that the kidneys of IgA nephropathy model-mice stained with the food dyes presented histopathological characteristics different from those observed in normal kidneys. The use of the food dyes enhances the quality of tissue images obtained by MPM and offers the potential to contribute to a clinical real-time diagnosis of kidney diseases. © 2015 Wiley Periodicals, Inc.

  13. High Deformability and Particle Size Distribution of Monodisperse Phytoglycogen Nanoparticles Revealed By Atomic Force Microscopy Imaging

    Science.gov (United States)

    Baylis, Benjamin; Dutcher, John

    We have used atomic force microscopy (AFM) imaging in water to determine the volume of hydrated monodisperse phytoglycogen nanoparticles adsorbed onto mica surfaces. By significantly reducing the interaction between the AFM tip and the ``sticky'' nanoparticles, we were able to obtain high quality images. We found that the adsorbed particles are highly deformed, forming pancake-like objects on the hydrophilic mica surface. By measuring the distribution of particle volumes, we calculated the average effective spherical radius of the hydrated particles, and compared this value with that measured in solution using small angle neutron scattering. These measurements illustrate the distinct advantages of AFM imaging over other imaging techniques, namely the ability to measure the height of objects in a liquid environment.

  14. Ex vivo imaging of human thyroid pathology using integrated optical coherence tomography and optical coherence microscopy

    Science.gov (United States)

    Zhou, Chao; Wang, Yihong; Aguirre, Aaron D.; Tsai, Tsung-Han; Cohen, David W.; Connolly, James L.; Fujimoto, James G.

    2010-01-01

    We evaluate the feasibility of optical coherence tomography (OCT) and optical coherence microscopy (OCM) for imaging of benign and malignant thyroid lesions ex vivo using intrinsic optical contrast. 34 thyroid gland specimens are imaged from 17 patients, covering a spectrum of pathology ranging from normal thyroid to benign disease/neoplasms (multinodular colloid goiter, Hashimoto's thyroiditis, and follicular adenoma) and malignant thyroid tumors (papillary carcinoma and medullary carcinoma). Imaging is performed using an integrated OCT and OCM system, with thyroid tissues. With further development of needle-based imaging probes, OCT and OCM could be promising techniques to use for the screening of thyroid nodules and to improve the diagnostic specificity of fine needle aspiration evaluation.

  15. 3D in vivo imaging with extended-focus optical coherence microscopy.

    Science.gov (United States)

    Chen, Yu; Trinh, Le A; Fingler, Jeff; Fraser, Scott E

    2017-11-01

    Optical coherence microscopy (OCM) has unique advantages of non-invasive 3D imaging without the need of exogenous labels for studying biological samples. However, the imaging depth of this technique is limited by the tradeoff between the depth of focus (DOF) and high lateral resolution in Gaussian optics. To overcome this limitation, we have developed an extended-focus OCM (xf-OCM) imaging system using quasi-Bessel beam illumination to extend the DOF to ∼100 μm, about 3-fold greater than standard OCM. High lateral resolution of 1.6 μm ensured detailed identification of structures within live animal samples. The insensitivity to spherical aberrations strengthened the capability of our xf-OCM system in 3D biological imaging. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Micro patterned surfaces: an effective tool for long term digital holographic microscopy cell imaging

    Science.gov (United States)

    Mues, Sarah; Lilge, Inga; Schönherr, Holger; Kemper, Björn; Schnekenburger, Jürgen

    2017-02-01

    The major problem of Digital Holographic Microscopy (DHM) long term live cell imaging is that over time most of the tracked cells move out of the image area and other ones move in. Therefore, most of the cells are lost for the evaluation of individual cellular processes. Here, we present an effective solution for this crucial problem of long-term microscopic live cell analysis. We have generated functionalized slides containing areas of 250 μm per 200 μm. These micropatterned biointerfaces consist of passivating polyaclrylamide brushes (PAAm). Inner areas are backfilled with octadecanthiol (ODT), which allows cell attachment. The fouling properties of these surfaces are highly controllable and therefore the defined areas designed for the size our microscopic image areas were effective in keeping all cells inside the rectangles over the selected imaging period.

  17. First-Principles Atomic Force Microscopy Image Simulations with Density Embedding Theory.

    Science.gov (United States)

    Sakai, Yuki; Lee, Alex J; Chelikowsky, James R

    2016-05-11

    We present an efficient first-principles method for simulating noncontact atomic force microscopy (nc-AFM) images using a "frozen density" embedding theory. Frozen density embedding theory enables one to efficiently compute the tip-sample interaction by considering a sample as a frozen external field. This method reduces the extensive computational load of first-principles AFM simulations by avoiding consideration of the entire tip-sample system and focusing on the tip alone. We demonstrate that our simulation with frozen density embedding theory accurately reproduces full density functional theory simulations of freestanding hydrocarbon molecules while the computational time is significantly reduced. Our method also captures the electronic effect of a Cu(111) substrate on the AFM image of pentacene and reproduces the experimental AFM image of Cu2N on a Cu(100) surface. This approach is applicable for theoretical imaging applications on large molecules, two-dimensional materials, and materials surfaces.

  18. Label-free imaging of rat spinal cords based on multiphoton microscopy

    Science.gov (United States)

    Liao, Chenxi; Wang, Zhenyu; Zhou, Linquan; Zhu, Xiaoqin; Liu, Wenge; Chen, Jianxin

    2016-10-01

    As an integral part of the central nervous system, the spinal cord is a communication cable between the body and the brain. It mainly contains neurons, glial cells, nerve fibers and fiber tracts. The recent development of the optical imaging technique allows high-resolution imaging of biological tissues with the great potential for non-invasively looking inside the body. In this work, we evaluate the imaging capacity of multiphoton microscopy (MPM) based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) for the cells and extracellular matrix in the spinal cord at molecular level. Rat spinal cord tissues were sectioned and imaged by MPM to demonstrate that MPM is able to show the microstructure including white matter, gray matter, ventral horns, dorsal horns, and axons based on the distinct intrinsic sources in each region of spinal cord. In the high-resolution and high-contrast MPM images, the cell profile can be clearly identified as dark shadows caused by nuclei and encircled by cytoplasm. The nerve fibers in white matter region emitted both SHG and TPEF signals. The multiphoton microscopic imaging technique proves to be a fast and effective tool for label-free imaging spinal cord tissues, based on endogenous signals in biological tissue. It has the potential to extend this optical technique to clinical study, where the rapid and damage-free imaging is needed.

  19. Enhanced simulator software for image validation and interpretation for multimodal localization super-resolution fluorescence microscopy

    Science.gov (United States)

    Erdélyi, Miklós; Sinkó, József; Gajdos, Tamás.; Novák, Tibor

    2017-02-01

    Optical super-resolution techniques such as single molecule localization have become one of the most dynamically developed areas in optical microscopy. These techniques routinely provide images of fixed cells or tissues with sub-diffraction spatial resolution, and can even be applied for live cell imaging under appropriate circumstances. Localization techniques are based on the precise fitting of the point spread functions (PSF) to the measured images of stochastically excited, identical fluorescent molecules. These techniques require controlling the rate between the on, off and the bleached states, keeping the number of active fluorescent molecules at an optimum value, so their diffraction limited images can be detected separately both spatially and temporally. Because of the numerous (and sometimes unknown) parameters, the imaging system can only be handled stochastically. For example, the rotation of the dye molecules obscures the polarization dependent PSF shape, and only an averaged distribution - typically estimated by a Gaussian function - is observed. TestSTORM software was developed to generate image stacks for traditional localization microscopes, where localization meant the precise determination of the spatial position of the molecules. However, additional optical properties (polarization, spectra, etc.) of the emitted photons can be used for further monitoring the chemical and physical properties (viscosity, pH, etc.) of the local environment. The image stack generating program was upgraded by several new features, such as: multicolour, polarization dependent PSF, built-in 3D visualization, structured background. These features make the program an ideal tool for optimizing the imaging and sample preparation conditions.

  20. A comparative study of 2 computer-assisted methods of quantifying brightfield microscopy images.

    Science.gov (United States)

    Tse, George H; Marson, Lorna P

    2013-10-01

    Immunohistochemistry continues to be a powerful tool for the detection of antigens. There are several commercially available software packages that allow image analysis; however, these can be complex, require relatively high level of computer skills, and can be expensive. We compared 2 commonly available software packages, Adobe Photoshop CS6 and ImageJ, in their ability to quantify percentage positive area after picrosirius red (PSR) staining and 3,3'-diaminobenzidine (DAB) staining. On analysis of DAB-stained B cells in the mouse spleen, with a biotinylated primary rat anti-mouse-B220 antibody, there was no significant difference on converting images from brightfield microscopy to binary images to measure black and white pixels using ImageJ compared with measuring a range of brown pixels with Photoshop (Student t test, P=0.243, correlation r=0.985). When analyzing mouse kidney allografts stained with PSR, Photoshop achieved a greater interquartile range while maintaining a lower 10th percentile value compared with analysis with ImageJ. A lower 10% percentile reflects that Photoshop analysis is better at analyzing tissues with low levels of positive pixels; particularly relevant for control tissues or negative controls, whereas after ImageJ analysis the same images would result in spuriously high levels of positivity. Furthermore comparing the 2 methods by Bland-Altman plot revealed that these 2 methodologies did not agree when measuring images with a higher percentage of positive staining and correlation was poor (r=0.804). We conclude that for computer-assisted analysis of images of DAB-stained tissue there is no difference between using Photoshop or ImageJ. However, for analysis of color images where differentiation into a binary pattern is not easy, such as with PSR, Photoshop is superior at identifying higher levels of positivity while maintaining differentiation of low levels of positive staining.

  1. Automatic measurement of compression wood cell attributes in fluorescence microscopy images.

    Science.gov (United States)

    Selig, B; Luengo Hendriks, C L; Bardage, S; Daniel, G; Borgefors, G

    2012-06-01

    This paper presents a new automated method for analyzing compression wood fibers in fluorescence microscopy. Abnormal wood known as compression wood is present in almost every softwood tree harvested. Compression wood fibers show a different cell wall morphology and chemistry compared to normal wood fibers, and their mechanical and physical characteristics are considered detrimental for both construction wood and pulp and paper purposes. Currently there is the need for improved methodologies for characterization of lignin distribution in wood cell walls, such as from compression wood fibers, that will allow for a better understanding of fiber mechanical properties. Traditionally, analysis of fluorescence microscopy images of fiber cross-sections has been done manually, which is time consuming and subjective. Here, we present an automatic method, using digital image analysis, that detects and delineates softwood fibers in fluorescence microscopy images, dividing them into cell lumen, normal and highly lignified areas. It also quantifies the different areas, as well as measures cell wall thickness. The method is evaluated by comparing the automatic with a manual delineation. While the boundaries between the various fiber wall regions are detected using the automatic method with precision similar to inter and intra expert variability, the position of the boundary between lumen and the cell wall has a systematic shift that can be corrected. Our method allows for transverse structural characterization of compression wood fibers, which may allow for improved understanding of the micro-mechanical modeling of wood and pulp fibers. © 2012 The Authors Journal of Microscopy © 2012 Wadsworth Center, New York State Department of Health.

  2. Bright field microscopy as an alternative to whole cell fluorescence in automated analysis of macrophage images.

    Directory of Open Access Journals (Sweden)

    Jyrki Selinummi

    2009-10-01

    Full Text Available Fluorescence microscopy is the standard tool for detection and analysis of cellular phenomena. This technique, however, has a number of drawbacks such as the limited number of available fluorescent channels in microscopes, overlapping excitation and emission spectra of the stains, and phototoxicity.We here present and validate a method to automatically detect cell population outlines directly from bright field images. By imaging samples with several focus levels forming a bright field -stack, and by measuring the intensity variations of this stack over the -dimension, we construct a new two dimensional projection image of increased contrast. With additional information for locations of each cell, such as stained nuclei, this bright field projection image can be used instead of whole cell fluorescence to locate borders of individual cells, separating touching cells, and enabling single cell analysis. Using the popular CellProfiler freeware cell image analysis software mainly targeted for fluorescence microscopy, we validate our method by automatically segmenting low contrast and rather complex shaped murine macrophage cells.The proposed approach frees up a fluorescence channel, which can be used for subcellular studies. It also facilitates cell shape measurement in experiments where whole cell fluorescent staining is either not available, or is dependent on a particular experimental condition. We show that whole cell area detection results using our projected bright field images match closely to the standard approach where cell areas are localized using fluorescence, and conclude that the high contrast bright field projection image can directly replace one fluorescent channel in whole cell quantification. Matlab code for calculating the projections can be downloaded from the supplementary site: http://sites.google.com/site/brightfieldorstaining.

  3. Laboratory cryo x-ray microscopy for 3D cell imaging.

    Science.gov (United States)

    Fogelqvist, Emelie; Kördel, Mikael; Carannante, Valentina; Önfelt, Björn; Hertz, Hans M

    2017-10-18

    Water-window x-ray microscopy allows two- and three-dimensional (2D and 3D) imaging of intact unstained cells in their cryofixed near-native state with unique contrast and high resolution. Present operational biological water-window microscopes are based at synchrotron facilities, which limits their accessibility and integration with complementary methods. Laboratory-source microscopes have had difficulty addressing relevant biological tasks with proper resolution and contrast due to long exposure times and limited up-time. Here we report on laboratory cryo x-ray microscopy with the exposure time, contrast, and reliability to allow for routine high-spatial resolution 3D imaging of intact cells and cell-cell interactions. Stabilization of the laser-plasma source combined with new optics and sample preparation provide high-resolution cell imaging, both in 2D with ten-second exposures and in 3D with twenty-minute tomography. Examples include monitoring of the distribution of carbon-dense vesicles in starving HEK293T cells and imaging the interaction between natural killer cells and target cells.

  4. New light on ion channel imaging by total internal reflection fluorescence (TIRF) microscopy.

    Science.gov (United States)

    Yamamura, Hisao; Suzuki, Yoshiaki; Imaizumi, Yuji

    2015-05-01

    Ion channels play pivotal roles in a wide variety of cellular functions; therefore, their physiological characteristics, pharmacological responses, and molecular structures have been extensively investigated. However, the mobility of an ion channel itself in the cell membrane has not been examined in as much detail. A total internal reflection fluorescence (TIRF) microscope allows fluorophores to be imaged in a restricted region within an evanescent field of less than 200 nm from the interface of the coverslip and plasma membrane in living cells. Thus the TIRF microscope is useful for selectively visualizing the plasmalemmal surface and subplasmalemmal zone. In this review, we focused on a single-molecule analysis of the dynamic movement of ion channels in the plasma membrane using TIRF microscopy. We also described two single-molecule imaging techniques under TIRF microscopy: fluorescence resonance energy transfer (FRET) for the identification of molecules that interact with ion channels, and subunit counting for the determination of subunit stoichiometry in a functional channel. TIRF imaging can also be used to analyze spatiotemporal Ca(2+) events in the subplasmalemma. Single-molecule analyses of ion channels and localized Ca(2+) signals based on TIRF imaging provide beneficial pharmacological and physiological information concerning the functions of ion channels. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  5. BioEM: GPU-accelerated computing of Bayesian inference of electron microscopy images

    Science.gov (United States)

    Cossio, Pilar; Rohr, David; Baruffa, Fabio; Rampp, Markus; Lindenstruth, Volker; Hummer, Gerhard

    2017-01-01

    In cryo-electron microscopy (EM), molecular structures are determined from large numbers of projection images of individual particles. To harness the full power of this single-molecule information, we use the Bayesian inference of EM (BioEM) formalism. By ranking structural models using posterior probabilities calculated for individual images, BioEM in principle addresses the challenge of working with highly dynamic or heterogeneous systems not easily handled in traditional EM reconstruction. However, the calculation of these posteriors for large numbers of particles and models is computationally demanding. Here we present highly parallelized, GPU-accelerated computer software that performs this task efficiently. Our flexible formulation employs CUDA, OpenMP, and MPI parallelization combined with both CPU and GPU computing. The resulting BioEM software scales nearly ideally both on pure CPU and on CPU+GPU architectures, thus enabling Bayesian analysis of tens of thousands of images in a reasonable time. The general mathematical framework and robust algorithms are not limited to cryo-electron microscopy but can be generalized for electron tomography and other imaging experiments.

  6. Simultaneous morphological and functional imaging of the honeybee's brain by two-photon microscopy

    International Nuclear Information System (INIS)

    Haase, A.

    2011-01-01

    Thanks to its rather simply structured but highly performing brain, the honeybee (Apis mellifera) is an important model for neurobiological studies. Therefore there is a great need for new functional imaging modalities adapted to this species. Herein we give a detailed report on the development and performance of a platform for in vivo functional and morphological imaging of the honeybee's brain, focusing on its primary olfactory centres, the antennal lobes (ALs). The experimental setup consists of a two-photon microscope combined with a synchronized odour stimulus generator. Our imaging platform allows to simultaneously obtain both morphological measurements of the ALs functional units, the glomeruli, and in vivo calcium recording of their neural activity. We were able to record the characteristic glomerular response maps to odour stimuli applied to the bee's antennae. Our approach offers several advantages over the commonly used conventional fluorescence microscopy. Two-photon microscopy provides substantial enhancement in both spatial and temporal resolutions, while minimizing photo damage. Calcium recordings show a more than fourfold improvement in the functional signal with respect to the techniques available up to now. Finally, the extended penetration depth, thanks to the infrared excitation, allows the functional imaging of profound glomeruli which have not been optically accessible up to now.

  7. High-speed atomic force microscopy imaging of live mammalian cells.

    Science.gov (United States)

    Shibata, Mikihiro; Watanabe, Hiroki; Uchihashi, Takayuki; Ando, Toshio; Yasuda, Ryohei

    2017-01-01

    Direct imaging of morphological dynamics of live mammalian cells with nanometer resolution under physiological conditions is highly expected, but yet challenging. High-speed atomic force microscopy (HS-AFM) is a unique technique for capturing biomolecules at work under near physiological conditions. However, application of HS-AFM for imaging of live mammalian cells was hard to be accomplished because of collision between a huge mammalian cell and a cantilever during AFM scanning. Here, we review our recent improvements of HS-AFM for imaging of activities of live mammalian cells without significant damage to the cell. The improvement of an extremely long (~3 μm) AFM tip attached to a cantilever enables us to reduce severe damage to soft mammalian cells. In addition, a combination of HS-AFM with simple fluorescence microscopy allows us to quickly locate the cell in the AFM scanning area. After these improvements, we demonstrate that developed HS-AFM for live mammalian cells is possible to image morphogenesis of filopodia, membrane ruffles, pits open-close formations, and endocytosis in COS-7, HeLa cells as well as hippocampal neurons.

  8. Fast segmentation of stained nuclei in terabyte-scale, time resolved 3D microscopy image stacks.

    Directory of Open Access Journals (Sweden)

    Johannes Stegmaier

    Full Text Available Automated analysis of multi-dimensional microscopy images has become an integral part of modern research in life science. Most available algorithms that provide sufficient segmentation quality, however, are infeasible for a large amount of data due to their high complexity. In this contribution we present a fast parallelized segmentation method that is especially suited for the extraction of stained nuclei from microscopy images, e.g., of developing zebrafish embryos. The idea is to transform the input image based on gradient and normal directions in the proximity of detected seed points such that it can be handled by straightforward global thresholding like Otsu's method. We evaluate the quality of the obtained segmentation results on a set of real and simulated benchmark images in 2D and 3D and show the algorithm's superior performance compared to other state-of-the-art algorithms. We achieve an up to ten-fold decrease in processing times, allowing us to process large data sets while still providing reasonable segmentation results.

  9. Image-based calibration of a deformable mirror in wide-field microscopy.

    Science.gov (United States)

    Turaga, Diwakar; Holy, Timothy E

    2010-04-10

    Optical aberrations limit resolution in biological tissues, and their influence is particularly large for promising techniques such as light-sheet microscopy. In principle, image quality might be improved by adaptive optics (AO), in which aberrations are corrected by using a deformable mirror (DM). To implement AO in microscopy, one requires a method to measure wavefront aberrations, but the most commonly used methods have limitations for samples lacking point-source emitters. Here we implement an image-based wavefront-sensing technique, a variant of generalized phase-diverse imaging called multiframe blind deconvolution, and exploit it to calibrate a DM in a light-sheet microscope. We describe two methods of parameterizing the influence of the DM on aberrations: a traditional Zernike expansion requiring 1040 parameters, and a direct physical model of the DM requiring just 8 or 110 parameters. By randomizing voltages on all actuators, we show that the Zernike expansion successfully predicts wavefronts to an accuracy of approximately 30 nm (rms) even for large aberrations. We thus show that image-based wavefront sensing, which requires no additional optical equipment, allows a simple but powerful method to calibrate a deformable optical element in a microscope setting.

  10. Nanoscale simultaneous chemical and mechanical imaging via peak force infrared microscopy.

    Science.gov (United States)

    Wang, Le; Wang, Haomin; Wagner, Martin; Yan, Yong; Jakob, Devon S; Xu, Xiaoji G

    2017-06-01

    Nondestructive chemical and mechanical measurements of materials with ~10-nm spatial resolution together with topography provide rich information on the compositions and organizations of heterogeneous materials and nanoscale objects. However, multimodal nanoscale correlations are difficult to achieve because of the limitation on spatial resolution of optical microscopy and constraints from instrumental complexities. We report a novel noninvasive spectroscopic scanning probe microscopy method-peak force infrared (PFIR) microscopy-that allows chemical imaging, collection of broadband infrared spectra, and mechanical mapping at a spatial resolution of 10 nm. In our technique, chemical absorption information is directly encoded in the withdraw curve of the peak force tapping cycle after illumination with synchronized infrared laser pulses in a simple apparatus. Nanoscale phase separation in block copolymers and inhomogeneity in CH 3 NH 3 PbBr 3 perovskite crystals are studied with correlative infrared/mechanical nanoimaging. Furthermore, we show that the PFIR method is sensitive to the presence of surface phonon polaritons in boron nitride nanotubes. PFIR microscopy will provide a powerful analytical tool for explorations at the nanoscale across wide disciplines.

  11. Cell segmentation in phase contrast microscopy images via semi-supervised classification over optics-related features.

    Science.gov (United States)

    Su, Hang; Yin, Zhaozheng; Huh, Seungil; Kanade, Takeo

    2013-10-01

    Phase-contrast microscopy is one of the most common and convenient imaging modalities to observe long-term multi-cellular processes, which generates images by the interference of lights passing through transparent specimens and background medium with different retarded phases. Despite many years of study, computer-aided phase contrast microscopy analysis on cell behavior is challenged by image qualities and artifacts caused by phase contrast optics. Addressing the unsolved challenges, the authors propose (1) a phase contrast microscopy image restoration method that produces phase retardation features, which are intrinsic features of phase contrast microscopy, and (2) a semi-supervised learning based algorithm for cell segmentation, which is a fundamental task for various cell behavior analysis. Specifically, the image formation process of phase contrast microscopy images is first computationally modeled with a dictionary of diffraction patterns; as a result, each pixel of a phase contrast microscopy image is represented by a linear combination of the bases, which we call phase retardation features. Images are then partitioned into phase-homogeneous atoms by clustering neighboring pixels with similar phase retardation features. Consequently, cell segmentation is performed via a semi-supervised classification technique over the phase-homogeneous atoms. Experiments demonstrate that the proposed approach produces quality segmentation of individual cells and outperforms previous approaches. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Automated image analysis of atomic force microscopy images of rotavirus particles

    Energy Technology Data Exchange (ETDEWEB)

    Venkataraman, S. [Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (United States); Department of Electrical and Computer Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Allison, D.P. [Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (United States); Department of Biochemistry, Cellular, and Molecular Biology, University of Tennessee, Knoxville, TN 37996 (United States); Molecular Imaging Inc. Tempe, AZ, 85282 (United States); Qi, H. [Department of Electrical and Computer Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Morrell-Falvey, J.L. [Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (United States); Kallewaard, N.L. [Vanderbilt University Medical Center, Nashville, TN 37232-2905 (United States); Crowe, J.E. [Vanderbilt University Medical Center, Nashville, TN 37232-2905 (United States); Doktycz, M.J. [Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (United States)]. E-mail: doktyczmj@ornl.gov

    2006-06-15

    A variety of biological samples can be imaged by the atomic force microscope (AFM) under environments that range from vacuum to ambient to liquid. Generally imaging is pursued to evaluate structural features of the sample or perhaps identify some structural changes in the sample that are induced by the investigator. In many cases, AFM images of sample features and induced structural changes are interpreted in general qualitative terms such as markedly smaller or larger, rougher, highly irregular, or smooth. Various manual tools can be used to analyze images and extract more quantitative data, but this is usually a cumbersome process. To facilitate quantitative AFM imaging, automated image analysis routines are being developed. Viral particles imaged in water were used as a test case to develop an algorithm that automatically extracts average dimensional information from a large set of individual particles. The extracted information allows statistical analyses of the dimensional characteristics of the particles and facilitates interpretation related to the binding of the particles to the surface. This algorithm is being extended for analysis of other biological samples and physical objects that are imaged by AFM.

  13. Digital Holographic Microscopy: Quantitative Phase Imaging and Applications in Live Cell Analysis

    Science.gov (United States)

    Kemper, Björn; Langehanenberg, Patrik; Kosmeier, Sebastian; Schlichthaber, Frank; Remmersmann, Christian; von Bally, Gert; Rommel, Christina; Dierker, Christian; Schnekenburger, Jürgen

    The analysis of complex processes in living cells creates a high demand for fast and label-free methods for online monitoring. Widely used fluorescence methods require specific labeling and are often restricted to chemically fixated samples. Thus, methods that offer label-free and minimally invasive detection of live cell processes and cell state alterations are of particular interest. In combination with light microscopy, digital holography provides label-free, multi-focus quantitative phase imaging of living cells. In overview, several methods for digital holographic microscopy (DHM) are presented. First, different experimental setups for the recording of digital holograms and the modular integration of DHM into common microscopes are described. Then the numerical processing of digitally captured holograms is explained. This includes the description of spatial and temporal phase shifting techniques, spatial filtering based reconstruction, holographic autofocusing, and the evaluation of self-interference holograms. Furthermore, the usage of partial coherent light and multi-wavelength approaches is discussed. Finally, potentials of digital holographic microscopy for quantitative cell imaging are illustrated by results from selected applications. It is shown that DHM can be used for automated tracking of migrating cells and cell thickness monitoring as well as for refractive index determination of cells and particles. Moreover, the use of DHM for label-free analysis in fluidics and micro-injection monitoring is demonstrated. The results show that DHM is a highly relevant method that allows novel insights in dynamic cell biology, with applications in cancer research and for drugs and toxicity testing.

  14. Evaluation of agave fiber delignification by means of microscopy techniques and image analysis.

    Science.gov (United States)

    Hernández-Hernández, Hilda M; Chanona-Pérez, Jorge J; Calderón-Domínguez, Georgina; Perea-Flores, María J; Mendoza-Pérez, Jorge A; Vega, Alberto; Ligero, Pablo; Palacios-González, Eduardo; Farrera-Rebollo, Reynold R

    2014-10-01

    Recently, the use of different types of natural fibers to produce paper and textiles from agave plants has been proposed. Agave atrovirens can be a good source of cellulose and lignin; nevertheless, the microstructural changes that happen during delignification have scarcely been studied. The aim of this work was to study the microstructural changes that occur during the delignification of agave fibers by means of microscopy techniques and image analysis. The fibers of A. atrovirens were obtained from leaves using convective drying, milling, and sieving. Fibers were processed using the Acetosolv pulping method at different concentrations of acetic acid; increasing acid concentration promoted higher levels of delignification, structural damage, and the breakdown of fiber clumps. Delignification followed by spectrometric analysis and microstructural studies were carried out by light, confocal laser scanning and scanning electron microscopy and showed that the delignification process follows three stages: initial, bulk, and residual. Microscopy techniques and image analysis were efficient tools for microstructural characterization during delignification of agave fibers, allowing quantitative evaluation of the process and the development of linear prediction models. The data obtained integrated numerical and microstructural information that could be valuable for the study of pulping of lignocellulosic materials.

  15. Robust Nucleus/Cell Detection and Segmentation in Digital Pathology and Microscopy Images: A Comprehensive Review

    Science.gov (United States)

    Xing, Fuyong; Yang, Lin

    2016-01-01

    Digital pathology and microscopy image analysis is widely used for comprehensive studies of cell morphology or tissue structure. Manual assessment is labor intensive and prone to inter-observer variations. Computer-aided methods, which can significantly improve the objectivity and reproducibility, have attracted a great deal of interest in recent literatures. Among the pipeline of building a computer-aided diagnosis system, nucleus or cell detection and segmentation play a very important role to describe the molecular morphological information. In the past few decades, many efforts have been devoted to automated nucleus/cell detection and segmentation. In this review, we provide a comprehensive summary of the recent state-of-the-art nucleus/cell segmentation approaches on different types of microscopy images including bright-field, phase-contrast, differential interference contrast (DIC), fluorescence, and electron microscopies. In addition, we discuss the challenges for the current methods and the potential future work of nucleus/cell detection and segmentation. PMID:26742143

  16. Diatom Valve Three-Dimensional Representation: A New Imaging Method Based on Combined Microscopies

    Directory of Open Access Journals (Sweden)

    Maria Antonietta Ferrara

    2016-09-01

    Full Text Available The frustule of diatoms, unicellular microalgae, shows very interesting photonic features, generally related to its complicated and quasi-periodic micro- and nano-structure. In order to simulate light propagation inside and through this natural structure, it is important to develop three-dimensional (3D models for synthetic replica with high spatial resolution. In this paper, we present a new method that generates images of microscopic diatoms with high definition, by merging scanning electron microscopy and digital holography microscopy or atomic force microscopy data. Starting from two digital images, both acquired separately with standard characterization procedures, a high spatial resolution (Δz = λ/20, Δx = Δy ≅ 100 nm, at least 3D model of the object has been generated. Then, the two sets of data have been processed by matrix formalism, using an original mathematical algorithm implemented on a commercially available software. The developed methodology could be also of broad interest in the design and fabrication of micro-opto-electro-mechanical systems.

  17. Diatom Valve Three-Dimensional Representation: A New Imaging Method Based on Combined Microscopies.

    Science.gov (United States)

    Ferrara, Maria Antonietta; De Tommasi, Edoardo; Coppola, Giuseppe; De Stefano, Luca; Rea, Ilaria; Dardano, Principia

    2016-09-28

    The frustule of diatoms, unicellular microalgae, shows very interesting photonic features, generally related to its complicated and quasi-periodic micro- and nano-structure. In order to simulate light propagation inside and through this natural structure, it is important to develop three-dimensional (3D) models for synthetic replica with high spatial resolution. In this paper, we present a new method that generates images of microscopic diatoms with high definition, by merging scanning electron microscopy and digital holography microscopy or atomic force microscopy data. Starting from two digital images, both acquired separately with standard characterization procedures, a high spatial resolution (Δ z = λ/20, Δ x = Δ y ≅ 100 nm, at least) 3D model of the object has been generated. Then, the two sets of data have been processed by matrix formalism, using an original mathematical algorithm implemented on a commercially available software. The developed methodology could be also of broad interest in the design and fabrication of micro-opto-electro-mechanical systems.

  18. Multiscale imaging of human thyroid pathologies using integrated optical coherence tomography (OCT) and optical coherence microscopy (OCM)

    Science.gov (United States)

    Zhou, Chao; Wang, Yihong; Aguirre, Aaron D.; Tsai, Tsung-Han; Cohen, David W.; Connolly, James L.; Fujimoto, James G.

    2010-02-01

    We evaluate the feasibility of optical coherence tomography (OCT) and optical coherence microscopy (OCM) for imaging of benign and malignant thyroid lesions ex vivo using intrinsic optical contrast. Thirty four thyroid gland specimens were imaged from 17 patients, covering a spectrum of pathology, ranging from normal thyroid to neoplasia and benign disease. The integrated OCT and OCM imaging system allows seamlessly switching between low and high magnifications, in a way similar to traditional microscopy. Good correspondence was observed between optical images and histological sections. The results provide a basis for interpretation of future OCT and OCM images of the thyroid tissues and suggest the possibility of future in vivo evaluation of thyroid pathology.

  19. Analysis of Point Based Image Registration Errors With Applications in Single Molecule Microscopy.

    Science.gov (United States)

    Cohen, E A K; Ober, R J

    2013-12-15

    We present an asymptotic treatment of errors involved in point-based image registration where control point (CP) localization is subject to heteroscedastic noise; a suitable model for image registration in fluorescence microscopy. Assuming an affine transform, CPs are used to solve a multivariate regression problem. With measurement errors existing for both sets of CPs this is an errors-in-variable problem and linear least squares is inappropriate; the correct method being generalized least squares. To allow for point dependent errors the equivalence of a generalized maximum likelihood and heteroscedastic generalized least squares model is achieved allowing previously published asymptotic results to be extended to image registration. For a particularly useful model of heteroscedastic noise where covariance matrices are scalar multiples of a known matrix (including the case where covariance matrices are multiples of the identity) we provide closed form solutions to estimators and derive their distribution. We consider the target registration error (TRE) and define a new measure called the localization registration error (LRE) believed to be useful, especially in microscopy registration experiments. Assuming Gaussianity of the CP localization errors, it is shown that the asymptotic distribution for the TRE and LRE are themselves Gaussian and the parameterized distributions are derived. Results are successfully applied to registration in single molecule microscopy to derive the key dependence of the TRE and LRE variance on the number of CPs and their associated photon counts. Simulations show asymptotic results are robust for low CP numbers and non-Gaussianity. The method presented here is shown to outperform GLS on real imaging data.

  20. Local raster scanning for high-speed imaging of biopolymers in atomic force microscopy.

    Science.gov (United States)

    Chang, Peter I; Huang, Peng; Maeng, Jungyeoul; Andersson, Sean B

    2011-06-01

    A novel algorithm is described and illustrated for high speed imaging of biopolymers and other stringlike samples using atomic force microscopy. The method uses the measurements in real-time to steer the tip of the instrument to localize the scanning area over the sample of interest. Depending on the sample, the scan time can be reduced by an order of magnitude or more while maintaining image resolution. Images are generated by interpolating the non-raster data using a modified Kriging algorithm. The method is demonstrated using physical simulations that include actuator and cantilever dynamics, nonlinear tip-sample interactions, and measurement noise as well as through scanning experiments in which a two-axis nanopositioning stage is steered by the algorithm using simulated height data. © 2011 American Institute of Physics

  1. Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images.

    Science.gov (United States)

    Watson, Jeffrey R; Gainer, Christian F; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G Michael; Anton, Rein; Romanowski, Marek

    2015-10-01

    Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures.

  2. Microscopy Image Browser: A Platform for Segmentation and Analysis of Multidimensional Datasets.

    Directory of Open Access Journals (Sweden)

    Ilya Belevich

    2016-01-01

    Full Text Available Understanding the structure-function relationship of cells and organelles in their natural context requires multidimensional imaging. As techniques for multimodal 3-D imaging have become more accessible, effective processing, visualization, and analysis of large datasets are posing a bottleneck for the workflow. Here, we present a new software package for high-performance segmentation and image processing of multidimensional datasets that improves and facilitates the full utilization and quantitative analysis of acquired data, which is freely available from a dedicated website. The open-source environment enables modification and insertion of new plug-ins to customize the program for specific needs. We provide practical examples of program features used for processing, segmentation and analysis of light and electron microscopy datasets, and detailed tutorials to enable users to rapidly and thoroughly learn how to use the program.

  3. Fluorescence confocal laser scanning microscopy for in vivo imaging of epidermal reactions to two experimental irritants

    DEFF Research Database (Denmark)

    Suihko, C.; Serup, J.

    2008-01-01

    Background: Fibre-optic fluorescence confocal laser scanning microscopy (CLSM) is a novel non-invasive technique for in vivo imaging of skin. The cellular structure of the epidermis can be studied. A fluorophore, e.g. fluorescein sodium, is introduced by an intradermal injection or applied...... to the skin surface before scanning. Images are horizontal optical sections parallel to the skin surface. Fluorescence CLSM has hitherto not been applied to experimental contact dermatitis. Objective: The aim was to study the applicability of fluorescence CLSM for in situ imaging of irritant contact......, modified the physico-chemical properties of the skin surface and both disturbed epicutaneous labelling with the flurophore and immersion oil coupling between the skin surface and the optical system. Thus, SLS was technically more difficult to study by CLSM than PA. Conclusions: This preliminary study...

  4. Dual-polarization interference microscopy for advanced quantification of phase associated with the image field.

    Science.gov (United States)

    Bouchal, Petr; Chmelík, Radim; Bouchal, Zdeněk

    2018-02-01

    A new concept of dual-polarization spatial light interference microscopy (DPSLIM) is proposed and demonstrated experimentally. The method works with two orthogonally polarized modes in which signal and reference waves are combined to realize the polarization-sensitive phase-shifting, thus allowing advanced reconstruction of the phase associated with the image field. The image phase is reconstructed directly from four polarization encoded interference records by a single step processing. This is a progress compared with common methods, in which the phase of the image field is reconstructed using the optical path difference and the amplitudes of interfering waves, which are calculated in multiple-step processing of the records. The DPSLIM is implemented in a common-path configuration using a spatial light modulator, which is connected to a commercial microscope Nikon E200. The optical performance of the method is demonstrated in experiments using both polystyrene microspheres and live LW13K2 cells.

  5. A new image correction method for live cell atomic force microscopy

    International Nuclear Information System (INIS)

    Shen, Y; Sun, J L; Zhang, A; Hu, J; Xu, L X

    2007-01-01

    During live cell imaging via atomic force microscopy (AFM), the interactions between the AFM probe and the membrane yield distorted cell images. In this work, an image correction method was developed based on the force-distance curve and the modified Hertzian model. The normal loading and lateral forces exerted on the cell membrane by the AFM tip were both accounted for during the scanning. Two assumptions were made in modelling based on the experimental measurements: (1) the lateral force on the endothelial cells was linear to the height; (2) the cell membrane Young's modulus could be derived from the displacement measurement of a normal force curve. Results have shown that the model could be used to recover up to 30% of the actual cell height depending on the loading force. The accuracy of the model was also investigated with respect to the loading force and mechanical property of the cell membrane

  6. Studying inner ear protein–protein interactions using FRET and FLIM

    OpenAIRE

    Hallworth, Richard; Currall, Benjamin; Nichols, Michael G.; Wu, Xudong; Zuo, Jian

    2006-01-01

    Molecular genetic studies of the inner ear have recently revealed a large number of previously undescribed proteins, but their functions remain unclear. Optical methods such as FRET and FLIM are just beginning to be applied to the study of functional interactions between novel inner ear proteins. This review discusses the various methods for employing FRET and FLIM in protein–protein interaction studies, their advantages and pitfalls, with examples drawn from inner ear studies.

  7. Optimized computational imaging methods for small-target sensing in lens-free holographic microscopy

    Science.gov (United States)

    Xiong, Zhen; Engle, Isaiah; Garan, Jacob; Melzer, Jeffrey E.; McLeod, Euan

    2018-02-01

    Lens-free holographic microscopy is a promising diagnostic approach because it is cost-effective, compact, and suitable for point-of-care applications, while providing high resolution together with an ultra-large field-of-view. It has been applied to biomedical sensing, where larger targets like eukaryotic cells, bacteria, or viruses can be directly imaged without labels, and smaller targets like proteins or DNA strands can be detected via scattering labels like micro- or nano-spheres. Automated image processing routines can count objects and infer target concentrations. In these sensing applications, sensitivity and specificity are critically affected by image resolution and signal-to-noise ratio (SNR). Pixel super-resolution approaches have been shown to boost resolution and SNR by synthesizing a high-resolution image from multiple, partially redundant, low-resolution images. However, there are several computational methods that can be used to synthesize the high-resolution image, and previously, it has been unclear which methods work best for the particular case of small-particle sensing. Here, we quantify the SNR achieved in small-particle sensing using regularized gradient-descent optimization method, where the regularization is based on cardinal-neighbor differences, Bayer-pattern noise reduction, or sparsity in the image. In particular, we find that gradient-descent with sparsity-based regularization works best for small-particle sensing. These computational approaches were evaluated on images acquired using a lens-free microscope that we assembled from an off-the-shelf LED array and color image sensor. Compared to other lens-free imaging systems, our hardware integration, calibration, and sample preparation are particularly simple. We believe our results will help to enable the best performance in lens-free holographic sensing.

  8. Giga-pixel lensfree holographic microscopy and tomography using color image sensors.

    Directory of Open Access Journals (Sweden)

    Serhan O Isikman

    Full Text Available We report Giga-pixel lensfree holographic microscopy and tomography using color sensor-arrays such as CMOS imagers that exhibit Bayer color filter patterns. Without physically removing these color filters coated on the sensor chip, we synthesize pixel super-resolved lensfree holograms, which are then reconstructed to achieve ~350 nm lateral resolution, corresponding to a numerical aperture of ~0.8, across a field-of-view of ~20.5 mm(2. This constitutes a digital image with ~0.7 Billion effective pixels in both amplitude and phase channels (i.e., ~1.4 Giga-pixels total. Furthermore, by changing the illumination angle (e.g., ± 50° and scanning a partially-coherent light source across two orthogonal axes, super-resolved images of the same specimen from different viewing angles are created, which are then digitally combined to synthesize tomographic images of the object. Using this dual-axis lensfree tomographic imager running on a color sensor-chip, we achieve a 3D spatial resolution of ~0.35 µm × 0.35 µm × ~2 µm, in x, y and z, respectively, creating an effective voxel size of ~0.03 µm(3 across a sample volume of ~5 mm(3, which is equivalent to >150 Billion voxels. We demonstrate the proof-of-concept of this lensfree optical tomographic microscopy platform on a color CMOS image sensor by creating tomograms of micro-particles as well as a wild-type C. elegans nematode.

  9. Fluorescent Nano-Probes to Image Plant Cell Walls by Super-Resolution STED Microscopy.

    Science.gov (United States)

    Paës, Gabriel; Habrant, Anouck; Terryn, Christine

    2018-02-06

    Lignocellulosic biomass is a complex network of polymers making up the cell walls of plants. It represents a feedstock of sustainable resources to be converted into fuels, chemicals, and materials. Because of its complex architecture, lignocellulose is a recalcitrant material that requires some pretreatments and several types of catalysts to be transformed efficiently. Gaining more knowledge in the architecture of plant cell walls is therefore important to understand and optimize transformation processes. For the first time, super-resolution imaging of poplar wood samples has been performed using the Stimulated Emission Depletion (STED) technique. In comparison to standard confocal images, STED reveals new details in cell wall structure, allowing the identification of secondary walls and middle lamella with fine details, while keeping open the possibility to perform topochemistry by the use of relevant fluorescent nano-probes. In particular, the deconvolution of STED images increases the signal-to-noise ratio so that images become very well defined. The obtained results show that the STED super-resolution technique can be easily implemented by using cheap commercial fluorescent rhodamine-PEG nano-probes which outline the architecture of plant cell walls due to their interaction with lignin. Moreover, the sample preparation only requires easily-prepared plant sections of a few tens of micrometers, in addition to an easily-implemented post-treatment of images. Overall, the STED super-resolution technique in combination with a variety of nano-probes can provide a new vision of plant cell wall imaging by filling in the gap between classical photon microscopy and electron microscopy.

  10. Fluorescent Nano-Probes to Image Plant Cell Walls by Super-Resolution STED Microscopy

    Directory of Open Access Journals (Sweden)

    Gabriel Paës

    2018-02-01

    Full Text Available Lignocellulosic biomass is a complex network of polymers making up the cell walls of plants. It represents a feedstock of sustainable resources to be converted into fuels, chemicals, and materials. Because of its complex architecture, lignocellulose is a recalcitrant material that requires some pretreatments and several types of catalysts to be transformed efficiently. Gaining more knowledge in the architecture of plant cell walls is therefore important to understand and optimize transformation processes. For the first time, super-resolution imaging of poplar wood samples has been performed using the Stimulated Emission Depletion (STED technique. In comparison to standard confocal images, STED reveals new details in cell wall structure, allowing the identification of secondary walls and middle lamella with fine details, while keeping open the possibility to perform topochemistry by the use of relevant fluorescent nano-probes. In particular, the deconvolution of STED images increases the signal-to-noise ratio so that images become very well defined. The obtained results show that the STED super-resolution technique can be easily implemented by using cheap commercial fluorescent rhodamine-PEG nano-probes which outline the architecture of plant cell walls due to their interaction with lignin. Moreover, the sample preparation only requires easily-prepared plant sections of a few tens of micrometers, in addition to an easily-implemented post-treatment of images. Overall, the STED super-resolution technique in combination with a variety of nano-probes can provide a new vision of plant cell wall imaging by filling in the gap between classical photon microscopy and electron microscopy.

  11. Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy

    Science.gov (United States)

    Takahashi, Yasufumi; Shevchuk, Andrew I.; Novak, Pavel; Babakinejad, Babak; Macpherson, Julie; Unwin, Patrick R.; Shiku, Hitoshi; Gorelik, Julia; Klenerman, David; Korchev, Yuri E.; Matsue, Tomokazu

    2012-01-01

    We describe voltage-switching mode scanning electrochemical microscopy (VSM-SECM), in which a single SECM tip electrode was used to acquire high-quality topographical and electrochemical images of living cells simultaneously. This was achieved by switching the applied voltage so as to change the faradaic current from a hindered diffusion feedback signal (for distance control and topographical imaging) to the electrochemical flux measurement of interest. This imaging method is robust, and a single nanoscale SECM electrode, which is simple to produce, is used for both topography and activity measurements. In order to minimize the delay at voltage switching, we used pyrolytic carbon nanoelectrodes with 6.5–100 nm radii that rapidly reached a steady-state current, typically in less than 20 ms for the largest electrodes and faster for smaller electrodes. In addition, these carbon nanoelectrodes are suitable for convoluted cell topography imaging because the RG value (ratio of overall probe diameter to active electrode diameter) is typically in the range of 1.5–3.0. We first evaluated the resolution of constant-current mode topography imaging using carbon nanoelectrodes. Next, we performed VSM-SECM measurements to visualize membrane proteins on A431 cells and to detect neurotransmitters from a PC12 cells. We also combined VSM-SECM with surface confocal microscopy to allow simultaneous fluorescence and topographical imaging. VSM-SECM opens up new opportunities in nanoscale chemical mapping at interfaces, and should find wide application in the physical and biological sciences. PMID:22611191

  12. Label free imaging of cell-substrate contacts by holographic total internal reflection microscopy.

    Science.gov (United States)

    Mandracchia, Biagio; Gennari, Oriella; Marchesano, Valentina; Paturzo, Melania; Ferraro, Pietro

    2017-09-01

    The study of cell adhesion contacts is pivotal to understand cell mechanics and interaction at substrates or chemical and physical stimuli. We designed and built a HoloTIR microscope for label-free quantitative phase imaging of total internal reflection. Here we show for the first time that HoloTIR is a good choice for label-free study of focal contacts and of cell/substrate interaction as its sensitivity is enhanced in comparison with standard TIR microscopy. Finally, the simplicity of implementation and relative low cost, due to the requirement of less optical components, make HoloTIR a reasonable alternative, or even an addition, to TIRF microscopy for mapping cell/substratum topography. As a proof of concept, we studied the formation of focal contacts of fibroblasts on three substrates with different levels of affinity for cell adhesion. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Evaluation of In-Vacuum Imaging Plate Detector for X-Ray Diffraction Microscopy

    International Nuclear Information System (INIS)

    Nishino, Yoshinori; Takahashi, Yukio; Yamamoto, Masaki; Ishikawa, Tetsuya

    2007-01-01

    We performed evaluation tests of a newly developed in-vacuum imaging plate (IP) detector for x-ray diffraction microscopy. IP detectors have advantages over direct x-ray detection charge-coupled device (CCD) detectors, which have been commonly used in x-ray diffraction microscopy experiments, in the capabilities for a high photon count and for a wide area. The detector system contains two IPs to make measurement efficient by recording data with the one while reading or erasing the other. We compared speckled diffraction patterns of single particles taken with the IP and a direct x-ray detection CCD. The IP was inferior to the CCD in spatial resolution and in signal-to-noise ratio at a low photon count

  14. Evaluation of atlas selection strategies for atlas-based image segmentation with application to confocal microscopy images of bee brains.

    Science.gov (United States)

    Rohlfing, Torsten; Brandt, Robert; Menzel, Randolf; Maurer, Calvin R

    2004-04-01

    This paper evaluates strategies for atlas selection in atlas-based segmentation of three-dimensional biomedical images. Segmentation by intensity-based nonrigid registration to atlas images is applied to confocal microscopy images acquired from the brains of 20 bees. This paper evaluates and compares four different approaches for atlas image selection: registration to an individual atlas image (IND), registration to an average-shape atlas image (AVG), registration to the most similar image from a database of individual atlas images (SIM), and registration to all images from a database of individual atlas images with subsequent multi-classifier decision fusion (MUL). The MUL strategy is a novel application of multi-classifier techniques, which are common in pattern recognition, to atlas-based segmentation. For each atlas selection strategy, the segmentation performance of the algorithm was quantified by the similarity index (SI) between the automatic segmentation result and a manually generated gold standard. The best segmentation accuracy was achieved using the MUL paradigm, which resulted in a mean similarity index value between manual and automatic segmentation of 0.86 (AVG, 0.84; SIM, 0.82; IND, 0.81). The superiority of the MUL strategy over the other three methods is statistically significant (two-sided paired t test, P strategies performed better than the best possible SIM and IND strategies with optimal a posteriori atlas selection (mean similarity index for optimal SIM, 0.83; for optimal IND, 0.81). Our findings show that atlas selection is an important issue in atlas-based segmentation and that, in particular, multi-classifier techniques can substantially increase the segmentation accuracy.

  15. LORENTZ PHASE IMAGING AND IN-SITU LORENTZ MICROSCOPY OF PATTERNED CO-ARRAYS

    International Nuclear Information System (INIS)

    VOLKOV, V.V.; ZHU, Y.

    2003-01-01

    Understanding magnetic structures and properties of patterned and ordinary magnetic films at nanometer length-scale is the area of immense technological and fundamental scientific importance. The key feature to such success is the ability to achieve visual quantitative information on domain configurations with a maximum ''magnetic'' resolution. Several methods have been developed to meet these demands (Kerr and Faraday effects, differential phase contrast microscopy, magnetic force microscopy, SEMPA etc.). In particular, the modern off-axis electron holography allows retrieval of the electron-wave phase shifts down to 2π/N (with typical N = 10-20, approaching in the limit N ∼ 100) in TEM equipped with field emission gun, which is already successfully employed for studies of magnetic materials at nanometer scale. However, it remains technically demanding, sensitive to noise and needs highly coherent electron sources. As possible alternative we developed a new method of Lorentz phase microscopy [1,2] based on the Fourier solution [3] of magnetic transport-of-intensity (MTIE) equation. This approach has certain advantages, since it is less sensitive to noise and does not need high coherence of the source required by the holography. In addition, it can be realized in any TEM without basic hardware changes. Our approach considers the electron-wave refraction in magnetic materials (magnetic refraction) and became possible due to general progress in understanding of noninterferometric phase retrieval [4-6] dealing with optical refraction. This approach can also be treated as further development of Fresnel microscopy, used so far for imaging of in-situ magnetization process in magnetic materials studied by TEM. Figs. 1-3 show some examples of what kind information can be retrieved from the conventional Fresnel images using the new approach. Most of these results can be compared with electron-holographic data. Using this approach we can shed more light on fine details of

  16. Single myelin fiber imaging in living rodents without labeling by deep optical coherence microscopy.

    Science.gov (United States)

    Ben Arous, Juliette; Binding, Jonas; Léger, Jean-François; Casado, Mariano; Topilko, Piotr; Gigan, Sylvain; Boccara, A Claude; Bourdieu, Laurent

    2011-11-01

    Myelin sheath disruption is responsible for multiple neuropathies in the central and peripheral nervous system. Myelin imaging has thus become an important diagnosis tool. However, in vivo imaging has been limited to either low-resolution techniques unable to resolve individual fibers or to low-penetration imaging of single fibers, which cannot provide quantitative information about large volumes of tissue, as required for diagnostic purposes. Here, we perform myelin imaging without labeling and at micron-scale resolution with >300-μm penetration depth on living rodents. This was achieved with a prototype [termed deep optical coherence microscopy (deep-OCM)] of a high-numerical aperture infrared full-field optical coherence microscope, which includes aberration correction for the compensation of refractive index mismatch and high-frame-rate interferometric measurements. We were able to measure the density of individual myelinated fibers in the rat cortex over a large volume of gray matter. In the peripheral nervous system, deep-OCM allows, after minor surgery, in situ imaging of single myelinated fibers over a large fraction of the sciatic nerve. This allows quantitative comparison of normal and Krox20 mutant mice, in which myelination in the peripheral nervous system is impaired. This opens promising perspectives for myelin chronic imaging in demyelinating diseases and for minimally invasive medical diagnosis.

  17. New Insights on Subsurface Imaging of Carbon Nanotubes in Polymer Composites via Scanning Electron Microscopy

    Science.gov (United States)

    Zhao, Minhua; Ming, Bin; Kim, Jae-Woo; Gibbons, Luke J.; Gu, Xiaohong; Nguyen, Tinh; Park, Cheol; Lillehei, Peter T.; Villarrubia, J. S.; Vladar, Andras E.; hide

    2015-01-01

    Despite many studies of subsurface imaging of carbon nanotube (CNT)-polymer composites via scanning electron microscopy (SEM), significant controversy exists concerning the imaging depth and contrast mechanisms. We studied CNT-polyimide composites and, by threedimensional reconstructions of captured stereo-pair images, determined that the maximum SEM imaging depth was typically hundreds of nanometers. The contrast mechanisms were investigated over a broad range of beam accelerating voltages from 0.3 to 30 kV, and ascribed to modulation by embedded CNTs of the effective secondary electron (SE) emission yield at the polymer surface. This modulation of the SE yield is due to non-uniform surface potential distribution resulting from current flows due to leakage and electron beam induced current. The importance of an external electric field on SEM subsurface imaging was also demonstrated. The insights gained from this study can be generally applied to SEM nondestructive subsurface imaging of conducting nanostructures embedded in dielectric matrices such as graphene-polymer composites, silicon-based single electron transistors, high resolution SEM overlay metrology or e-beam lithography, and have significant implications in nanotechnology.

  18. A New Method for Automated Identification and Morphometry of Myelinated Fibers Through Light Microscopy Image Analysis.

    Science.gov (United States)

    Novas, Romulo Bourget; Fazan, Valeria Paula Sassoli; Felipe, Joaquim Cezar

    2016-02-01

    Nerve morphometry is known to produce relevant information for the evaluation of several phenomena, such as nerve repair, regeneration, implant, transplant, aging, and different human neuropathies. Manual morphometry is laborious, tedious, time consuming, and subject to many sources of error. Therefore, in this paper, we propose a new method for the automated morphometry of myelinated fibers in cross-section light microscopy images. Images from the recurrent laryngeal nerve of adult rats and the vestibulocochlear nerve of adult guinea pigs were used herein. The proposed pipeline for fiber segmentation is based on the techniques of competitive clustering and concavity analysis. The evaluation of the proposed method for segmentation of images was done by comparing the automatic segmentation with the manual segmentation. To further evaluate the proposed method considering morphometric features extracted from the segmented images, the distributions of these features were tested for statistical significant difference. The method achieved a high overall sensitivity and very low false-positive rates per image. We detect no statistical difference between the distribution of the features extracted from the manual and the pipeline segmentations. The method presented a good overall performance, showing widespread potential in experimental and clinical settings allowing large-scale image analysis and, thus, leading to more reliable results.

  19. PathGrid: a service-orientated architecture for microscopy image analysis.

    Science.gov (United States)

    Walton, N A; Brenton, J D; Caldas, C; Irwin, M J; Akram, A; Gonzalez-Solares, E; Lewis, J R; Maccallum, P H; Morris, L J; Rixon, G T

    2010-08-28

    This paper describes 'PathGrid'--an analysis and data integration system, developed initially to meet the demands in the analysis of medical microscopy imaging data. An overview of the current system is given, describing the techniques used in developing the data handling infrastructure and the analysis algorithm development. The use of software created in the context of systems designed for the astronomy domain is noted, specifically infrastructure from the astronomy virtual observatory movement for data discovery, access and workflow management, and astronomical image analysis software adapted for the analysis of high-throughput astronomy imaging surveys. This paper notes the applicability of the techniques from the astronomy domain. The testbed infrastructure deployment is described, emphasizing its speed and ease of use and support. The validity of the analysis techniques is confirmed through the pilot study described here--with the application to a large sample of immunohistochemistry microscopy data obtained in part for assessing the oestrogen receptor status of breast cancers. The analysis showed that the specificity and sensitivity values for the automatic scoring using PathGrid were within the errors of those obtained via a 'gold standard' manual pathologist scoring.

  20. Approaches to automatic parameter fitting in a microscopy image segmentation pipeline: An exploratory parameter space analysis

    Directory of Open Access Journals (Sweden)

    Christian Held

    2013-01-01

    Full Text Available Introduction: Research and diagnosis in medicine and biology often require the assessment of a large amount of microscopy image data. Although on the one hand, digital pathology and new bioimaging technologies find their way into clinical practice and pharmaceutical research, some general methodological issues in automated image analysis are still open. Methods: In this study, we address the problem of fitting the parameters in a microscopy image segmentation pipeline. We propose to fit the parameters of the pipeline′s modules with optimization algorithms, such as, genetic algorithms or coordinate descents, and show how visual exploration of the parameter space can help to identify sub-optimal parameter settings that need to be avoided. Results: This is of significant help in the design of our automatic parameter fitting framework, which enables us to tune the pipeline for large sets of micrographs. Conclusion: The underlying parameter spaces pose a challenge for manual as well as automated parameter optimization, as the parameter spaces can show several local performance maxima. Hence, optimization strategies that are not able to jump out of local performance maxima, like the hill climbing algorithm, often result in a local maximum.

  1. New analysis procedure for fast and reliable size measurement of nanoparticles from atomic force microscopy images

    International Nuclear Information System (INIS)

    Boyd, Robert D.; Cuenat, Alexandre

    2011-01-01

    Accurate size measurement during nanoparticle production is essential for the continuing innovation, quality and safety of nano-enabled products. Size measurement by analysing a number of separate particles individually has particular advantages over ensemble methods. In the latter case nanoparticles have to be well dispersed in a fluid and changes that may occur during analysis, such as agglomeration and degradation, will not be detected which could lead to misleading results. Atomic force microscopy (AFM) allows imaging of particles both in air and liquid, however, the strong interactions between the probe and the particle will cause the broadening of the lateral dimension in the final image. In this paper a new procedure to measure the size of spherical nanoparticles from AFM images via vertical height measurement is described. This procedure will quickly analyse hundred of particles simultaneously and reproduce the measurements obtained from electron microscopy (EM). Nanoparticles samples that were difficult, if not impossible, to analyse with EM were successfully measured using this method. The combination of this procedure with the use of a metrological AFM moves closer to true traceable measurements of nanoparticle dispersions.

  2. Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging.

    Science.gov (United States)

    Abrahamsson, Sara; Ilic, Rob; Wisniewski, Jan; Mehl, Brian; Yu, Liya; Chen, Lei; Davanco, Marcelo; Oudjedi, Laura; Fiche, Jean-Bernard; Hajj, Bassam; Jin, Xin; Pulupa, Joan; Cho, Christine; Mir, Mustafa; El Beheiry, Mohamed; Darzacq, Xavier; Nollmann, Marcelo; Dahan, Maxime; Wu, Carl; Lionnet, Timothée; Liddle, J Alexander; Bargmann, Cornelia I

    2016-03-01

    Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cells nuclei to entire embryos. We here describe pattern designs and nanofabrication methods for diffractive optics that optimize the light-efficiency of the central optical component of MFM: the diffractive multifocus grating (MFG). We also implement a "precise color" MFM layout with MFGs tailored to individual fluorophores in separate optical arms. The reported advancements enable faster and brighter volumetric time-lapse imaging of biological samples. In live microscopy applications, photon budget is a critical parameter and light-efficiency must be optimized to obtain the fastest possible frame rate while minimizing photodamage. We provide comprehensive descriptions and code for designing diffractive optical devices, and a detailed methods description for nanofabrication of devices. Theoretical efficiencies of reported designs is ≈90% and we have obtained efficiencies of > 80% in MFGs of our own manufacture. We demonstrate the performance of a multi-phase MFG in 3D functional neuronal imaging in living C. elegans.

  3. Systems and methods for selective detection and imaging in coherent Raman microscopy by spectral excitation shaping

    Science.gov (United States)

    Xie, Xiaoliang Sunney; Freudiger, Christian; Min, Wei

    2016-03-15

    A microscopy imaging system is disclosed that includes a light source system, a spectral shaper, a modulator system, an optics system, an optical detector and a processor. The light source system is for providing a first train of pulses and a second train of pulses. The spectral shaper is for spectrally modifying an optical property of at least some frequency components of the broadband range of frequency components such that the broadband range of frequency components is shaped producing a shaped first train of pulses to specifically probe a spectral feature of interest from a sample, and to reduce information from features that are not of interest from the sample. The modulator system is for modulating a property of at least one of the shaped first train of pulses and the second train of pulses at a modulation frequency. The optical detector is for detecting an integrated intensity of substantially all optical frequency components of a train of pulses of interest transmitted or reflected through the common focal volume. The processor is for detecting a modulation at the modulation frequency of the integrated intensity of substantially all of the optical frequency components of the train of pulses of interest due to the non-linear interaction of the shaped first train of pulses with the second train of pulses as modulated in the common focal volume, and for providing an output signal for a pixel of an image for the microscopy imaging system.

  4. Multispectral confocal microscopy images and artificial neural nets to monitor the photosensitizer uptake and degradation in Candida albicans cells

    Science.gov (United States)

    Romano, Renan A.; Pratavieira, Sebastião.; da Silva, Ana P.; Kurachi, Cristina; Guimarães, Francisco E. G.

    2017-07-01

    This study clearly demonstrates that multispectral confocal microscopy images analyzed by artificial neural networks provides a powerful tool to real-time monitoring photosensitizer uptake, as well as photochemical transformations occurred.

  5. Real-time Image Processing for Microscopy-based Label-free Imaging Flow Cytometry in a Microfluidic Chip.

    Science.gov (United States)

    Heo, Young Jin; Lee, Donghyeon; Kang, Junsu; Lee, Keondo; Chung, Wan Kyun

    2017-09-14

    Imaging flow cytometry (IFC) is an emerging technology that acquires single-cell images at high-throughput for analysis of a cell population. Rich information that comes from high sensitivity and spatial resolution of a single-cell microscopic image is beneficial for single-cell analysis in various biological applications. In this paper, we present a fast image-processing pipeline (R-MOD: Real-time Moving Object Detector) based on deep learning for high-throughput microscopy-based label-free IFC in a microfluidic chip. The R-MOD pipeline acquires all single-cell images of cells in flow, and identifies the acquired images as a real-time process with minimum hardware that consists of a microscope and a high-speed camera. Experiments show that R-MOD has the fast and reliable accuracy (500 fps and 93.3% mAP), and is expected to be used as a powerful tool for biomedical and clinical applications.

  6. A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images

    Science.gov (United States)

    Afshar, Yaser; Sbalzarini, Ivo F.

    2016-01-01

    Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 1010 pixels), but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments. PMID:27046144

  7. A workflow for the automatic segmentation of organelles in electron microscopy image stacks

    Directory of Open Access Journals (Sweden)

    Alex Joseph Perez

    2014-11-01

    Full Text Available Electron microscopy (EM facilitates analysis of the form, distribution, and functional status of key organelle systems in various pathological processes, including those associated with neurodegenerative disease. Such EM data often provide important new insights into the underlying disease mechanisms. The development of more accurate and efficient methods to quantify changes in subcellular microanatomy has already proven key to understanding the pathogenesis of Parkinson’s and Alzheimer’s diseases, as well as glaucoma. While our ability to acquire large volumes of 3D EM data is progressing rapidly, more advanced analysis tools are needed to assist in measuring precise three-dimensional morphologies of organelles within data sets that can include hundreds to thousands of whole cells. Although new imaging instrument throughputs can exceed teravoxels of data per day, image segmentation and analysis remain significant bottlenecks to achieving quantitative descriptions of whole cell structural organellomes. Here, we present a novel method for the automatic segmentation of organelles in 3D EM image stacks. Segmentations are generated using only 2D image information, making the method suitable for anisotropic imaging techniques such as serial block-face scanning electron microscopy (SBEM. Additionally, no assumptions about 3D organelle morphology are made, ensuring the method can be easily expanded to any number of structurally and functionally diverse organelles. Following the presentation of our algorithm, we validate its performance by assessing the segmentation accuracy of different organelle targets in an example SBEM dataset and demonstrate that it can be efficiently parallelized on supercomputing resources, resulting in a dramatic reduction in runtime.

  8. Image processing of transmission ion microscopy webcam video with Iris software

    Science.gov (United States)

    Newton, Nicole; Pallone, Arthur

    2011-03-01

    Scientists and engineers build simple, low-cost, webcam-based instruments for use in many disciplines. Analysis of the optical signal received through the three broadband color filters -- red, green and blue -- form the basis of many of those instruments. The CMOS sensors in webcam pixels also produce signals in response to ionizing radiations -- such as alpha particles from a radioactive source. Simple alpha radiography has been demonstrated with a commercially available antistatic Po-210 source and a webcam modified to expose the pixel sensors. Analysis of webcam video with the Iris software is presented as a way to demonstrate other transmission ion microscopy imaging modes.

  9. Time-resolved imaging refractometry of microbicidal films using quantitative phase microscopy.

    Science.gov (United States)

    Rinehart, Matthew T; Drake, Tyler K; Robles, Francisco E; Rohan, Lisa C; Katz, David; Wax, Adam

    2011-12-01

    Quantitative phase microscopy is applied to image temporal changes in the refractive index (RI) distributions of solutions created by microbicidal films undergoing hydration. We present a novel method of using an engineered polydimethylsiloxane structure as a static phase reference to facilitate calibration of the absolute RI across the entire field. We present a study of dynamic structural changes in microbicidal films during hydration and subsequent dissolution. With assumptions about the smoothness of the phase changes induced by these films, we calculate absolute changes in the percentage of film in regions across the field of view.

  10. Microstructural orientation of isotactic polypropylene studied by computerized scanning eletron microscopy image analysis

    Directory of Open Access Journals (Sweden)

    Machado Giovanna

    2001-01-01

    Full Text Available We evaluate the orientation of microstructural elements of isotatic polypropylene (i-PP before and after deformation using computerized "Quantikov" software analysis of Scanning Electron Microscopy (SEM images. We observed that before deformation through uniaxial compression, the polymeric material doesn?t exhibit any significant orientation. After deformation at 1349 MPa the material clearly showed preferential orientation that was attested by the orientation axis seen between two petals of the rose of the number of intercepts. This effect was more pronounced after deformation at 2699 MPa.

  11. Optimized cobalt nanowires for domain wall manipulation imaged by in situ Lorentz microscopy

    International Nuclear Information System (INIS)

    Rodríguez, L. A.; Magén, C.; Snoeck, E.; Gatel, C.; Serrano-Ramón, L.

    2013-01-01

    Direct observation of domain wall (DW) nucleation and propagation in focused electron beam induced deposited Co nanowires as a function of their dimensions was carried out by Lorentz microscopy (LTEM) upon in situ application of magnetic field. Optimal dimensions favoring the unambiguous DW nucleation/propagation required for applications were found in 500-nm-wide and 13-nm-thick Co nanowires, with a maximum nucleation field and the largest gap between nucleation and propagation fields. The internal DW structures were resolved using the transport-of-intensity equation formalism in LTEM images and showed that the optimal nanowire dimensions correspond to the crossover between the nucleation of transverse and vortex walls.

  12. Wide-field computational imaging of pathology slides using lens-free on-chip microscopy.

    Science.gov (United States)

    Greenbaum, Alon; Zhang, Yibo; Feizi, Alborz; Chung, Ping-Luen; Luo, Wei; Kandukuri, Shivani R; Ozcan, Aydogan

    2014-12-17

    Optical examination of microscale features in pathology slides is one of the gold standards to diagnose disease. However, the use of conventional light microscopes is partially limited owing to their relatively high cost, bulkiness of lens-based optics, small field of view (FOV), and requirements for lateral scanning and three-dimensional (3D) focus adjustment. We illustrate the performance of a computational lens-free, holographic on-chip microscope that uses the transport-of-intensity equation, multi-height iterative phase retrieval, and rotational field transformations to perform wide-FOV imaging of pathology samples with comparable image quality to a traditional transmission lens-based microscope. The holographically reconstructed image can be digitally focused at any depth within the object FOV (after image capture) without the need for mechanical focus adjustment and is also digitally corrected for artifacts arising from uncontrolled tilting and height variations between the sample and sensor planes. Using this lens-free on-chip microscope, we successfully imaged invasive carcinoma cells within human breast sections, Papanicolaou smears revealing a high-grade squamous intraepithelial lesion, and sickle cell anemia blood smears over a FOV of 20.5 mm(2). The resulting wide-field lens-free images had sufficient image resolution and contrast for clinical evaluation, as demonstrated by a pathologist's blinded diagnosis of breast cancer tissue samples, achieving an overall accuracy of ~99%. By providing high-resolution images of large-area pathology samples with 3D digital focus adjustment, lens-free on-chip microscopy can be useful in resource-limited and point-of-care settings. Copyright © 2014, American Association for the Advancement of Science.

  13. Remote microscopy and volumetric imaging on the surface of icy satellites

    Science.gov (United States)

    Soto, Alejandro; Nowicki, Keith; Howett, Carly; Feldkhun, Daniel; Retherford, Kurt D.

    2017-10-01

    With NASA PIDDP support we have applied recent advancements in Fourier-domain microscopy to develop an instrument capable of microscopic imaging from meter-scale distances for use on a planetary lander on the surface of an icy satellite or other planetary bodies. Without moving parts, our instrument projects dynamic patterns of laser light onto a distant target using a lightweight large-aperture reflector, which then collects the light scattered or fluoresced by the target on a fast photon-bucket detector. Using Fourier Transform based techniques, we reconstruct an image from the detected light. The remote microscope has been demonstrated to produce 2D images with better than 15 micron lateral resolution for targets at a distance of 5 meters and is capable of linearly proportionally higher resolution at shorter distances. The remote microscope is also capable of providing three-dimensional (3D) microscopic imaging capabilities, allowing future surface scientists to explore the morphology of microscopic features in surface ices, for example. The instrument enables microscopic in-situ imaging during day or night without the use of a robotic arm, greatly facilitating the surface operations for a lander or rover while expanding the area of investigation near a landing site for improved science targeting. We are developing this remote microscope for in-situ planetary exploration as a collaboration between the Southwest Research Institute, LambdaMetrics, and the University of Colorado.

  14. Virus Particle Detection by Convolutional Neural Network in Transmission Electron Microscopy Images.

    Science.gov (United States)

    Ito, Eisuke; Sato, Takaaki; Sano, Daisuke; Utagawa, Etsuko; Kato, Tsuyoshi

    2018-01-19

    A new computational method for the detection of virus particles in transmission electron microscopy (TEM) images is presented. Our approach is to use a convolutional neural network that transforms a TEM image to a probabilistic map that indicates where virus particles exist in the image. Our proposed approach automatically and simultaneously learns both discriminative features and classifier for virus particle detection by machine learning, in contrast to existing methods that are based on handcrafted features that yield many false positives and require several postprocessing steps. The detection performance of the proposed method was assessed against a dataset of TEM images containing feline calicivirus particles and compared with several existing detection methods, and the state-of-the-art performance of the developed method for detecting virus was demonstrated. Since our method is based on supervised learning that requires both the input images and their corresponding annotations, it is basically used for detection of already-known viruses. However, the method is highly flexible, and the convolutional networks can adapt themselves to any virus particles by learning automatically from an annotated dataset.

  15. Joint denoising and distortion correction of atomic scale scanning transmission electron microscopy images

    Science.gov (United States)

    Berkels, Benjamin; Wirth, Benedikt

    2017-09-01

    Nowadays, modern electron microscopes deliver images at atomic scale. The precise atomic structure encodes information about material properties. Thus, an important ingredient in the image analysis is to locate the centers of the atoms shown in micrographs as precisely as possible. Here, we consider scanning transmission electron microscopy (STEM), which acquires data in a rastering pattern, pixel by pixel. Due to this rastering combined with the magnification to atomic scale, movements of the specimen even at the nanometer scale lead to random image distortions that make precise atom localization difficult. Given a series of STEM images, we derive a Bayesian method that jointly estimates the distortion in each image and reconstructs the underlying atomic grid of the material by fitting the atom bumps with suitable bump functions. The resulting highly non-convex minimization problems are solved numerically with a trust region approach. Existence of minimizers and the model behavior for faster and faster rastering are investigated using variational techniques. The performance of the method is finally evaluated on both synthetic and real experimental data.

  16. Clean localization super-resolution microscopy for 3D biological imaging

    Energy Technology Data Exchange (ETDEWEB)

    Mondal, Partha P., E-mail: partha@iap.iisc.ernet.in [Nanobioimaging Laboratory, Department of Instrumentation and Applied Physics, Indian Institute of Science, Bangalore 560012 (India); Curthoys, Nikki M.; Hess, Samuel T. [Department of Physics and Astronomy, University of Maine, Orono, Maine 04469 (United States)

    2016-01-15

    We propose clean localization microscopy (a variant of fPALM) using a molecule filtering technique. Localization imaging involves acquiring a large number of images containing single molecule signatures followed by one-to-one mapping to render a super-resolution image. In principle, this process can be repeated for other z-planes to construct a 3D image. But, single molecules observed from off-focal planes result in false representation of their presence in the focal plane, resulting in incorrect quantification and analysis. We overcome this with a single molecule filtering technique that imposes constraints on the diffraction limited spot size of single molecules in the image plane. Calibration with sub-diffraction size beads puts a natural cutoff on the actual diffraction-limited size of single molecules in the focal plane. This helps in distinguishing beads present in the focal plane from those in the off-focal planes thereby providing an estimate of the single molecules in the focal plane. We study the distribution of actin (labeled with a photoactivatable CAGE 552 dye) in NIH 3T3 mouse fibroblast cells.

  17. Automated detection of synapses in serial section transmission electron microscopy image stacks.

    Directory of Open Access Journals (Sweden)

    Anna Kreshuk

    Full Text Available We describe a method for fully automated detection of chemical synapses in serial electron microscopy images with highly anisotropic axial and lateral resolution, such as images taken on transmission electron microscopes. Our pipeline starts from classification of the pixels based on 3D pixel features, which is followed by segmentation with an Ising model MRF and another classification step, based on object-level features. Classifiers are learned on sparse user labels; a fully annotated data subvolume is not required for training. The algorithm was validated on a set of 238 synapses in 20 serial 7197×7351 pixel images (4.5×4.5×45 nm resolution of mouse visual cortex, manually labeled by three independent human annotators and additionally re-verified by an expert neuroscientist. The error rate of the algorithm (12% false negative, 7% false positive detections is better than state-of-the-art, even though, unlike the state-of-the-art method, our algorithm does not require a prior segmentation of the image volume into cells. The software is based on the ilastik learning and segmentation toolkit and the vigra image processing library and is freely available on our website, along with the test data and gold standard annotations (http://www.ilastik.org/synapse-detection/sstem.

  18. Hyperspectral and differential CARS microscopy for quantitative chemical imaging in human adipocytes.

    Science.gov (United States)

    Di Napoli, Claudia; Pope, Iestyn; Masia, Francesco; Watson, Peter; Langbein, Wolfgang; Borri, Paola

    2014-05-01

    In this work, we demonstrate the applicability of coherent anti-Stokes Raman scattering (CARS) micro-spectroscopy for quantitative chemical imaging of saturated and unsaturated lipids in human stem-cell derived adipocytes. We compare dual-frequency/differential CARS (D-CARS), which enables rapid imaging and simple data analysis, with broadband hyperspectral CARS microscopy analyzed using an unsupervised phase-retrieval and factorization method recently developed by us for quantitative chemical image analysis. Measurements were taken in the vibrational fingerprint region (1200-2000/cm) and in the CH stretch region (2600-3300/cm) using a home-built CARS set-up which enables hyperspectral imaging with 10/cm resolution via spectral focussing from a single broadband 5 fs Ti:Sa laser source. Through a ratiometric analysis, both D-CARS and phase-retrieved hyperspectral CARS determine the concentration of unsaturated lipids with comparable accuracy in the fingerprint region, while in the CH stretch region D-CARS provides only a qualitative contrast owing to its non-linear behavior. When analyzing hyperspectral CARS images using the blind factorization into susceptibilities and concentrations of chemical components recently demonstrated by us, we are able to determine vol:vol concentrations of different lipid components and spatially resolve inhomogeneities in lipid composition with superior accuracy compared to state-of-the art ratiometric methods.

  19. Hyperspectral and differential CARS microscopy for quantitative chemical imaging in human adipocytes

    Science.gov (United States)

    Di Napoli, Claudia; Pope, Iestyn; Masia, Francesco; Watson, Peter; Langbein, Wolfgang; Borri, Paola

    2014-01-01

    In this work, we demonstrate the applicability of coherent anti-Stokes Raman scattering (CARS) micro-spectroscopy for quantitative chemical imaging of saturated and unsaturated lipids in human stem-cell derived adipocytes. We compare dual-frequency/differential CARS (D-CARS), which enables rapid imaging and simple data analysis, with broadband hyperspectral CARS microscopy analyzed using an unsupervised phase-retrieval and factorization method recently developed by us for quantitative chemical image analysis. Measurements were taken in the vibrational fingerprint region (1200–2000/cm) and in the CH stretch region (2600–3300/cm) using a home-built CARS set-up which enables hyperspectral imaging with 10/cm resolution via spectral focussing from a single broadband 5 fs Ti:Sa laser source. Through a ratiometric analysis, both D-CARS and phase-retrieved hyperspectral CARS determine the concentration of unsaturated lipids with comparable accuracy in the fingerprint region, while in the CH stretch region D-CARS provides only a qualitative contrast owing to its non-linear behavior. When analyzing hyperspectral CARS images using the blind factorization into susceptibilities and concentrations of chemical components recently demonstrated by us, we are able to determine vol:vol concentrations of different lipid components and spatially resolve inhomogeneities in lipid composition with superior accuracy compared to state-of-the art ratiometric methods. PMID:24877002

  20. Functional differentiation of spider hemocytes by light and transmission electron microscopy, and MALDI-MS-imaging.

    Science.gov (United States)

    Kuhn-Nentwig, Lucia; Kopp, Lukas S; Nentwig, Wolfgang; Haenni, Beat; Streitberger, Kathrin; Schürch, Stefan; Schaller, Johann

    2014-03-01

    The most abundant cell types in the hemolymph of Cupiennius salei are plasmatocytes (70-80%) and granulocytes (20-30%). Both cells differ in shape, cytochemical and transmission electron microscopy staining of their cytoplasma and granules. According to MALDI-IMS (matrix-assisted laser desorption ionisation-mass spectrometry imaging), granulocytes exhibit ctenidin 1 (9510 Da) and ctenidin 3 (9568 Da), SIBD-1 (8675 Da), and unknown peptides with masses of 2207 and 6239 Da. Plasmatocytes exhibit mainly a mass of 6908 Da. Unknown peptides with masses of 1546 and 1960 Da were detected in plasmatocytes and granulocytes. Transmission electron microscopy confirms the presence of two compounds in one granule and cytochemical staining (light microscopy) tends to support this view. Two further hemocyte types (cyanocytes containing hemocyanin and prehemocytes as stem cells) are only rarely detected in the hemolymph. These four hemocyte types constitute the cellular part of the spider immune system and this is discussed in view of arachnid hemocyte evolution. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Structured illumination microscopy and automatized image processing as a rapid diagnostic tool for podocyte effacement.

    Science.gov (United States)

    Siegerist, Florian; Ribback, Silvia; Dombrowski, Frank; Amann, Kerstin; Zimmermann, Uwe; Endlich, Karlhans; Endlich, Nicole

    2017-09-13

    The morphology of podocyte foot processes is obligatory for renal function. Here we describe a method for the superresolution-visualization of podocyte foot processes using structured illumination microscopy of the slit diaphragm, which before has only been achieved by electron microscopy. As a proof of principle, we measured a mean foot process width of 0.249 ± 0.068 µm in healthy kidneys and a significant higher mean foot process width of 0.675 ± 0.256 µm in minimal change disease patients indicating effacement of foot processes. We then hypothesized that the slit length per glomerular capillary surface area (slit diaphragm density) could be used as an equivalent for the diagnosis of effacement. Using custom-made software we measured a mean value of 3.10 ± 0.27 µm -1 in healthy subjects and 1.83 ± 0.49 µm -1 in the minimal change disease patients. As foot process width was highly correlated with slit diaphragm density (R 2  = 0.91), we concluded that our approach is a valid method for the diagnosis of foot process effacement. In summary, we present a new technique to quantify podocyte damage, which combines superresolution microscopy with automatized image processing. Due to its diverse advantages, we propose this technique to be included into routine diagnostics of glomerular histopathology.

  2. Evaluation of the metastatic potential of malignant cells by image processing of digital holographic microscopy data.

    Science.gov (United States)

    Calin, Violeta L; Mihailescu, Mona; Scarlat, Eugen I; Baluta, Alexandra V; Calin, Daniel; Kovacs, Eugenia; Savopol, Tudor; Moisescu, Mihaela G

    2017-10-01

    The cell refractive index has been proposed as a putative cancer biomarker of great potential, being correlated with cell content and morphology, cell division rate and membrane permeability. We used digital holographic microscopy to compare the refractive index and dry mass density of two B16 murine melanoma sublines of different metastatic potential. Using statistical methods, the distribution of phase shifts within the reconstructed quantitative phase images was analyzed by the method of bimodality coefficients. The observed correlation of refractive index, dry mass density and bimodality profile with the metastatic potential of the cells was validated by real time impedance-based assay and clonogenic tests. We suggest that the refractive index and bimodality analysis of quantitative phase image histograms could be developed as optical biomarkers useful in label-free detection and quantitative evaluation of cell metastatic potential.

  3. A Novel Intravital Imaging Window for Longitudinal Microscopy of the Mouse Ovary

    Science.gov (United States)

    Bochner, Filip; Fellus-Alyagor, Liat; Kalchenko, Vyacheslav; Shinar, Shiri; Neeman, Michal

    2015-01-01

    The ovary is a dynamic organ that undergoes dramatic remodeling throughout the ovulatory cycle. Maturation of the ovarian follicle, release of the oocyte in the course of ovulation as well as formation and degradation of corpus luteum involve tightly controlled remodeling of the extracellular matrix and vasculature. Ovarian tumors, regardless of their tissue of origin, dynamically interact with the ovarian microenvironment. Their activity in the tissue encompasses recruitment of host stroma and immune cells, attachment of tumor cells to mesothelial layer, degradation of the extracellular matrix and tumor cell migration. High-resolution dynamic imaging of such processes is particularly challenging for internal organs. The implementation of a novel imaging window as reported here enabled longitudinal microscopy of ovarian physiology and orthotopic tumor invasion. PMID:26207832

  4. Identification of antibiotic mycelia residue in protein rich feed using on near-infrared microscopy imaging.

    Science.gov (United States)

    Lin, Yufei; Yang, Zengling; Liang, Hao; Li, Shouxue; Fan, Xia; Xiao, Zhiming

    2018-03-12

    Antibiotic mycelial residues (AMRs) added to animal feeds easily lead to drug resistance that affects human health and environment. However, there is a lack of effective detection methods, especially a fast and convenient detection technology, to distinguish AMRs from other components in animal feeds. To develop effective detection methods, two types of global Mahalanobis distance (GH) algorithms based on near-infrared microscopy (NIRM) imaging are proposed. The aim of this study is to investigate the feasibility of using NIRM imaging to identify AMRs in soybean meals. We prepared 15 mixed samples containing 5% AMRs using three types of soybean meals and four types of AMRs. The GH algorithm was used to identify non-soybean meals among the mixed samples. The hierarchical cluster analysis was employed to verify the recognition accuracy. The results indicate that use of the GH algorithm could identify soybean meals with AMR at a level as low as 5%.

  5. Femtosecond few- to single-electron point-projection microscopy for nanoscale dynamic imaging

    Science.gov (United States)

    Bainbridge, A. R.; Barlow Myers, C. W.; Bryan, W. A.

    2016-01-01

    Femtosecond electron microscopy produces real-space images of matter in a series of ultrafast snapshots. Pulses of electrons self-disperse under space-charge broadening, so without compression, the ideal operation mode is a single electron per pulse. Here, we demonstrate femtosecond single-electron point projection microscopy (fs-ePPM) in a laser-pump fs-e-probe configuration. The electrons have an energy of only 150 eV and take tens of picoseconds to propagate to the object under study. Nonetheless, we achieve a temporal resolution with a standard deviation of 114 fs (equivalent to a full-width at half-maximum of 269 ± 40 fs) combined with a spatial resolution of 100 nm, applied to a localized region of charge at the apex of a nanoscale metal tip induced by 30 fs 800 nm laser pulses at 50 kHz. These observations demonstrate real-space imaging of reversible processes, such as tracking charge distributions, is feasible whilst maintaining femtosecond resolution. Our findings could find application as a characterization method, which, depending on geometry, could resolve tens of femtoseconds and tens of nanometres. Dynamically imaging electric and magnetic fields and charge distributions on sub-micron length scales opens new avenues of ultrafast dynamics. Furthermore, through the use of active compression, such pulses are an ideal seed for few-femtosecond to attosecond imaging applications which will access sub-optical cycle processes in nanoplasmonics. PMID:27158637

  6. Femtosecond few- to single-electron point-projection microscopy for nanoscale dynamic imaging

    Directory of Open Access Journals (Sweden)

    A. R. Bainbridge

    2016-03-01

    Full Text Available Femtosecond electron microscopy produces real-space images of matter in a series of ultrafast snapshots. Pulses of electrons self-disperse under space-charge broadening, so without compression, the ideal operation mode is a single electron per pulse. Here, we demonstrate femtosecond single-electron point projection microscopy (fs-ePPM in a laser-pump fs-e-probe configuration. The electrons have an energy of only 150 eV and take tens of picoseconds to propagate to the object under study. Nonetheless, we achieve a temporal resolution with a standard deviation of 114 fs (equivalent to a full-width at half-maximum of 269 ± 40 fs combined with a spatial resolution of 100 nm, applied to a localized region of charge at the apex of a nanoscale metal tip induced by 30 fs 800 nm laser pulses at 50 kHz. These observations demonstrate real-space imaging of reversible processes, such as tracking charge distributions, is feasible whilst maintaining femtosecond resolution. Our findings could find application as a characterization method, which, depending on geometry, could resolve tens of femtoseconds and tens of nanometres. Dynamically imaging electric and magnetic fields and charge distributions on sub-micron length scales opens new avenues of ultrafast dynamics. Furthermore, through the use of active compression, such pulses are an ideal seed for few-femtosecond to attosecond imaging applications which will access sub-optical cycle processes in nanoplasmonics.

  7. Quantitative Imaging of Cell Membrane-associated Effective Mass Density Using Photonic Crystal Enhanced Microscopy (PCEM)

    Science.gov (United States)

    Zhuo, Yue; Choi, Ji Sun; Marin, Thibault; Yu, Hojeong; Harley, Brendan A.; Cunningham, Brian T.

    2017-01-01

    Adhesion is a critical cellular process that contributes to migration, apoptosis, differentiation, and division. It is followed by the redistribution of cellular materials at the cell membrane or at the cell-surface interface for cells interacting with surfaces, such as basement membranes. Dynamic and quantitative tracking of changes in cell adhesion mass redistribution is challenging because cells are rapidly moving, inhomogeneous, and nonequilibrium objects, whose physical and mechanical properties are difficult to measure or predict. Here, we report a novel biosensor based microscopy approach termed Photonic Crystal Enhanced Microscopy (PCEM) that enables the movement of cellular materials at the plasma membrane of individual live cells to be dynamically monitored and quantitatively imaged. PCEM utilizes a photonic crystal biosensor surface, which can be coated with arbitrary extracellular matrix materials to facilitate cellular interactions, within a modified brightfield microscope with a low intensity non-coherent light source. Benefiting from the high sensitivity, narrow resonance peak, and tight spatial confinement of the evanescent field atop the photonic crystal biosensor, PCEM enables label-free live cell imaging with high sensitivity and high lateral and axial spatial-resolution, thereby allowing dynamic adhesion phenotyping of single cells without the use of fluorescent tags or stains. We apply PCEM to investigate adhesion and the early stage migration of different types of stem cells and cancer cells. By applying image processing algorithms to analyze the complex spatiotemporal information generated by PCEM, we offer insight into how the plasma membrane of anchorage dependent cells is dynamically organized during cell adhesion. The imaging and analysis results presented here provide a new tool for biologists to gain a deeper understanding of the fundamental mechanisms involved with cell adhesion and concurrent or subsequent migration events. PMID

  8. Diagnosis of basal cell carcinoma by two photon excited fluorescence combined with lifetime imaging

    Science.gov (United States)

    Fan, Shunping; Peng, Xiao; Liu, Lixin; Liu, Shaoxiong; Lu, Yuan; Qu, Junle

    2014-02-01

    Basal cell carcinoma (BCC) is the most common type of human skin cancer. The traditional diagnostic procedure of BCC is histological examination with haematoxylin and eosin staining of the tissue biopsy. In order to reduce complexity of the diagnosis procedure, a number of noninvasive optical methods have been applied in skin examination, for example, multiphoton tomography (MPT) and fluorescence lifetime imaging microscopy (FLIM). In this study, we explored two-photon optical tomography of human skin specimens using two-photon excited autofluorescence imaging and FLIM. There are a number of naturally endogenous fluorophores in skin sample, such as keratin, melanin, collagen, elastin, flavin and porphyrin. Confocal microscopy was used to obtain structures of the sample. Properties of epidermic and cancer cells were characterized by fluorescence emission spectra, as well as fluorescence lifetime imaging. Our results show that two-photon autofluorescence lifetime imaging can provide accurate optical biopsies with subcellular resolution and is potentially a quantitative optical diagnostic method in skin cancer diagnosis.

  9. From the physics of secondary electron emission to image contrasts in scanning electron microscopy.

    Science.gov (United States)

    Cazaux, Jacques

    2012-01-01

    Image formation in scanning electron microscopy (SEM) is a combination of physical processes, electron emissions from the sample, and of a technical process related to the detection of a fraction of these electrons. For the present survey of image contrasts in SEM, simplified considerations in the physics of the secondary electron emission yield, δ, are combined with the effects of a partial collection of the emitted secondary electrons. Although some consideration is initially given to the architecture of modern SEM, the main attention is devoted to the material contrasts with the respective roles of the sub-surface and surface compositions of the sample, as well as with the roles of the field effects in the vacuum gap. The recent trends of energy filtering in normal SEM and the reduction of the incident energy to a few electron volts in very low-energy electron microscopy are also considered. For an understanding by the SEM community, the mathematical expressions are explained with simple physical arguments.

  10. Nanoscale imaging of the Candida-macrophage interaction using correlated fluorescence-atomic force microscopy.

    Science.gov (United States)

    El-Kirat-Chatel, Sofiane; Dufrêne, Yves F

    2012-12-21

    Knowledge of the molecular bases underlying the interaction of fungal pathogens with immune cells is critical to our understanding of fungal infections and offers exciting perspectives for controlling immune responses for therapy. Although fluorescence microscopy is a valuable tool to visualize pathogen-host interactions, the spatial resolution is low, meaning the fine structural details of the interacting cells cannot be observed. Here, we demonstrate the ability of correlated fluorescence-atomic force microscopy (AFM) to image the various steps of the interaction between fungal pathogens and macrophages with nanoscale resolution. We focus on Candida albicans, known to grow as two morphological forms (yeast cells, filamentous hyphae) that play important roles in modulating the interaction with macrophages. We observe the main steps of macrophage infection, including initial intercellular contact, phagocytosis by internalization of yeast cells, intracellular hyphal growth leading to mechanical stretching, and piercing of the macrophage membrane resulting in pathogen escape. While fluorescence imaging clearly distinguishes fungal cells from macrophages during the various steps of the process, AFM captures nanoscale structural features of the macrophage surface that are of high biological relevance, including ruffles, lamellipodia, filopodia, membrane remnants, and phagocytic cups. As fungal pathogenesis is mainly controlled by the ability of fungi to escape from immune cells, the nanoimaging platform established here has great potential in nanomedicine for understanding and controlling fungal infections.

  11. Structural analysis of nano structured carbon by transmission electron microscopy and image processing

    Energy Technology Data Exchange (ETDEWEB)

    Oshida, K., E-mail: oshida@nagano-nct.ac.jp [Nagano National College of Technology, Nagano 381-8550 (Japan); Murata, M.; Fujiwara, K.; Itaya, T. [Nagano National College of Technology, Nagano 381-8550 (Japan); Yanagisawa, T.; Kimura, K. [GSI Creos Corporation, Kanagawa 210-0855 (Japan); Nakazawa, T.; Kim, Y.A.; Endo, M. [Shinshu University, Nagano 380-8553 (Japan); Kim, B.-H.; Yang, K.S. [Chonnam National University, Gwangju 500-757 (Korea, Republic of)

    2013-06-15

    Transmission electron microscopy (TEM) is one of the highest resolution analysis methods of materials. The three dimensional recognition of the materials is difficult by TEM because the observation data is projection images through the materials. In this study, space structure of carbon nanotubes loaded with metal particles was analyzed by three dimensional TEM (3D-TEM) [1,2]. The nano structured carbons are also observed by high resolution transmission electron microscopy (HRTEM) with Cs corrector. Cup-stack type carbon nanotubes (CSCNTs) loaded with Pt particles (2–3 nm in diameter) prepared by GSI Creos Corporation were analyzed by these methods. Pt particles are bound selectively to the edges of hexagonal carbon layers of inside and outer surface of CSCNTs efficiently and can be expected to work well as catalysts of electrodes of fuel cell. It is sometimes difficult that the nano sized area is analyzed by selected area electron diffraction (SAD) because the selected area aperture cannot be so small. The HRTEM and image processing technique give similar results of SAD when it works and revealed to be useful to analyze nano structured carbons.

  12. 4D Lorentz electron microscopy imaging: magnetic domain wall nucleation, reversal, and wave velocity.

    Science.gov (United States)

    Park, Hyun Soon; Baskin, J Spencer; Zewail, Ahmed H

    2010-09-08

    Magnetization reversal is an important topic of research in the fields of both basic and applied ferromagnetism. For the study of magnetization reversal dynamics and magnetic domain wall (DW) motion in ferromagnetic thin films, imaging techniques are indispensable. Here, we report 4D imaging of DWs by the out-of-focus Fresnel method in Lorentz ultrafast electron microscopy (UEM), with in situ spatial and temporal resolutions. The temporal change in magnetization, as revealed by changes in image contrast, is clocked using an impulsive optical field to produce structural deformation of the specimen, thus modulating magnetic field components in the specimen plane. Directly visualized are DW nucleation and subsequent annihilation and oscillatory reappearance (periods of 32 and 45 ns) in nickel films on two different substrates. For the case of Ni films on a Ti/Si(3)N(4) substrate, under conditions of minimum residual external magnetic field, the oscillation is associated with a unique traveling wave train of periodic magnetization reversal. The velocity of DW propagation in this wave train is measured to be 172 m/s with a wavelength of 7.8 microm. The success of this study demonstrates the promise of Lorentz UEM for real-space imaging of spin switching, ferromagnetic resonance, and laser-induced demagnetization in ferromagnetic nanostructures.

  13. Live endothelial cells imaged by Scanning Near-field Optical Microscopy (SNOM): capabilities and challenges.

    Science.gov (United States)

    Bulat, Katarzyna; Rygula, Anna; Szafraniec, Ewelina; Ozaki, Yukihiro; Baranska, Malgorzata

    2017-06-01

    The scanning near-field optical microscopy (SNOM) shows a potential to study details of biological samples, since it provides the optical images of objects with nanometric spatial resolution (50-200 nm) and the topographic information at the same time. The goal of this work is to demonstrate the capabilities of SNOM in transmission configuration to study human endothelial cells and their morphological changes, sometimes very subtle, upon inflammation. Various sample preparations were tested for SNOM measurements and promising results are collected to show: 1) the influence of α tumor necrosis factor (TNF-α) on EA.hy 926 cells (measurements of the fixed cells); 2) high resolution images of various endothelial cell lines, i.e. EA.hy 926 and HLMVEC (investigations of the fixed cells in buffer environment); 3) imaging of live endothelial cells in physiological buffers. The study demonstrate complementarity of the SNOM measurements performed in air and in liquid environments, on fixed as well as on living cells. Furthermore, it is proved that the SNOM is a very useful method for analysis of cellular morphology and topography. Changes in the cell shape and nucleus size, which are the symptoms of inflammatory reaction, were noticed in TNF-α activated EA.hy 926 cells. The cellular structures of submicron size were observed in high resolution optical images of cells from EA.hy 926 and HLMVEC lines. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. High throughput imaging of blood smears using white light diffraction phase microscopy

    Science.gov (United States)

    Majeed, Hassaan; Kandel, Mikhail E.; Bhaduri, Basanta; Han, Kevin; Luo, Zelun; Tangella, Krishnarao; Popescu, Gabriel

    2015-03-01

    While automated blood cell counters have made great progress in detecting abnormalities in blood, the lack of specificity for a particular disease, limited information on single cell morphology and intrinsic uncertainly due to high throughput in these instruments often necessitates detailed inspection in the form of a peripheral blood smear. Such tests are relatively time consuming and frequently rely on medical professionals tally counting specific cell types. These assays rely on the contrast generated by chemical stains, with the signal intensity strongly related to staining and preparation techniques, frustrating machine learning algorithms that require consistent quantities to denote the features in question. Instead we opt to use quantitative phase imaging, understanding that the resulting image is entirely due to the structure (intrinsic contrast) rather than the complex interplay of stain and sample. We present here our first steps to automate peripheral blood smear scanning, in particular a method to generate the quantitative phase image of an entire blood smear at high throughput using white light diffraction phase microscopy (wDPM), a single shot and common path interferometric imaging technique.

  15. Optimization of digital image processing to determine quantum dots' height and density from atomic force microscopy.

    Science.gov (United States)

    Ruiz, J E; Paciornik, S; Pinto, L D; Ptak, F; Pires, M P; Souza, P L

    2018-01-01

    An optimized method of digital image processing to interpret quantum dots' height measurements obtained by atomic force microscopy is presented. The method was developed by combining well-known digital image processing techniques and particle recognition algorithms. The properties of quantum dot structures strongly depend on dots' height, among other features. Determination of their height is sensitive to small variations in their digital image processing parameters, which can generate misleading results. Comparing the results obtained with two image processing techniques - a conventional method and the new method proposed herein - with the data obtained by determining the height of quantum dots one by one within a fixed area, showed that the optimized method leads to more accurate results. Moreover, the log-normal distribution, which is often used to represent natural processes, shows a better fit to the quantum dots' height histogram obtained with the proposed method. Finally, the quantum dots' height obtained were used to calculate the predicted photoluminescence peak energies which were compared with the experimental data. Again, a better match was observed when using the proposed method to evaluate the quantum dots' height. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Imaging the surface stress and vibration modes of a microcantilever by laser beam deflection microscopy.

    Science.gov (United States)

    Tamayo, Javier; Pini, Valerio; Kosaka, Prisicila; Martinez, Nicolas F; Ahumada, Oscar; Calleja, Montserrat

    2012-08-10

    There is a need for noninvasive techniques for simultaneous imaging of the stress and vibration mode shapes of nanomechanical systems in the fields of scanning probe microscopy, nanomechanical biological and chemical sensors and the semiconductor industry. Here we show a novel technique that combines a scanning laser, the beam deflection method and digital multifrequency excitation and analysis for simultaneous imaging of the static out-of-plane displacement and the shape of five vibration modes of nanomechanical systems. The out-of-plane resolution is at least 100 pm Hz⁻¹/² and the lateral resolution, which is determined by the laser spot size, is 1-1.5 μm. The capability of the technique is demonstrated by imaging the residual surface stress of a microcantilever together with the shape of the first 22 vibration modes. The vibration behavior is compared with rigorous finite element simulations. The technique is suitable for major improvements in the imaging of liquids, such as higher bandwidth and enhanced spatial resolution.

  17. 3D imaging by serial block face scanning electron microscopy for materials science using ultramicrotomy.

    Science.gov (United States)

    Hashimoto, Teruo; Thompson, George E; Zhou, Xiaorong; Withers, Philip J

    2016-04-01

    Mechanical serial block face scanning electron microscopy (SBFSEM) has emerged as a means of obtaining three dimensional (3D) electron images over volumes much larger than possible by focused ion beam (FIB) serial sectioning and at higher spatial resolution than achievable with conventional X-ray computed tomography (CT). Such high resolution 3D electron images can be employed for precisely determining the shape, volume fraction, distribution and connectivity of important microstructural features. While soft (fixed or frozen) biological samples are particularly well suited for nanoscale sectioning using an ultramicrotome, the technique can also produce excellent 3D images at electron microscope resolution in a time and resource-efficient manner for engineering materials. Currently, a lack of appreciation of the capabilities of ultramicrotomy and the operational challenges associated with minimising artefacts for different materials is limiting its wider application to engineering materials. Consequently, this paper outlines the current state of the art for SBFSEM examining in detail how damage is introduced during slicing and highlighting strategies for minimising such damage. A particular focus of the study is the acquisition of 3D images for a variety of metallic and coated systems. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Free Form Deformation-Based Image Registration Improves Accuracy of Traction Force Microscopy.

    Directory of Open Access Journals (Sweden)

    Alvaro Jorge-Peñas

    Full Text Available Traction Force Microscopy (TFM is a widespread method used to recover cellular tractions from the deformation that they cause in their surrounding substrate. Particle Image Velocimetry (PIV is commonly used to quantify the substrate's deformations, due to its simplicity and efficiency. However, PIV relies on a block-matching scheme that easily underestimates the deformations. This is especially relevant in the case of large, locally non-uniform deformations as those usually found in the vicinity of a cell's adhesions to the substrate. To overcome these limitations, we formulate the calculation of the deformation of the substrate in TFM as a non-rigid image registration process that warps the image of the unstressed material to match the image of the stressed one. In particular, we propose to use a B-spline -based Free Form Deformation (FFD algorithm that uses a connected deformable mesh to model a wide range of flexible deformations caused by cellular tractions. Our FFD approach is validated in 3D fields using synthetic (simulated data as well as with experimental data obtained using isolated endothelial cells lying on a deformable, polyacrylamide substrate. Our results show that FFD outperforms PIV providing a deformation field that allows a better recovery of the magnitude and orientation of tractions. Together, these results demonstrate the added value of the FFD algorithm for improving the accuracy of traction recovery.

  19. Direct electron imaging in electron microscopy with monolithic active pixel sensors

    Energy Technology Data Exchange (ETDEWEB)

    Deptuch, G. [Brookhaven National Laboratory, Upton, NY 11973 (United States)], E-mail: deptuch@ieee.org; Besson, A. [IPHC, CNRS-IN2P3/ULP, 23 rue du Loess, BP 28, 67037 Strasbourg Cedex 02 (France); Rehak, P. [Brookhaven National Laboratory, Upton, NY 11973 (United States); Szelezniak, M. [IPHC, CNRS-IN2P3/ULP, 23 rue du Loess, BP 28, 67037 Strasbourg Cedex 02 (France); Wall, J. [Brookhaven National Laboratory, Upton, NY 11973 (United States); Winter, M. [IPHC, CNRS-IN2P3/ULP, 23 rue du Loess, BP 28, 67037 Strasbourg Cedex 02 (France); Zhu, Y. [Brookhaven National Laboratory, Upton, NY 11973 (United States)

    2007-08-15

    A new imaging device for dynamic electron microscopy is in great demand. The detector should provide the experimenter with images having sufficient spatial resolution at high speed. Immunity to radiation damage, accumulated during exposures, is critical. Photographic film, a traditional medium, is not adequate for studies that require large volumes of data or rapid recording and charge coupled device (CCD) cameras have limited resolution, due to phosphor screen coupling. CCD chips are not suitable for direct recording due to their extreme sensitivity to radiation damage. This paper discusses characterization of monolithic active pixel sensors (MAPS) in a scanning electron microscope (SEM) as well as in a transmission electron microscope (TEM). The tested devices were two versions of the MIMOSA V (MV) chip. This 1 M pixel device features pixel size of 17x17 {mu}m{sup 2} and was designed in a 0.6 {mu}m CMOS process. The active layer for detection is a thin (less than 20 {mu}m) epitaxial layer, limiting the broadening of the electron beam. The first version of the detector was a standard imager with electronics, passivation and interconnection layers on top of the active region; the second one was bottom-thinned, reaching the epitaxial layer from the bottom. The electron energies used range from a few keV to 30 keV for SEM and from 40 to 400 keV for TEM. Deterioration of the image resolution due to backscattering was quantified for different energies and both detector versions.

  20. Non parametric denoising methods based on wavelets: Application to electron microscopy images in low exposure time

    International Nuclear Information System (INIS)

    Soumia, Sid Ahmed; Messali, Zoubeida; Ouahabi, Abdeldjalil; Trepout, Sylvain; Messaoudi, Cedric; Marco, Sergio

    2015-01-01

    The 3D reconstruction of the Cryo-Transmission Electron Microscopy (Cryo-TEM) and Energy Filtering TEM images (EFTEM) hampered by the noisy nature of these images, so that their alignment becomes so difficult. This noise refers to the collision between the frozen hydrated biological samples and the electrons beam, where the specimen is exposed to the radiation with a high exposure time. This sensitivity to the electrons beam led specialists to obtain the specimen projection images at very low exposure time, which resulting the emergence of a new problem, an extremely low signal-to-noise ratio (SNR). This paper investigates the problem of TEM images denoising when they are acquired at very low exposure time. So, our main objective is to enhance the quality of TEM images to improve the alignment process which will in turn improve the three dimensional tomography reconstructions. We have done multiple tests on special TEM images acquired at different exposure time 0.5s, 0.2s, 0.1s and 1s (i.e. with different values of SNR)) and equipped by Golding beads for helping us in the assessment step. We herein, propose a structure to combine multiple noisy copies of the TEM images. The structure is based on four different denoising methods, to combine the multiple noisy TEM images copies. Namely, the four different methods are Soft, the Hard as Wavelet-Thresholding methods, Bilateral Filter as a non-linear technique able to maintain the edges neatly, and the Bayesian approach in the wavelet domain, in which context modeling is used to estimate the parameter for each coefficient. To ensure getting a high signal-to-noise ratio, we have guaranteed that we are using the appropriate wavelet family at the appropriate level. So we have chosen âĂIJsym8âĂİ wavelet at level 3 as the most appropriate parameter. Whereas, for the bilateral filtering many tests are done in order to determine the proper filter parameters represented by the size of the filter, the range parameter and the

  1. Asymmetric-detection time-stretch optical microscopy (ATOM) for ultrafast high-contrast cellular imaging in flow

    Science.gov (United States)

    Wong, Terence T. W.; Lau, Andy K. S.; Ho, Kenneth K. Y.; Tang, Matthew Y. H.; Robles, Joseph D. F.; Wei, Xiaoming; Chan, Antony C. S.; Tang, Anson H. L.; Lam, Edmund Y.; Wong, Kenneth K. Y.; Chan, Godfrey C. F.; Shum, Ho Cheung; Tsia, Kevin K.

    2014-01-01

    Accelerating imaging speed in optical microscopy is often realized at the expense of image contrast, image resolution, and detection sensitivity – a common predicament for advancing high-speed and high-throughput cellular imaging. We here demonstrate a new imaging approach, called asymmetric-detection time-stretch optical microscopy (ATOM), which can deliver ultrafast label-free high-contrast flow imaging with well delineated cellular morphological resolution and in-line optical image amplification to overcome the compromised imaging sensitivity at high speed. We show that ATOM can separately reveal the enhanced phase-gradient and absorption contrast in microfluidic live-cell imaging at a flow speed as high as ~10 m/s, corresponding to an imaging throughput of ~100,000 cells/sec. ATOM could thus be the enabling platform to meet the pressing need for intercalating optical microscopy in cellular assay, e.g. imaging flow cytometry – permitting high-throughput access to the morphological information of the individual cells simultaneously with a multitude of parameters obtained in the standard assay. PMID:24413677

  2. Imaging of radiation damage using complementary field ion microscopy and atom probe tomography

    International Nuclear Information System (INIS)

    Dagan, Michal; Hanna, Luke R.; Xu, Alan; Roberts, Steve G.; Smith, George D.W.; Gault, Baptiste; Edmondson, Philip D.; Bagot, Paul A.J.; Moody, Michael P.

    2015-01-01

    Radiation damage in tungsten and a tungsten–tantalum alloy, both of relevance to nuclear fusion research, has been characterized using a combination of field ion microscopy (FIM) imaging and atom probe tomography (APT). While APT provides 3D analytical imaging with sub-nanometer resolution, FIM is capable of imaging the arrangements of single atoms on a crystal lattice and has the potential to provide insights into radiation induced crystal damage, all the way down to its smallest manifestation – a single vacancy. This paper demonstrates the strength of combining these characterization techniques. In ion implanted tungsten, it was found that atomic scale lattice damage is best imaged using FIM. In certain cases, APT reveals an identifiable imprint in the data via the segregation of solute and impurities and trajectory aberrations. In a W–5 at%Ta alloy, a combined APT–FIM study was able to determine the atomic distribution of tantalum inside the tungsten matrix. An indirect method was implemented to identify tantalum atoms inside the tungsten matrix in FIM images. By tracing irregularities in the evaporation sequence of atoms imaged with FIM, this method enables the benefit of FIM's atomic resolution in chemical distinction between the two species. - Highlights: • Complementary FIM and APT was used to study nanoscale radiation damage in tungsten. • Trajectory aberrations in APT revealed extended lattice damage which FIM confirmed. • Nano scale features were detected indirectly with APT via segregation of impurities. • Induced damage at the scale of a single vacancy could only be detected via FIM. • 3DFIM characterized W–5Ta atomic distribution with improved detection efficiency.

  3. High-Resolution 3 T MR Microscopy Imaging of Arterial Walls

    International Nuclear Information System (INIS)

    Sailer, Johannes; Rand, Thomas; Berg, Andreas; Sulzbacher, Irene; Peloschek, P.; Hoelzenbein, Thomas; Lammer, Johannes

    2006-01-01

    Purpose. To achieve a high spatial resolution in MR imaging that allows for clear visualization of anatomy and even histology and documentation of plaque morphology in in vitro samples from patients with advanced atherosclerosis. A further objective of our study was to evaluate whether T2-weighted high-resolution MR imaging can provide accurate classification of atherosclerotic plaque according to a modified American Heart Association classification. Methods. T2-weighted images of arteries were obtained in 13 in vitro specimens using a 3 T MR unit (Medspec 300 Avance/Bruker, Ettlingen, Germany) combined with a dedicated MR microscopy system. Measurement parameters were: T2-weighted sequences with TR 3.5 sec, TE 15-120 msec; field of view (FOV) 1.4 x 1.4; NEX 8; matrix 192; and slice thickness 600 μm. MR measurements were compared with corresponding histologic sections. Results. We achieved excellent spatial and contrast resolution in all specimens. We found high agreement between MR images and histology with regard to the morphology and extent of intimal proliferations in all but 2 specimens. We could differentiate fibrous caps and calcifications from lipid plaque components based on differences in signal intensity in order to differentiate hard and soft atheromatous plaques. Hard plaques with predominantly intimal calcifications were found in 7 specimens, and soft plaques with a cholesterol/lipid content in 5 cases. In all specimens, hemorrhage or thrombus formation, and fibrotic and hyalinized tissue could be detected on both MR imaging and histopathology. Conclusion. High-resolution, high-field MR imaging of arterial walls demonstrates the morphologic features, volume, and extent of intimal proliferations with high spatial and contrast resolution in in vitro specimens and can differentiate hard and soft plaques

  4. Flow Microscopy Imaging Is Sensitive to Characteristics of Subvisible Particles in Peginesatide Formulations Associated With Severe Adverse Reactions.

    Science.gov (United States)

    Daniels, Austin L; Randolph, Theodore W

    2018-02-01

    The presence of subvisible particles in formulations of therapeutic proteins is a risk factor for adverse immune responses. Although the immunogenic potential of particulate contaminants likely depends on particle structural characteristics (e.g., composition, size, and shape), exact structure-immunogenicity relationships are unknown. Images recorded by flow imaging microscopy reflect information about particle morphology, but flow microscopy is typically used to determine only particle size distributions, neglecting information on particle morphological features that may be immunologically relevant. We recently developed computational techniques that utilize the Kullback-Leibler divergence and multidimensional scaling to compare the morphological properties of particles in sets of flow microscopy images. In the current work, we combined these techniques with expectation maximization cluster analyses and used them to compare flow imaging microscopy data sets that had been collected by the U.S. Food and Drug Administration after severe adverse drug reactions (including 7 fatalities) were observed in patients who had been administered some lots of peginesatide formulations. Flow microscopy images of particle populations found in the peginesatide lots associated with severe adverse reactions in patients were readily distinguishable from images of particles in lots where severe adverse reactions did not occur. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  5. Quantitative neuroanatomy of all Purkinje cells with light sheet microscopy and high-throughput image analysis

    Directory of Open Access Journals (Sweden)

    Ludovico eSilvestri

    2015-05-01

    Full Text Available Characterizing the cytoarchitecture of mammalian central nervous system on a brain-wide scale is becoming a compelling need in neuroscience. For example, realistic modeling of brain activity requires the definition of quantitative features of large neuronal populations in the whole brain. Quantitative anatomical maps will also be crucial to classify the cytoarchtitectonic abnormalities associated with neuronal pathologies in a high reproducible and reliable manner. In this paper, we apply recent advances in optical microscopy and image analysis to characterize the spatial distribution of Purkinje cells across the whole cerebellum. Light sheet microscopy was used to image with micron-scale resolution a fixed and cleared cerebellum of an L7-GFP transgenic mouse, in which all Purkinje cells are fluorescently labeled. A fast and scalable algorithm for fully automated cell identification was applied on the image to extract the position of all the fluorescent Purkinje cells. This vectorized representation of the cell population allows a thorough characterization of the complex three-dimensional distribution of the neurons, highlighting the presence of gaps inside the lamellar organization of Purkinje cells, whose density is believed to play a significant role in autism spectrum disorders. Furthermore, clustering analysis of the localized somata permits dividing the whole cerebellum in groups of Purkinje cells with high spatial correlation, suggesting new possibilities of anatomical partition. The quantitative approach presented here can be extended to study the distribution of different types of cell in many brain regions and across the whole encephalon, providing a robust base for building realistic computational models of the brain, and for unbiased morphological tissue screening in presence of pathologies and/or drug treatments.

  6. Rapid diagnostic imaging and pathologic evaluation of whole core biopsies at the point-of-care using structured illumination microscopy

    Science.gov (United States)

    Wang, Mei; Sholl, Andrew B.; Kimbrell, Hillary; Tulman, David B.; Elfer, Katherine N.; Brown, J. Quincy

    2015-07-01

    Video-rate structured illumination microscopy (VR-SIM) of fluorescently stained prostate biopsies is demonstrated as a potential tool for rapid diagnosis of prostate biopsies at the point of care. Images of entire biopsies at 1.3 micron lateral resolution are rendered in seconds, and pathologist review of the resulting images achieves 90% accuracy as compared to gold standard histopathology.

  7. Massively parallel data processing for quantitative total flow imaging with optical coherence microscopy and tomography

    Science.gov (United States)

    Sylwestrzak, Marcin; Szlag, Daniel; Marchand, Paul J.; Kumar, Ashwin S.; Lasser, Theo

    2017-08-01

    We present an application of massively parallel processing of quantitative flow measurements data acquired using spectral optical coherence microscopy (SOCM). The need for massive signal processing of these particular datasets has been a major hurdle for many applications based on SOCM. In view of this difficulty, we implemented and adapted quantitative total flow estimation algorithms on graphics processing units (GPU) and achieved a 150 fold reduction in processing time when compared to a former CPU implementation. As SOCM constitutes the microscopy counterpart to spectral optical coherence tomography (SOCT), the developed processing procedure can be applied to both imaging modalities. We present the developed DLL library integrated in MATLAB (with an example) and have included the source code for adaptations and future improvements. Catalogue identifier: AFBT_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AFBT_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GNU GPLv3 No. of lines in distributed program, including test data, etc.: 913552 No. of bytes in distributed program, including test data, etc.: 270876249 Distribution format: tar.gz Programming language: CUDA/C, MATLAB. Computer: Intel x64 CPU, GPU supporting CUDA technology. Operating system: 64-bit Windows 7 Professional. Has the code been vectorized or parallelized?: Yes, CPU code has been vectorized in MATLAB, CUDA code has been parallelized. RAM: Dependent on users parameters, typically between several gigabytes and several tens of gigabytes Classification: 6.5, 18. Nature of problem: Speed up of data processing in optical coherence microscopy Solution method: Utilization of GPU for massively parallel data processing Additional comments: Compiled DLL library with source code and documentation, example of utilization (MATLAB script with raw data) Running time: 1,8 s for one B-scan (150 × faster in comparison to the CPU

  8. Composition quantification of electron-transparent samples by backscattered electron imaging in scanning electron microscopy.

    Science.gov (United States)

    Müller, E; Gerthsen, D

    2017-02-01

    The contrast of backscattered electron (BSE) images in scanning electron microscopy (SEM) depends on material parameters which can be exploited for composition quantification if some information on the material system is available. As an example, the In-concentration in thin In x Ga 1-x As layers embedded in a GaAs matrix is analyzed in this work. The spatial resolution of the technique is improved by using thin electron-transparent specimens instead of bulk samples. Although the BSEs are detected in a comparably small angular range by an annular semiconductor detector, the image intensity can be evaluated to determine the composition and local thickness of the specimen. The measured intensities are calibrated within one single image to eliminate the influence of the detection and amplification system. Quantification is performed by comparison of experimental and calculated data. Instead of using time-consuming Monte-Carlo simulations, an analytical model is applied for BSE-intensity calculations which considers single electron scattering and electron diffusion. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Automatic identification and characterization of radial files in light microscopy images of wood.

    Science.gov (United States)

    Brunel, Guilhem; Borianne, Philippe; Subsol, Gérard; Jaeger, Marc; Caraglio, Yves

    2014-09-01

    Analysis of anatomical sections of wood provides important information for understanding the secondary growth and development of plants. This study reports on a new method for the automatic detection and characterization of cell files in wood images obtained by light microscopy. To facilitate interpretation of the results, reliability coefficients have been determined, which characterize the files, their cells and their respective measurements. Histological sections and blocks of the gymnosperms Pinus canariensis, P. nigra and Abies alba were used, together with histological sections of the angiosperm mahogany (Swietenia spp.). Samples were scanned microscopically and mosaic images were built up. After initial processing to reduce noise and enhance contrast, cells were identified using a 'watershed' algorithm and then cell files were built up by the successive aggregation of cells taken from progressively enlarged neighbouring regions. Cell characteristics such as thickness and size were calculated, and a method was developed to determine the reliability of the measurements relative to manual methods. Image analysis using this method can be performed in less than 20 s, which compares with a time of approx. 40 min to produce the same results manually. The results are accompanied by a reliability indicator that can highlight specific configurations of cells and also potentially erroneous data. The method provides a fast, economical and reliable tool for the identification of cell files. The reliability indicator characterizing the files permits quick filtering of data for statistical analysis while also highlighting particular biological configurations present in the wood sections.

  10. [Imaging of corneal dystrophies: Correlations between en face anterior segment OCT and in vivo confocal microscopy].

    Science.gov (United States)

    Ghouali, W; Tahiri Joutei Hassani, R; Liang, H; Dupont-Monod, S; Auclin, F; Baudouin, C; Labbé, A

    2015-05-01

    To evaluate the usefulness of en face Optical Coherence Tomography (OCT) for evaluation of corneal dystrophies and to describe correlations with in vivo confocal microscopy (IVCM). Thirty-two eyes of 16 patients with 4 types of corneal dystrophies (epithelial basement membrane dystrophy, Fuchs dystrophy, Reis-Bücklers corneal dystrophy and Crocodile Shagreen dystrophy) were enrolled in this study. Axial and reconstructed en face scans were acquired using OCT. Images were then correlated to IVCM findings. En face OCT provided new insights into the structure, size and depth of corneal tissue alterations in various corneal dystrophies. OCT en face images were well correlated with IVCM features. Despite lower resolution than IVCM, en face OCT offers the advantages of being non-invasive and allowing the analysis of larger corneal areas. En face OCT provides useful new information in corneal dystrophies. This imaging technique will probably increase in popularity in the near future for the assessment of various anterior segment diseases. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  11. Crystal lattice imaging of the silica and alumina faces of kaolinite using atomic force microscopy.

    Science.gov (United States)

    Gupta, Vishal; Hampton, Marc A; Nguyen, Anh V; Miller, Jan D

    2010-12-01

    The crystal lattice images of the two faces of kaolinite (the silica face and the alumina face) have been obtained using contact-mode atomic force microscopy (AFM) under ambient conditions. Lattice resolution images reveal the hexagonal surface lattice of these two faces of kaolinite. Analysis of the silica face of kaolinite showed that the hexagonal surface lattice ring of oxygen atoms had a periodicity of 0.50±0.04nm between neighboring oxygen atoms, which is in good agreement with the surface lattice structure of the mica basal plane. The center of the hexagonal ring of oxygen atoms is vacant. Analysis of the alumina face of kaolinite showed that the hexagonal surface lattice ring of hydroxyls surround a hydroxyl in the center of the ring. The atomic spacing between neighboring hydroxyls was determined as 0.36±0.04nm. Ordering of the kaolinite particles for examination of the silica and alumina surfaces was accomplished using different substrates, a procedure previously established. Crystal lattice imaging supports previous results and independently confirms that the two faces of kaolinite have been properly identified. Copyright © 2010 Elsevier Inc. All rights reserved.

  12. Reconstruction of Kelvin probe force microscopy image with experimentally calibrated point spread function.

    Science.gov (United States)

    Lan, Fei; Jiang, Minlin; Tao, Quan; Wei, Fanan; Li, Guangyong

    2017-03-01

    A Kelvin probe force microscopy (KPFM) image is sometimes difficult to interpret because it is a blurred representation of the true surface potential (SP) distribution of the materials under test. The reason for the blurring is that KPFM relies on the detection of electrostatic force, which is a long-range force compared to other surface forces. Usually, KPFM imaging model is described as the convolution of the true SP distribution of the sample with an intrinsic point spread function (PSF) of the measurement system. To restore the true SP signals from the blurred ones, the intrinsic PSF of the system is needed. In this work, we present a way to experimentally calibrate the PSF of the KPFM system. Taking the actual probe shape and experimental parameters into consideration, this calibration method leads to a more accurate PSF than the ones obtained from simulations. Moreover, a nonlinear reconstruction algorithm based on total variation (TV) regularization is applied to KPFM measurement to reverse the blurring caused by PSF during KPFM imaging process; as a result, noises are reduced and the fidelity of SP signals is improved.

  13. In vivo imaging of spinal cord in contusion injury model mice by multi-photon microscopy

    Science.gov (United States)

    Oshima, Y.; Horiuchi, H.; Ogata, T.; Hikita, A.; Miura, H.; Imamura, T.

    2014-03-01

    Fluorescent imaging technique is a promising method and has been developed for in vivo applications in cellular biology. In particular, nonlinear optical imaging technique, multi-photon microscopy has make it possible to analyze deep portion of tissues in living animals such as axons of spinal code. Traumatic spinal cord injuries (SCIs) are usually caused by contusion damages. Therefore, observation of spinal cord tissue after the contusion injury is necessary for understanding cellular dynamics in response to traumatic SCI and development of the treatment for traumatic SCI. Our goal is elucidation of mechanism for degeneration of axons after contusion injuries by establishing SCI model and chronic observation of injured axons in the living animals. Firstly we generated and observed acute SCI model by contusion injury. By using a multi-photon microscope, axons in dorsal cord were visualized approximately 140 micron in depth from the surface. Immediately after injury, minimal morphological change of spinal cord was observed. At 3 days after injury, spinal cord was swelling and the axons seem to be fragmented. At 7 days after injury, increased degradation of axons could be observed, although the image was blurred due to accumulation of the connective tissue. In the present study, we successfully observed axon degeneration after the contusion SCI in a living animal in vivo. Our final goal is to understand molecular mechanisms and cellular dynamics in response to traumatic SCIs in acute and chronic stage.

  14. Dimensionality reduction, segmentation, and quantification of multidimensional images: application to fluorescence microscopy

    Science.gov (United States)

    Bonnet, Noel; Raby, Beatrice; Zahm, Jean-Marie

    2000-03-01

    Fluorescence microscopy is rapidly becoming a multi- dimensional technique. Many applications generate similar data analysis problems. Whatever the non-spatial dimension (time, energy), users have to make the choice between local analysis and global analysis. For local analysis, the evolution of pixels (or regions of interest) is modeled as a function of the external parameter. Results are displayed as parametric images. For global analysis, multivariate statistical analysis can be used to extract and interpret the significant information (in the presence of redundancy and noise) in the form of eigenimages and eigenfactors. Automatic classification methods start to play a role for the co-location problem, in which pixels are classified into regions corresponding to positive, null or negative correlation. With two or three images, the scatterplot (an estimation of the joint probability density function), can be built. Interactive and automatic correlation partitioning (ICP, ACP) can then be performed. The method we have developed (Parzen estimate of the probability density function followed by the watersheds mathematical morphology approach) does not make assumptions about the shape of clusters. With more than three images, dimensionality reduction must be applied, for visualization purposes and for simplifying classification. This can be done by linear or non-linear methods such as Multi-Dimensional Scaling, Auto-Associative Neural Networks or Self-Organizing Mapping.

  15. Imaging and size measurement of nanoparticles in aqueous medium by use of atomic force microscopy.

    Science.gov (United States)

    Takechi-Haraya, Yuki; Goda, Yukihiro; Sakai-Kato, Kumiko

    2018-02-01

    Size control of nanoparticles in nanotechnology-based drug products is crucial for their successful development, since the in vivo pharmacokinetics of nanoparticles are size-dependent. In this study, we evaluated the use of atomic force microscopy (AFM) for imaging and size measurement of nanoparticles in aqueous medium. The height sizes of rigid polystyrene nanoparticles and soft liposomes were measured by AFM and were compared with the hydrodynamic sizes measured by dynamic light scattering (DLS). The lipid compositions of the studied liposomes were similar to those of commercial products. AFM proved to be a viable method for obtaining images of both polystyrene nanoparticles and liposomes in aqueous medium. For the polystyrene nanoparticles, the average height size observed by AFM was similar to the average number-weighted diameter obtained by DLS, indicating the usefulness of AFM for measuring the sizes of nanoparticles in aqueous medium. For the liposomes, the height sizes obtained by AFM differed depending upon the procedures of immobilizing the liposomes onto a solid substrate. In addition, the resultant average height sizes of the liposomes were smaller than those obtained by DLS. This knowledge will help the correct use of AFM as a powerful tool for imaging and size measurement of nanotechnology-based drug products for clinical use.

  16. Simulation of image formation in x-ray coded aperture microscopy with polycapillary optics.

    Science.gov (United States)

    Korecki, P; Roszczynialski, T P; Sowa, K M

    2015-04-06

    In x-ray coded aperture microscopy with polycapillary optics (XCAMPO), the microstructure of focusing polycapillary optics is used as a coded aperture and enables depth-resolved x-ray imaging at a resolution better than the focal spot dimensions. Improvements in the resolution and development of 3D encoding procedures require a simulation model that can predict the outcome of XCAMPO experiments. In this work we introduce a model of image formation in XCAMPO which enables calculation of XCAMPO datasets for arbitrary positions of the object relative to the focal plane as well as to incorporate optics imperfections. In the model, the exit surface of the optics is treated as a micro-structured x-ray source that illuminates a periodic object. This makes it possible to express the intensity of XCAMPO images as a convolution series and to perform simulations by means of fast Fourier transforms. For non-periodic objects, the model can be applied by enforcing artificial periodicity and setting the spatial period larger then the field-of-view. Simulations are verified by comparison with experimental data.

  17. Metabolic changes of cultured DRG neurons induced by adenosine using confocal microscopy imaging

    Science.gov (United States)

    Zheng, Liqin; Huang, Yimei; Chen, Jiangxu; Wang, Yuhua; Yang, Hongqin; Zhang, Yanding; Xie, Shusen

    2012-12-01

    Adenosine exerts multiple effects on pain transmission in the peripheral nervous system. This study was performed to use confocal microscopy to evaluate whether adenosine could affect dorsal root ganglia (DRG) neurons in vitro and test which adenosine receptor mediates the effect of adenosine on DRG neurons. After adding adenosine with different concentration, we compared the metabolic changes by the real time imaging of calcium and mitochondria membrane potential using confocal microscopy. The results showed that the effect of 500 μM adenosine on the metabolic changes of DRG neurons was more significant than others. Furthermore, four different adenosine receptor antagonists were used to study which receptor mediated the influences of adenosine on the cultured DRG neurons. All adenosine receptor antagonists especially A1 receptor antagonist (DPCPX) had effect on the Ca2+ and mitochondria membrane potential dynamics of DRG neurons. The above studies demonstrated that the effect of adenosine which may be involved in the signal transmission on the sensory neurons was dose-dependent, and all the four adenosine receptors especially the A1R may mediate the transmission.

  18. In vivo ocular imaging of the cornea of the normal female laboratory beagle using confocal microscopy.

    Science.gov (United States)

    Strom, Ann R; Cortés, Dennis E; Thomasy, Sara M; Kass, Philip H; Mannis, Mark J; Murphy, Christopher J

    2016-01-01

    To obtain normative data for the normal laboratory beagle cornea using high-resolution in vivo confocal microscopy (IVCM). Sixteen eyes of eight healthy young female intact beagles. The central cornea was imaged using IVCM. Mixed effects linear regression was used for statistical analysis. in vivo confocal microscopy allowed detailed visualization and quantification of epithelial cells (superficial epithelial cell diameter: 43.25 ± 6.64 μm, basal cell diameter: 4.43 ± 0.67 μm), and nerve fibers (subepithelial nerve fiber diameter: 2.38 ± 0.69 μm, anterior stromal nerve fiber diameter: 16.93 ± 4.55 μm). Keratocyte density (anterior stroma 993.38 ± 134.24 cells/mm(2) , posterior stroma 789.38 ± 87.13 cells/mm(2) ) and endothelial cell density (2815.18 ± 212.59 cells/mm(2) ) were also measured. High-resolution IVCM provides detailed noninvasive evaluation of the cornea in the normal laboratory beagle. © 2015 American College of Veterinary Ophthalmologists.

  19. Dual-view plane illumination microscopy for rapid and spatially isotropic imaging.

    Science.gov (United States)

    Kumar, Abhishek; Wu, Yicong; Christensen, Ryan; Chandris, Panagiotis; Gandler, William; McCreedy, Evan; Bokinsky, Alexandra; Colón-Ramos, Daniel A; Bao, Zhirong; McAuliffe, Matthew; Rondeau, Gary; Shroff, Hari

    2014-11-01

    We describe the construction and use of a compact dual-view inverted selective plane illumination microscope (diSPIM) for time-lapse volumetric (4D) imaging of living samples at subcellular resolution. Our protocol enables a biologist with some prior microscopy experience to assemble a diSPIM from commercially available parts, to align optics and test system performance, to prepare samples, and to control hardware and data processing with our software. Unlike existing light sheet microscopy protocols, our method does not require the sample to be embedded in agarose; instead, samples are prepared conventionally on glass coverslips. Tissue culture cells and Caenorhabditis elegans embryos are used as examples in this protocol; successful implementation of the protocol results in isotropic resolution and acquisition speeds up to several volumes per s on these samples. Assembling and verifying diSPIM performance takes ∼6 d, sample preparation and data acquisition take up to 5 d and postprocessing takes 3-8 h, depending on the size of the data.

  20. Biological Atomic Force Microscopy for Imaging Gold-Labeled Liposomes on Human Coronary Artery Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Ana-María Zaske

    2013-01-01

    Full Text Available Although atomic force microscopy (AFM has been used extensively to characterize cell membrane structure and cellular processes such as endocytosis and exocytosis, the corrugated surface of the cell membrane hinders the visualization of extracellular entities, such as liposomes, that may interact with the cell. To overcome this barrier, we used 90 nm nanogold particles to label FITC liposomes and monitor their endocytosis on human coronary artery endothelial cells (HCAECs in vitro. We were able to study the internalization process of gold-coupled liposomes on endothelial cells, by using AFM. We found that the gold-liposomes attached to the HCAEC cell membrane during the first 15–30 min of incubation, liposome cell internalization occurred from 30 to 60 min, and most of the gold-labeled liposomes had invaginated after 2 hr of incubation. Liposomal uptake took place most commonly at the periphery of the nuclear zone. Dynasore monohydrate, an inhibitor of endocytosis, obstructed the internalization of the gold-liposomes. This study showed the versatility of the AFM technique, combined with fluorescent microscopy, for investigating liposome uptake by endothelial cells. The 90 nm colloidal gold nanoparticles proved to be a noninvasive contrast agent that efficiently improves AFM imaging during the investigation of biological nanoprocesses.

  1. New approaches to intracellular drug imaging by stimulated Raman scattering microscopy

    Science.gov (United States)

    Lee, M.; Tipping, W. J.; Serrels, A.; Hulme, A. N.; Brunton, V. G.

    2017-02-01

    A range of proposed alkyne Raman tags are examined in-silico for activity and then synthesised generating a library of analogues of the natural product, anisomycin. We report the use of bisaryl butadiyne-anisomycin (BADY-anisomycin) in intracellular SRS microscopy studies of uptake and localisation within live and fixed cells. Following rational design and synthesis, BADY-anisomycin was shown to produce an intense Raman band at 2219 cm-1, that is centrally located within the cellular silent region and is approximately 60 times more Raman active than the corresponding propargylanisomycin. Finally, we demonstrate two-colour imaging utilising EdU, an alkyne-containing proliferation probe and BADY-anisomycin.

  2. Atomic force microscopy imaging of polyurethane nanoparticles onto different solid substrates

    Energy Technology Data Exchange (ETDEWEB)

    Beddin Fritzen-Garcia, Mauricia [Laboratorio de Bioenergetica e Bioquimica de Macromoleculas, Departamento de Ciencias Farmaceuticas, Universidade Federal de Santa Catarina, 88040-900, Florianopolis, SC (Brazil); POLIMAT, Departamento de Quimica, Universidade Federal de Santa Catarina, 88040-900, Florianopolis, SC (Brazil)], E-mail: maurifritzen@hotmail.com; Giehl Zanetti-Ramos, Betina [Laboratorio de Bioenergetica e Bioquimica de Macromoleculas, Departamento de Ciencias Farmaceuticas, Universidade Federal de Santa Catarina, 88040-900, Florianopolis, SC (Brazil); Schweitzer de Oliveira, Cristian [Laboratorio de Filmes Finos e Superficies, Departamento de Fisica, Universidade Federal de Santa Catarina, 88040-900, Florianopolis, SC (Brazil); Soldi, Valdir [POLIMAT, Departamento de Quimica, Universidade Federal de Santa Catarina, 88040-900, Florianopolis, SC (Brazil); Avelino Pasa, Andre [Laboratorio de Filmes Finos e Superficies, Departamento de Fisica, Universidade Federal de Santa Catarina, 88040-900, Florianopolis, SC (Brazil); Creczynski-Pasa, Tania Beatriz [Laboratorio de Bioenergetica e Bioquimica de Macromoleculas, Departamento de Ciencias Farmaceuticas, Universidade Federal de Santa Catarina, 88040-900, Florianopolis, SC (Brazil)

    2009-03-01

    Atomic force microscopy (AFM) is a technique suited for characterizing nanoparticles on solid surfaces because it offers the capability of 3D visualization and quantitative information about the topography of the samples. In the present work, contact-mode AFM has been applied to imaging polyurethane nanoparticles formulated from a natural triol and isophorone diisocyanate (IPDI) in the presence of poly(ethylene glycol) (PEG). The colloidal polymeric system was deposited on mica, hydrophilic and hydrophobic silicon solid substrates to evaluate the size and shape of the nanoparticles. Our data showed that the nanoparticles were better distributed on mica and hydrophilic silicon. From the analysis of line-scan profiles we obtained different values for the ratio between the diameter and the height of the nanoparticles, indicating that the shape of the particles depends on the interaction between the nanoparticles and the substrate.

  3. Microtubules in Plant Cells: Strategies and Methods for Immunofluorescence, Transmission Electron Microscopy, and Live Cell Imaging.

    Science.gov (United States)

    Celler, Katherine; Fujita, Miki; Kawamura, Eiko; Ambrose, Chris; Herburger, Klaus; Holzinger, Andreas; Wasteneys, Geoffrey O

    2016-01-01

    Microtubules (MTs) are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labeling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high-pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography.

  4. Principal component and spatial correlation analysis of spectroscopic-imaging data in scanning probe microscopy

    International Nuclear Information System (INIS)

    Jesse, Stephen; Kalinin, Sergei V

    2009-01-01

    An approach for the analysis of multi-dimensional, spectroscopic-imaging data based on principal component analysis (PCA) is explored. PCA selects and ranks relevant response components based on variance within the data. It is shown that for examples with small relative variations between spectra, the first few PCA components closely coincide with results obtained using model fitting, and this is achieved at rates approximately four orders of magnitude faster. For cases with strong response variations, PCA allows an effective approach to rapidly process, de-noise, and compress data. The prospects for PCA combined with correlation function analysis of component maps as a universal tool for data analysis and representation in microscopy are discussed.

  5. Computerized thermogravimetric reactor with video microscopy imaging system for coal pyrolysis and combustion studies

    Science.gov (United States)

    Matzakos, Andreas N.; Zygourakis, Kyriacos

    1993-06-01

    We present the development of a novel thermogravimetric reactor with in situ video microscopy imaging capabilities (TGA/VMI) and discuss its use for studying coal pyrolysis and combustion. Our design combines a long working distance optical microscope and video recording equipment with a computer-automated microbalance reactor. The TGA/VMI apparatus allows for simultaneous thermogravimetric measurement of reaction rates and direct viewing or video taping of structural transformations that may accompany the heterogeneous reactions. Reactor temperature is controlled by a digital algorithm combining feedback with model-based feedforward control. With its computer control and custom-built furnace, our reactor can achieve high heating rates (up to 50 °C/s) and temperatures (1000 °C) with excellent accuracy, stability, and reproducibility. We present some experimental results from our studies on coal pyrolysis and gasification to demonstrate the capabilities of the apparatus and suggest other potential applications.

  6. Lattice Light Sheet Microscopy: Imaging Molecules to Embryos at High Spatiotemporal Resolution

    Science.gov (United States)

    Chen, Bi-Chang; Legant, Wesley R.; Wang, Kai; Shao, Lin; Milkie, Daniel E.; Davidson, Michael W.; Janetopoulos, Chris; Wu, Xufeng S.; Hammer, John A.; Liu, Zhe; English, Brian P.; Mimori-Kiyosue, Yuko; Romero, Daniel P.; Ritter, Alex T.; Lippincott-Schwartz, Jennifer; Fritz-Laylin, Lillian; Mullins, R. Dyche; Mitchell, Diana M.; Bembenek, Joshua N.; Reymann, Anne-Cecile; Böhme, Ralph; Grill, Stephan W.; Wang, Jennifer T.; Seydoux, Geraldine; Tulu, U. Serdar; Kiehart, Daniel P.; Betzig, Eric

    2015-01-01

    Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, too small, or occur too rapidly to see clearly with existing tools. We crafted ultra-thin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at sub-second intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and complexity of living systems. PMID:25342811

  7. Atomic force microscopy imaging of polyurethane nanoparticles onto different solid substrates

    International Nuclear Information System (INIS)

    Beddin Fritzen-Garcia, Mauricia; Giehl Zanetti-Ramos, Betina; Schweitzer de Oliveira, Cristian; Soldi, Valdir; Avelino Pasa, Andre; Creczynski-Pasa, Tania Beatriz

    2009-01-01

    Atomic force microscopy (AFM) is a technique suited for characterizing nanoparticles on solid surfaces because it offers the capability of 3D visualization and quantitative information about the topography of the samples. In the present work, contact-mode AFM has been applied to imaging polyurethane nanoparticles formulated from a natural triol and isophorone diisocyanate (IPDI) in the presence of poly(ethylene glycol) (PEG). The colloidal polymeric system was deposited on mica, hydrophilic and hydrophobic silicon solid substrates to evaluate the size and shape of the nanoparticles. Our data showed that the nanoparticles were better distributed on mica and hydrophilic silicon. From the analysis of line-scan profiles we obtained different values for the ratio between the diameter and the height of the nanoparticles, indicating that the shape of the particles depends on the interaction between the nanoparticles and the substrate

  8. Fluorescence confocal laser scanning microscopy for in vivo imaging of epidermal reactions to two experimental irritants

    DEFF Research Database (Denmark)

    Suihko, C.; Serup, J.

    2008-01-01

    Background: Fibre-optic fluorescence confocal laser scanning microscopy (CLSM) is a novel non-invasive technique for in vivo imaging of skin. The cellular structure of the epidermis can be studied. A fluorophore, e.g. fluorescein sodium, is introduced by an intradermal injection or applied...... dermatitis reactions caused by established model irritants, e.g. sodium lauryl sulphate (SLS) and pelargonic acid (PA). Methods: Twelve healthy individuals volunteered. The flexor aspect of the right and the left forearm was exposed to SLS in water and PA in isopropanol and occluded under Finn Chambers...... for 24 h. The reactions were rated clinically and, following epicutaneous and intra-dermal application of fluorescein sodium, studied by fluorescence CLSM, magnification x 1000. Results: Both irritants disturbed the epidermal intercellular borders, which became blurred, thickened and variably altered...

  9. Three-dimensional molecular imaging using mass spectrometry and atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Wucher, Andreas [Department of Physics, University of Duisburg-Essen, D-47048 Duisburg (Germany)], E-mail: andreas.wucher@uni-due.de; Cheng Juan; Zheng Leiliang; Willingham, David; Winograd, Nicholas [Department of Chemistry, Pennsylvania State University, University Park, PA 16802 (United States)

    2008-12-15

    We combine imaging ToF-SIMS depth profiling and wide area atomic force microscopy to analyze a test structure consisting of a 300 nm trehalose film deposited on a Si substrate and pre-structured by means of a focused 15-keV Ga{sup +} ion beam. Depth profiling is performed using a 40-keV C{sub 60}{sup +} cluster ion beam for erosion and mass spectral data acquisition. A generic protocol for depth axis calibration is described which takes into account both lateral and in-depth variations of the erosion rate. By extrapolation towards zero analyzed lateral area, an 'intrinsic' depth resolution of about 8 nm is found which appears to be characteristic of the cluster-surface interaction process.

  10. Spectroscopic imaging with spectral domain visible light optical coherence microscopy in Alzheimer's disease brain samples.

    Science.gov (United States)

    Lichtenegger, Antonia; Harper, Danielle J; Augustin, Marco; Eugui, Pablo; Muck, Martina; Gesperger, Johanna; Hitzenberger, Christoph K; Woehrer, Adelheid; Baumann, Bernhard

    2017-09-01

    A visible light spectral domain optical coherence microscopy system was developed. A high axial resolution of 0.88 μm in tissue was achieved using a broad visible light spectrum (425 - 685 nm ). Healthy human brain tissue was imaged to quantify the difference between white (WM) and grey matter (GM) in intensity and attenuation. The high axial resolution enables the investigation of amyloid-beta plaques of various sizes in human brain tissue and animal models of Alzheimer's disease (AD). By performing a spectroscopic analysis of the OCM data, differences in the characteristics for WM, GM, and neuritic amyloid-beta plaques were found. To gain additional contrast, Congo red stained AD brain tissue was investigated. A first effort was made to investigate optically cleared mouse brain tissue to increase the penetration depth and visualize hyperscattering structures in deeper cortical regions.

  11. X-ray intravital microscopy for functional imaging in rat hearts using synchrotron radiation coronary microangiography.

    Science.gov (United States)

    Umetani, K; Fukushima, K

    2013-03-01

    An X-ray intravital microscopy technique was developed to enable in vivo visualization of the coronary, cerebral, and pulmonary arteries in rats without exposure of organs and with spatial resolution in the micrometer range and temporal resolution in the millisecond range. We have refined the system continually in terms of the spatial resolution and exposure time. X-rays transmitted through an object are detected by an X-ray direct-conversion type detector, which incorporates an X-ray SATICON pickup tube. The spatial resolution has been improved to 6 μm, yielding sharp images of small arteries. The exposure time has been shortened to around 2 ms using a new rotating-disk X-ray shutter, enabling imaging of beating rat hearts. Quantitative evaluations of the X-ray intravital microscopy technique were extracted from measurements of the smallest-detectable vessel size and detection of the vessel function. The smallest-diameter vessel viewed for measurements is determined primarily by the concentration of iodinated contrast material. The iodine concentration depends on the injection technique. We used ex vivo rat hearts under Langendorff perfusion for accurate evaluation. After the contrast agent is injected into the origin of the aorta in an isolated perfused rat heart, the contrast agent is delivered directly into the coronary arteries with minimum dilution. The vascular internal diameter response of coronary arterial circulation is analyzed to evaluate the vessel function. Small blood vessels of more than about 50 μm diameters were visualized clearly at heart rates of around 300 beats/min. Vasodilation compared to the control was observed quantitatively using drug manipulation. Furthermore, the apparent increase in the number of small vessels with diameters of less than about 50 μm was observed after the vasoactive agents increased the diameters of invisible small blood vessels to visible sizes. This technique is expected to offer the potential for direct

  12. Segmentation and Quantitative Analysis of Apoptosis of Chinese Hamster Ovary Cells from Fluorescence Microscopy Images.

    Science.gov (United States)

    Du, Yuncheng; Budman, Hector M; Duever, Thomas A

    2017-06-01

    Accurate and fast quantitative analysis of living cells from fluorescence microscopy images is useful for evaluating experimental outcomes and cell culture protocols. An algorithm is developed in this work to automatically segment and distinguish apoptotic cells from normal cells. The algorithm involves three steps consisting of two segmentation steps and a classification step. The segmentation steps are: (i) a coarse segmentation, combining a range filter with a marching square method, is used as a prefiltering step to provide the approximate positions of cells within a two-dimensional matrix used to store cells' images and the count of the number of cells for a given image; and (ii) a fine segmentation step using the Active Contours Without Edges method is applied to the boundaries of cells identified in the coarse segmentation step. Although this basic two-step approach provides accurate edges when the cells in a given image are sparsely distributed, the occurrence of clusters of cells in high cell density samples requires further processing. Hence, a novel algorithm for clusters is developed to identify the edges of cells within clusters and to approximate their morphological features. Based on the segmentation results, a support vector machine classifier that uses three morphological features: the mean value of pixel intensities in the cellular regions, the variance of pixel intensities in the vicinity of cell boundaries, and the lengths of the boundaries, is developed for distinguishing apoptotic cells from normal cells. The algorithm is shown to be efficient in terms of computational time, quantitative analysis, and differentiation accuracy, as compared with the use of the active contours method without the proposed preliminary coarse segmentation step.

  13. Chemical Reactions of Molecules Promoted and Simultaneously Imaged by the Electron Beam in Transmission Electron Microscopy.

    Science.gov (United States)

    Skowron, Stephen T; Chamberlain, Thomas W; Biskupek, Johannes; Kaiser, Ute; Besley, Elena; Khlobystov, Andrei N

    2017-08-15

    The main objective of this Account is to assess the challenges of transmission electron microscopy (TEM) of molecules, based on over 15 years of our work in this field, and to outline the opportunities in studying chemical reactions under the electron beam (e-beam). During TEM imaging of an individual molecule adsorbed on an atomically thin substrate, such as graphene or a carbon nanotube, the e-beam transfers kinetic energy to atoms of the molecule, displacing them from equilibrium positions. Impact of the e-beam triggers bond dissociation and various chemical reactions which can be imaged concurrently with their activation by the e-beam and can be presented as stop-frame movies. This experimental approach, which we term ChemTEM, harnesses energy transferred from the e-beam to the molecule via direct interactions with the atomic nuclei, enabling accurate predictions of bond dissociation events and control of the type and rate of chemical reactions. Elemental composition and structure of the reactant molecules as well as the operating conditions of TEM (particularly the energy of the e-beam) determine the product formed in ChemTEM processes, while the e-beam dose rate controls the reaction rate. Because the e-beam of TEM acts simultaneously as a source of energy for the reaction and as an imaging tool monitoring the same reaction, ChemTEM reveals atomic-level chemical information, such as pathways of reactions imaged for individual molecules, step-by-step and in real time; structures of illusive reaction intermediates; and direct comparison of catalytic activity of different transition metals filmed with atomic resolution. Chemical transformations in ChemTEM often lead to previously unforeseen products, demonstrating the potential of this method to become not only an analytical tool for studying reactions, but also a powerful instrument for discovery of materials that can be synthesized on preparative scale.

  14. Integrated local binary pattern texture features for classification of breast tissue imaged by optical coherence microscopy.

    Science.gov (United States)

    Wan, Sunhua; Lee, Hsiang-Chieh; Huang, Xiaolei; Xu, Ting; Xu, Tao; Zeng, Xianxu; Zhang, Zhan; Sheikine, Yuri; Connolly, James L; Fujimoto, James G; Zhou, Chao

    2017-05-01

    This paper proposes a texture analysis technique that can effectively classify different types of human breast tissue imaged by Optical Coherence Microscopy (OCM). OCM is an emerging imaging modality for rapid tissue screening and has the potential to provide high resolution microscopic images that approach those of histology. OCM images, acquired without tissue staining, however, pose unique challenges to image analysis and pattern classification. We examined multiple types of texture features and found Local Binary Pattern (LBP) features to perform better in classifying tissues imaged by OCM. In order to improve classification accuracy, we propose novel variants of LBP features, namely average LBP (ALBP) and block based LBP (BLBP). Compared with the classic LBP feature, ALBP and BLBP features provide an enhanced encoding of the texture structure in a local neighborhood by looking at intensity differences among neighboring pixels and among certain blocks of pixels in the neighborhood. Fourty-six freshly excised human breast tissue samples, including 27 benign (e.g. fibroadenoma, fibrocystic disease and usual ductal hyperplasia) and 19 breast carcinoma (e.g. invasive ductal carcinoma, ductal carcinoma in situ and lobular carcinoma in situ) were imaged with large field OCM with an imaging area of 10 × 10 mm 2 (10, 000 × 10, 000 pixels) for each sample. Corresponding H&E histology was obtained for each sample and used to provide ground truth diagnosis. 4310 small OCM image blocks (500 × 500 pixels) each paired with corresponding H&E histology was extracted from large-field OCM images and labeled with one of the five different classes: adipose tissue (n = 347), fibrous stroma (n = 2,065), breast lobules (n = 199), carcinomas (pooled from all sub-types, n = 1,127), and background (regions outside of the specimens, n = 572). Our experiments show that by integrating a selected set of LBP and the two new variant (ALBP and BLBP) features at multiple scales, the

  15. Fluorescein Derivatives in Intravital Fluorescence Imaging

    Directory of Open Access Journals (Sweden)

    Michael S. Roberts

    2013-08-01

    Full Text Available Intravital fluorescence microscopy enables the direct imaging of fluorophores in vivo and advanced techniques such as fluorescence lifetime imaging (FLIM enable the simultaneous detection of multiple fluorophores. Consequently, it is now possible to record distribution and metabolism of a chemical in vivo and to optimise the delivery of fluorophores in vivo. Recent clinical applications with fluorescein and other intravital fluorescent stains have occurred in neurosurgery, dermatology [including photodynamic therapy (PDT] and endomicroscopy. Potential uses have been identified in periodontal disease, skin graft and cancer surgery. Animal studies have demonstrated that diseased tissue can be specifically stained with fluorophore conjugates. This review focuses on the fluorescein derived fluorophores in common clinical use and provides examples of novel applications from studies in tissue samples.

  16. Alignment of large image series using cubic B-splines tessellation: application to transmission electron microscopy data.

    Science.gov (United States)

    Dauguet, Julien; Bock, Davi; Reid, R Clay; Warfield, Simon K

    2007-01-01

    3D reconstruction from serial 2D microscopy images depends on non-linear alignment of serial sections. For some structures, such as the neuronal circuitry of the brain, very large images at very high resolution are necessary to permit reconstruction. These very large images prevent the direct use of classical registration methods. We propose in this work a method to deal with the non-linear alignment of arbitrarily large 2D images using the finite support properties of cubic B-splines. After initial affine alignment, each large image is split into a grid of smaller overlapping sub-images, which are individually registered using cubic B-splines transformations. Inside the overlapping regions between neighboring sub-images, the coefficients of the knots controlling the B-splines deformations are blended, to create a virtual large grid of knots for the whole image. The sub-images are resampled individually, using the new coefficients, and assembled together into a final large aligned image. We evaluated the method on a series of large transmission electron microscopy images and our results indicate significant improvements compared to both manual and affine alignment.

  17. Fast imaging with inelastically scattered electrons by off-axis chromatic confocal electron microscopy.

    Science.gov (United States)

    Zheng, Changlin; Zhu, Ye; Lazar, Sorin; Etheridge, Joanne

    2014-04-25

    We introduce off-axis chromatic scanning confocal electron microscopy, a technique for fast mapping of inelastically scattered electrons in a scanning transmission electron microscope without a spectrometer. The off-axis confocal mode enables the inelastically scattered electrons to be chromatically dispersed both parallel and perpendicular to the optic axis. This enables electrons with different energy losses to be separated and detected in the image plane, enabling efficient energy filtering in a confocal mode with an integrating detector. We describe the experimental configuration and demonstrate the method with nanoscale core-loss chemical mapping of silver (M4,5) in an aluminium-silver alloy and atomic scale imaging of the low intensity core-loss La (M4,5@840  eV) signal in LaB6. Scan rates up to 2 orders of magnitude faster than conventional methods were used, enabling a corresponding reduction in radiation dose and increase in the field of view. If coupled with the enhanced depth and lateral resolution of the incoherent confocal configuration, this offers an approach for nanoscale three-dimensional chemical mapping.

  18. Toward Fourier interferometry fluorescence excitation/emission imaging of malignant cells combined with photoacoustic microscopy

    Science.gov (United States)

    Kohen, Elli; Hirschberg, Joseph G.; Berry, John P.; Ozkutuk, Nuri; Ornek, Ceren; Monti, Marco; Leblanc, Roger M.; Schachtschabel, Dietrich O.; Haroon, Sumaira

    2003-10-01

    Dual excitation fluorescence imaging has been used as a first step towards multi-wavelength excitation/emission fluorescence spectral imaging. Target cells are transformed keratinocytes, and other osteosarcoma, human breast and color cancer cells. Mitochondrial membrane potential probes, e.g. TMRM (tetramethylrhodamine methyl ester), Mitotracker Green (Molecular Probes, Inc., Eugene OR,USA; a recently synthesized mitochondrial oxygen probe, [PRE,P1"- pyrene butyl)-2-rhodamine ester] allow dual excitation in the UV plus in teh blue-green spectral regions. Also, using the natural endogenous probe NAD(P)H, preliminary results indicate mitochondrial responses to metabolic challenges (e.g. glucose addition), plus changes in mitochonrial distribution and morphology. In terms of application to biomedicine (for diagnostiscs, prognostsics and drug trials) three parameters have been selected in addition to the natural probe NAD(P)H, i.e. vital fluorescence probing of mitochondria, lysosomes and Golgi apparatus. It is hoped that such a multiparameter approach will allow malignant cell characterization and grading. A new area being introduced is the use of similar methodology for biotechnical applications such as the study of the hydrogen-producing alga Chlamydomonas Reinhardtii, and possible agricultural applications, such as Saccharomyces yeast for oenology. Complementation by Photoacoustic Microscopy is also contemplated, to study the internal conversion component which follows the excitation by photons.

  19. Photo-excited hot carrier dynamics in hydrogenated amorphous silicon imaged by 4D electron microscopy

    Science.gov (United States)

    Liao, Bolin; Najafi, Ebrahim; Li, Heng; Minnich, Austin J.; Zewail, Ahmed H.

    2017-09-01

    Charge carrier dynamics in amorphous semiconductors has been a topic of intense research that has been propelled by modern applications in thin-film solar cells, transistors and optical sensors. Charge transport in these materials differs fundamentally from that in crystalline semiconductors owing to the lack of long-range order and high defect density. Despite the existence of well-established experimental techniques such as photoconductivity time-of-flight and ultrafast optical measurements, many aspects of the dynamics of photo-excited charge carriers in amorphous semiconductors remain poorly understood. Here, we demonstrate direct imaging of carrier dynamics in space and time after photo-excitation in hydrogenated amorphous silicon (a-Si:H) by scanning ultrafast electron microscopy (SUEM). We observe an unexpected regime of fast diffusion immediately after photoexcitation, together with spontaneous electron-hole separation and charge trapping induced by the atomic disorder. Our findings demonstrate the rich dynamics of hot carrier transport in amorphous semiconductors that can be revealed by direct imaging based on SUEM.

  20. Transmission x-ray microscopy at Diamond-Manchester I13 Imaging Branchline

    Energy Technology Data Exchange (ETDEWEB)

    Vila-Comamala, Joan, E-mail: joan.vila.comamala@gmail.com; Wagner, Ulrich; Bodey, Andrew J.; Garcia-Fernandez, Miryam; Rau, Christoph [Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Bosgra, Jeroen; David, Christian [Paul Scherrer Institut, 5232 PSI-Villigen (Switzerland); Eastwood, David S. [Manchester X-ray Imaging Facility, School of Materials, University of Manchester, Manchester M13 9PL, UK and Research Complex at Harwell, Harwell Campus, Didcot OX11 0FA (United Kingdom)

    2016-01-28

    Full-field Transmission X-ray Microscopy (TXM) has been shown to be a powerful method for obtaining quantitative internal structural and chemical information from materials at the nanoscale. The installation of a Full-field TXM station will extend the current microtomographic capabilities of the Diamond-Manchester I13 Imaging Branchline at Diamond Light Source (UK) into the sub-100 nm spatial resolution range using photon energies from 8 to 14 keV. The dedicated Full-field TXM station will be built in-house with contributions of Diamond Light Source support divisions and via collaboration with the X-ray Optics Group of Paul Scherrer Institut (Switzerland) which will develop state-of-the-art diffractive X-ray optical elements. Preliminary results of the I13 Full-field TXM station are shown. The Full-field TXM will become an important Diamond Light Source direct imaging asset for material science, energy science and biology at the nanoscale.