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Sample records for imaging microscopy flim

  1. Analysis of human aorta using fluorescence lifetime imaging microscopy (FLIM)

    Science.gov (United States)

    Vieira-Damiani, Gislaine; Adur, J.; Ferro, D. P.; Adam, R. L.; Pelegati, V.; Thomáz, A.; Cesar, C. L.; Metze, K.

    2012-03-01

    The use of photonics has improved our understanding of biologic phenomena. For the study of the normal and pathologic architecture of the aorta the use of Two-Photon Excited Fluorescence (TPEF) and Second Harmonic Generation showed interesting details of morphologic changes of the elastin-collagen architecture during aging or development of hypertension in previous studies. In this investigation we tried to apply fluorescence lifetime imaging (FLIM) for the morphologic analysis of human aortas. The aim of our study was to use FLIM in non-stained formalin-fixed and paraffin-embedded samples of the aorta ascendants in hypertensive and normotensive patients of various ages, examining two different topographical regions. The FLIM-spectra of collagen and elastic fibers were clearly distinguishable, thus permitting an exact analysis of unstained material on the microscopic level. Moreover the FLIM spectrum of elastic fibers revealed variations between individual cases, which indicate modifications on a molecular level and might be related to FLIM age or diseases states and reflect modifications on a molecular level.

  2. Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase.

    Science.gov (United States)

    Okkelman, Irina A; Dmitriev, Ruslan I; Foley, Tara; Papkovsky, Dmitri B

    2016-01-01

    Incorporation of thymidine analogues in replicating DNA, coupled with antibody and fluorophore staining, allows analysis of cell proliferation, but is currently limited to monolayer cultures, fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation, S phase progression over several division cycles, effects of anti-proliferative drugs and other applications. It is based on the prominent (~ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain, Hoechst 33342 upon the incorporation of 5-bromo-2'-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content, multi-parametric dynamic analyses, far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures, complex 3D tissue models of tumor cell spheroids and intestinal organoids, and in physiological study with metformin treatment.

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

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    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. Signal peptide peptidase (SPP dimer formation as assessed by fluorescence lifetime imaging microscopy (FLIM in intact cells

    Directory of Open Access Journals (Sweden)

    Nyborg Andrew C

    2006-11-01

    Full Text Available Abstract Background Signal peptide peptidase (SPP is an intramembrane cleaving protease identified by its cleavage of several type II membrane signal peptides. Conservation of intramembrane active site residues demonstrates that SPP, SPP family members, and presenilins (PSs make up a family of intramembrane cleaving proteases. Because SPP appears to function without additional protein cofactors, the study of SPP may provide structural insights into the mechanism of intramembrane proteolysis by this biomedically important family of proteins. Previous studies have shown that SPP isolated from cells appears to be a homodimer, but some evidence exists that in vitro SPP may be active as a monomer. We have conducted additional experiments to determine if SPP exists as a monomer or dimer in vivo. Results Fluorescence lifetime imaging microscopy (FLIM can be is used to determine intra- or intermolecular interactions by fluorescently labeling epitopes on one or two different molecules. If the donor and acceptor fluorophores are less than 10 nm apart, the donor fluorophore lifetime shortens proportionally to the distance between the fluorophores. In this study, we used two types of fluorescence energy transfer (FRET pairs; cyan fluorescent protein (CFP with yellow fluorescent protein (YFP or Alexa 488 with Cy3 to differentially label the NH2- or COOH-termini of SPP molecules. A cell based SPP activity assay was used to show that all tagged SPP proteins are proteolytically active. Using FLIM we were able to show that the donor fluorophore lifetime of the CFP tagged SPP construct in living cells significantly decreases when either a NH2- or COOH-terminally YFP tagged SPP construct is co-transfected, indicating close proximity between two different SPP molecules. These data were then confirmed in cell lines stably co-expressing V5- and FLAG-tagged SPP constructs. Conclusion Our FLIM data strongly suggest dimer formation between two separate SPP proteins

  5. Fluorescence and fluorescence-lifetime imaging microscopy (FLIM) to characterize yeast strains by autofluorescence

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    Bhatta, H.; Goldys, E. M.; Ma, J.

    2006-02-01

    We characterised populations of wild type baking and brewing yeast cells using intrinsic fluorescence and fluorescence lifetime microscopy, in order to obtain quantitative identifiers of different strains. The cell autofluorescence was excited at 405 nm and observed within 440-540 nm range where strong cell to cell variability was observed. The images were analyzed using customised public domain software, which provided information on cell size, intensity and texture-related features. In light of significant diversity of the data, statistical methods were utilized to assess the validity of the proposed quantitative identifiers for strain differentiation. The Kolmogorov-Smirnov test was applied to confirm that empirical distribution functions for size, intensity and entropy for different strains were statistically different. These characteristics were followed with culture age of 24, 48 and 72 h, (the latter corresponding to a stationary growth phase) and size, and to some extent entropy, were found to be independent of age. The fluorescence intensity presented a distinctive evolution with age, different for each of the examined strains. The lifetime analysis revealed a short decay time component of 1.4 ns and a second, longer one with the average value of 3.5 ns and a broad distribution. High variability of lifetime values within cells was observed however a lifetime texture feature in the studied strains was statistically different.

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

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    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.

  7. A STED-FLIM microscope applied to imaging the natural killer cell immune synapse

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    Lenz, M. O.; Brown, A. C. N.; Auksorius, E.; Davis, D. M.; Dunsby, C.; Neil, M. A. A.; French, P. M. W.

    2011-03-01

    We present a stimulated emission depletion (STED) fluorescence lifetime imaging (FLIM) microscope, excited by a microstructured optical fibre supercontinuum source that is pumped by a femtosecond Ti:Sapphire-laser, which is also used for depletion. Implemented using a piezo-scanning stage on a laser scanning confocal fluorescence microscope system with FLIM realised using time correlated single photon counting (TCSPC), this provides convenient switching between confocal and STED-FLIM with spatial resolution down to below 60 nm. We will present our design considerations to make a robust instrument for biological applications including a comparison between fixed phase plate and spatial light modulator (SLM) approaches to shape the STED beam and the correlation of STED and confocal FLIM microscopy. Following our previous application of FLIM-FRET to study intercellular signalling at the immunological synapse (IS), we are employing STED microscopy to characterize the spatial distribution of cellular molecules with subdiffraction resolution at the IS. In particular, we are imaging cytoskeletal structure at the Natural Killer cell activated immune synapse. We will also present our progress towards multilabel STED microscopy to determine how relative spatial molecular organization, previously undetectable by conventional microscopy techniques, is important for NK cell cytotoxic function. Keywords: STED, Stimulated Emission Depletion Microscopy, Natural Killer (NK) cell, Fluorescence lifetime imaging, FLIM, Super resolution microscopy.

  8. Use of fluorescence lifetime imaging (FLIM) for latent fingerprints detection

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    Wang, Peng; Chao, Zhi Xia; Seah, Leong K.; Murukeshan, Vadakke M.

    2005-04-01

    Fluorescence lifetime imaging (FLIM) in frequency domain enables the mapping of the spatial distribution of fluorescence lifetimes of a specimen. FLIM can provide unique information about fluorophores and hence is widely used in biology and for medical diagnostics. In this paper, a theoretical analysis for the fluorescence lifetime determination of latent fingerprint samples is described, which is followed by the feasibility study of using FLIM in frequency domain for latent fingerprints detection. Experiments are carried out with fingerprint on green paper substrate and postcard substrate treated with certain fluorescent powder. The total phase lag and demodulation factor are calculated to determine the lifetimes pixel by pixel. The resulting fluorescence lifetime image of fingerprint revealed an improvement in the contrast, and was able to detect the latent fingerprint clearly.

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

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    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.

  10. Photon budget analysis for fluorescence lifetime imaging microscopy

    NARCIS (Netherlands)

    Zhao, Q.; Young, I.T.; De Jong, J.G.S.

    2011-01-01

    We have constructed a mathematical model to analyze the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy (FLIM). The power of the light source needed for illumination in a FLIM system and the signal-to-noise ratio of the detector have led us to a photon “budget.” These

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

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    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.

  12. Two-photon-excited fluorescence (TPEF) and fluorescence lifetime imaging (FLIM) with sub-nanosecond pulses and a high analog bandwidth signal detection

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    Eibl, Matthias; Karpf, Sebastian; Hakert, Hubertus; Weng, Daniel; Huber, Robert

    2017-02-01

    Two-photon excited fluorescence (TPEF) microscopy and fluorescence lifetime imaging (FLIM) are powerful imaging techniques in bio-molecular science. The need for elaborate light sources for TPEF and speed limitations for FLIM, however, hinder an even wider application. 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 synchronized to a high analog bandwidth signal detection for single shot TPEF- and single shot FLIM imaging. The actively modulated pulses at 1064nm from the fiber laser are adjustable from 50ps to 5ns with kW of peak power. At a typically applied pulse lengths and repetition rates, the duty cycle is comparable to typically used femtosecond pulses and thus the peak power is also comparable at same cw-power. Hence, both types of excitation should yield the same number of fluorescence photons per time on average when used for TPEF imaging. However, in the 100ps configuration, a thousand times more fluorescence photons are generated per pulse. In this paper, we now show that the 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 the performance of our system, we acquired FLIM images of a Convallaria sample with pixel rates of 1 MHz where the lifetime information is directly measured with a fast real time digitizer. With the presented results, we show that longer pulses in the many-10ps to nanosecond regime can be readily applied for TPEF imaging and enable new imaging modalities like single pulse FLIM.

  13. Rapid FLIM: The new and innovative method for ultra-fast imaging of biological processes (Conference Presentation)

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    Orthaus-Mueller, Sandra; Kraemer, Benedikt; Tannert, Astrid; Roehlicke, Tino; Wahl, Michael; Rahn, Hans-Juergen; Koberling, Felix; Erdmann, Rainer

    2017-02-01

    Over the last two decades, time-resolved fluorescence microscopy has become an essential tool in Life Sciences thanks to measurement procedures such as Fluorescence Lifetime Imaging (FLIM), lifetime based Foerster Resonance Energy Transfer (FRET), and Fluorescence (Lifetime) Correlation Spectroscopy (F(L)CS) down to the single molecule level. Today, complete turn-key systems are available either as stand-alone units or as upgrades for confocal laser scanning microscopes (CLSM). Data acquisition on such systems is typically based on Time-Correlated Single Photon Counting (TCSPC) electronics along with picosecond pulsed diode lasers as excitation sources and highly sensitive, single photon counting detectors. Up to now, TCSPC data acquisition is considered a somewhat slow process as a large number of photons per pixel is required for reliable data analysis, making it difficult to use FLIM for following fast FRET processes, such as signal transduction pathways in cells or fast moving sub-cellular structures. We present here a novel and elegant solution to tackle this challenge. Our approach, named rapidFLIM, exploits recent hardware developments such as TCSPC modules with ultra short dead times and hybrid photomultiplier detector assemblies enabling significantly higher detection count rates. Thanks to these improved components, it is possible to achieve much better photon statistics in significantly shorter time spans while being able to perform FLIM imaging for fast processes in a qualitative manner and with high optical resolution. FLIM imaging can now be performed with up to several frames per second making it possible to study fast processes such as protein interactions involved in endosome trafficking.

  14. Multiphoton Microscopy for Ophthalmic Imaging

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    Emily A. Gibson

    2011-01-01

    Full Text Available We review multiphoton microscopy (MPM including two-photon autofluorescence (2PAF, second harmonic generation (SHG, third harmonic generation (THG, fluorescence lifetime (FLIM, and coherent anti-Stokes Raman Scattering (CARS with relevance to clinical applications in ophthalmology. The different imaging modalities are discussed highlighting the particular strength that each has for functional tissue imaging. MPM is compared with current clinical ophthalmological imaging techniques such as reflectance confocal microscopy, optical coherence tomography, and fluorescence imaging. In addition, we discuss the future prospects for MPM in disease detection and clinical monitoring of disease progression, understanding fundamental disease mechanisms, and real-time monitoring of drug delivery.

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

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    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.

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

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    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.

  17. Temporal Data Set Reduction Based on D-Optimality for Quantitative FLIM-FRET Imaging.

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    Omer, Travis; Intes, Xavier; Hahn, Juergen

    2015-01-01

    Fluorescence lifetime imaging (FLIM) when paired with Förster resonance energy transfer (FLIM-FRET) enables the monitoring of nanoscale interactions in living biological samples. FLIM-FRET model-based estimation methods allow the quantitative retrieval of parameters such as the quenched (interacting) and unquenched (non-interacting) fractional populations of the donor fluorophore and/or the distance of the interactions. The quantitative accuracy of such model-based approaches is dependent on multiple factors such as signal-to-noise ratio and number of temporal points acquired when sampling the fluorescence decays. For high-throughput or in vivo applications of FLIM-FRET, it is desirable to acquire a limited number of temporal points for fast acquisition times. Yet, it is critical to acquire temporal data sets with sufficient information content to allow for accurate FLIM-FRET parameter estimation. Herein, an optimal experimental design approach based upon sensitivity analysis is presented in order to identify the time points that provide the best quantitative estimates of the parameters for a determined number of temporal sampling points. More specifically, the D-optimality criterion is employed to identify, within a sparse temporal data set, the set of time points leading to optimal estimations of the quenched fractional population of the donor fluorophore. Overall, a reduced set of 10 time points (compared to a typical complete set of 90 time points) was identified to have minimal impact on parameter estimation accuracy (≈5%), with in silico and in vivo experiment validations. This reduction of the number of needed time points by almost an order of magnitude allows the use of FLIM-FRET for certain high-throughput applications which would be infeasible if the entire number of time sampling points were used.

  18. 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.

  19. Timing and Operating Mode Design for Time-Gated Fluorescence Lifetime Imaging Microscopy

    OpenAIRE

    Chao Liu; Xinwei Wang; Yan Zhou; Yuliang Liu

    2013-01-01

    Steady-state fluorence imaging and time-resolved fluorescence imaging are two important areas in fluorescence imaging research. Fluorescence lifetime imaging is an absolute measurement method which is independent of excitation laser intensity, fluorophore concentration, and photobleaching compared to fluorescence intensity imaging techniques. Time-gated fluorescence lifetime imaging microscopy (FLIM) can provide high resolution and high imaging frame during mature FLIM methods. An abstract ti...

  20. Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM

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    George Chennell

    2016-08-01

    Full Text Available We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP Activated Protein Kinase (AMPK FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR, for fluorescence lifetime imaging (FLIM readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP, in the original FRET biosensor, AMPK activity reporter (AMPKAR, with mTurquoise2 (mTq2FP, increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation.

  1. Clinical multiphoton FLIM tomography

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    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. Time-gated FLIM microscope for corneal metabolic imaging

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    Silva, Susana F.; Batista, Ana; Domingues, José Paulo; Quadrado, Maria João.; Morgado, António Miguel

    2016-03-01

    Detecting corneal cells metabolic alterations may prove a valuable tool in the early diagnosis of corneal diseases. Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent metabolic co-factors that allow the assessment of metabolic changes through non-invasive optical methods. These co-factors exhibit double-exponential fluorescence decays, with well-separated short and lifetime components, which are related to their protein-bound and free-states. Corneal metabolism can be assessed by measuring the relative contributions of these two components. For that purpose, we have developed a wide-field time-gated fluorescence lifetime microscope based on structured illumination and one-photon excitation to record FAD lifetime images from corneas. NADH imaging was not considered as its UV excitation peak is regarded as not safe for in vivo measurements. The microscope relies on a pulsed blue diode laser (λ=443 nm) as excitation source, an ultra-high speed gated image intensifier coupled to a CCD camera to acquire fluorescence signals and a Digital Micromirror Device (DMD) to implement the Structured Illumination technique. The system has a lateral resolution better than 2.4 μm, a field of view of 160 per 120 μm and an optical sectioning of 6.91 +/- 0.45 μm when used with a 40x, 0.75 NA, Water Immersion Objective. With this setup we were able to measure FAD contributions from ex-vivo chicken corneas collected from a local slaughterhouse..

  3. Fluorescence liftime imaging (FLIM) using ps-pulsed diode lasers in laser scanning microscopes

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    Ruck, Angelika C.; Dolp, Frank; Happ, Claudia; Steiner, Rudolf; Beil, Michael

    2003-06-01

    A setup consisting on a laser scanning microscope equipped with appropriate detection units was developed for time-resolved intracellular fluorescence spectroscopy and fluorescence lifetime imaging (FLIM) for on-line detection of structural changes of various biomolecules. Short-pulsed excitation was performed with a diode laser which emits pulses at 398 nm with 70 ps duration. The laser was coupled to the laser scanning microscope. For time resolved spectroscopy a setup consisting of an Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in "spot scan" mode. In addition, a time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images (τ-mapping). The time-resolved fluorescence characteristics of 5-ALA (5-aminolevulinic-acid), as well as 5-ALAhe (5-aminolevulinic-acid-hexylester)- induced protoporphyrine IX (PPIX) were investigated before and during PDT with subcellular resolution. For cells which were incubated with 5-ALA, a component with a fluorescence lifetime of about 7 ns was correlated with a structured fluorescence, which probably coincides with mitochondria, whereas a shorter lifetime was found in the cytoplasm. In the case of 5-ALAhe the lifetime of PPIX was longer, which could be due to different localization. During PDT the component with the longer lifetime completely vanished, whereas the shorter liftime was retained. It seems that FLIM is a valuable method to selectively identify and localize the photodynamically active photosensitizer.

  4. Combination of a spinning disc confocal unit with frequency-domain fluorescence lifetime imaging microscopy.

    NARCIS (Netherlands)

    van Munster, E.B.; Goedhart, J.; Kremers, G.J.; Manders, E.M.M.; Gadella, Th.W.J.

    2007-01-01

    BACKGROUND: Wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM) is an established technique to determine fluorescence lifetimes. Disadvantage of wide-field imaging is that measurements are compromised by out-of-focus blur. Conventional scanning confocal typically means long

  5. High-speed confocal fluorescence lifetime imaging microscopy by analog mean-delay method

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    Won, Youngjae; Kim, Donguk; Yang, Wenzhong; Kim, Dug Y.

    2010-02-01

    We have demonstrated the high-speed confocal fluorescence lifetime imaging microscopy (FLIM) by analog mean-delay (AMD) method. The AMD method is a new signal processing technique for calculation of fluorescence lifetime and it is very suitable for the high-speed confocal FLIM with good accuracy and photon economy. We achieved the acquisition speed of 7.7 frames per second for confocal FLIM imaging. Here, the highest photon detection rate for one pixel was larger than 125 MHz and averaged photon detection rate was more than 62.5 MHz. Based on our system, we successfully obtained a sequence of confocal fluorescence lifetime images of RBL-2H3 cell labeled with Fluo-3/AM and excited by 4αPDD (TRPV channel agonist) within one second.

  6. Visualizing heterogeneity of photosynthetic properties of plant leaves with two-photon fluorescence lifetime imaging microscopy

    NARCIS (Netherlands)

    Iermak, Ievgeniia; Vink, Jochem; Bader, Arjen N.; Wientjes, Emilie; Amerongen, van Herbert

    2016-01-01

    Two-photon fluorescence lifetime imaging microscopy (FLIM) was used to analyse the distribution and properties of Photosystem I (PSI) and Photosystem II (PSII) in palisade and spongy chloroplasts of leaves from the C3 plant Arabidopsis thaliana and the C4 plant Miscanthus x giganteus. This was ac

  7. Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy

    NARCIS (Netherlands)

    Manen, van Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; Berg, van den 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 gp91phox, which are both subunits of the phagocyte NADPH oxidase

  8. A phasor approach analysis of multiphoton FLIM measurements of three-dimensional cell culture models

    Science.gov (United States)

    Lakner, P. H.; Möller, Y.; Olayioye, M. A.; Brucker, S. Y.; Schenke-Layland, K.; Monaghan, M. G.

    2016-03-01

    Fluorescence lifetime imaging microscopy (FLIM) is a useful approach to obtain information regarding the endogenous fluorophores present in biological samples. The concise evaluation of FLIM data requires the use of robust mathematical algorithms. In this study, we developed a user-friendly phasor approach for analyzing FLIM data and applied this method on three-dimensional (3D) Caco-2 models of polarized epithelial luminal cysts in a supporting extracellular matrix environment. These Caco-2 based models were treated with epidermal growth factor (EGF), to stimulate proliferation in order to determine if FLIM could detect such a change in cell behavior. Autofluorescence from nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) in luminal Caco-2 cysts was stimulated by 2-photon laser excitation. Using a phasor approach, the lifetimes of involved fluorophores and their contribution were calculated with fewer initial assumptions when compared to multiexponential decay fitting. The phasor approach simplified FLIM data analysis, making it an interesting tool for non-experts in numerical data analysis. We observed that an increased proliferation stimulated by EGF led to a significant shift in fluorescence lifetime and a significant alteration of the phasor data shape. Our data demonstrates that multiphoton FLIM analysis with the phasor approach is a suitable method for the non-invasive analysis of 3D in vitro cell culture models qualifying this method for monitoring basic cellular features and the effect of external factors.

  9. Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells.

    Science.gov (United States)

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-09-22

    In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

  10. Photothermal imaging scanning microscopy

    Science.gov (United States)

    Chinn, Diane; Stolz, Christopher J.; Wu, Zhouling; Huber, Robert; Weinzapfel, Carolyn

    2006-07-11

    Photothermal Imaging Scanning Microscopy produces a rapid, thermal-based, non-destructive characterization apparatus. Also, a photothermal characterization method of surface and subsurface features includes micron and nanoscale spatial resolution of meter-sized optical materials.

  11. 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 inte

  12. 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

  13. Frequency-domain fluorescence lifetime imaging system (pco.flim) based on a in-pixel dual tap control CMOS image sensor

    Science.gov (United States)

    Franke, Robert; Holst, Gerhard A.

    2015-03-01

    The luminescence lifetime as a beneficial analytical parameter is known for many years and is well described by a large variety of publications. Many instruments including 2D measuring systems with cameras have been developed and applied in the past years. However, since the current instrumentation to perform either time- or frequency-domain lifetime measurements is rather complex, new developments in CMOS image sensor technology have achieved to create new image sensors, which can efficiently be integrated into easier-to-handle luminescence lifetime measuring systems. The principle of these modulatable CMOS image sensors, while initially being designed for distance measurements, shows a clear analogy to frequency-domain FLIM measurements, which was proven by researchers [1, 2]. Based on this principle a new CMOS image sensor has been developed, integrated into a camera system and has been investigated within a research project. The image sensor has a resolution of 1024 × 1024 pixels with a 5.6 μm pitch and can be modulated up to 50 MHz. First measurements show an effective dynamic range of larger than 1:1024 (corresponding to 10 bit dynamic). The maximum frame rate is in the range of 90 frames/s in dual-tap mode, resulting in an effective lifetime image frame rate for realistic measurements of approximately 22 frames/s. The camera system pco.flim, featuring that image sensor, generates all required modulation signals from 5 kHz to 50 MHz (sinusoidal and rectangular). It performs advanced pixel correction to generate linear and high-quality images, while the basic lifetime image processing is done in the computer. The modulation frequency can be freely adjusted within the specified range. The characteristics of the camera systems are presented, and first results are discussed using different representations of the data like for example the phasor approach [3], which has been established to provide a more global view to pixelwise fluorescence lifetime data and

  14. Optical imaging. Expansion microscopy.

    Science.gov (United States)

    Chen, Fei; Tillberg, Paul W; Boyden, Edward S

    2015-01-30

    In optical microscopy, fine structural details are resolved by using refraction to magnify images of a specimen. We discovered that by synthesizing a swellable polymer network within a specimen, it can be physically expanded, resulting in physical magnification. By covalently anchoring specific labels located within the specimen directly to the polymer network, labels spaced closer than the optical diffraction limit can be isotropically separated and optically resolved, a process we call expansion microscopy (ExM). Thus, this process can be used to perform scalable superresolution microscopy with diffraction-limited microscopes. We demonstrate ExM with apparent ~70-nanometer lateral resolution in both cultured cells and brain tissue, performing three-color superresolution imaging of ~10(7) cubic micrometers of the mouse hippocampus with a conventional confocal microscope.

  15. Fluorescence lifetime imaging microscopy of nanodiamonds in vivo

    Science.gov (United States)

    Kuo, Yung; Hsu, Tsung-Yuan; Wu, Yi-Chun; Hsu, Jui-Hung; Chang, Huan-Cheng

    2013-03-01

    The negatively charged nitrogen-vacancy (NV-) center in bulk diamond is a photostable fluorophore with a radiative lifetime of 11.6 ns at room temperature. The lifetime substantially increases to ~20 ns for diamond nanoparticles (size ~ 100 nm) suspended in water due to the change in refractive index of the surrounding medium of the NV- centers. This fluorescence decay time is much longer than that (typically 1 - 4 ns) of endogenous and exogenous fluorophores commonly used in biological imaging, making it possible to detect NV--containing nanodiamonds in vivo at the single particle level by fluorescence lifetime imaging microscopy (FLIM). We demonstrate the feasibility of this approach using Caenorhabditis elegans (C. elegans) as a model organism.

  16. Quantitative mapping of aqueous microfluidic temperature with sub-degree resolution using fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Graham, Emmelyn M; Iwai, Kaoru; Uchiyama, Seiichi; de Silva, A Prasanna; Magennis, Steven W; Jones, Anita C

    2010-05-21

    The use of a water-soluble, thermo-responsive polymer as a highly sensitive fluorescence-lifetime probe of microfluidic temperature is demonstrated. The fluorescence lifetime of poly(N-isopropylacrylamide) labelled with a benzofurazan fluorophore is shown to have a steep dependence on temperature around the polymer phase transition and the photophysical origin of this response is established. The use of this unusual fluorescent probe in conjunction with fluorescence lifetime imaging microscopy (FLIM) enables the spatial variation of temperature in a microfluidic device to be mapped, on the micron scale, with a resolution of less than 0.1 degrees C. This represents an increase in temperature resolution of an order of magnitude over that achieved previously by FLIM of temperature-sensitive dyes.

  17. Exciton-polaron quenching in organic thin-film transistors studied by fluorescence lifetime imaging microscopy

    DEFF Research Database (Denmark)

    Jensen, Per Baunegaard With; Leißner, Till; Osadnik, Andreas

    Organic semiconductors show great potential in electronic and optical applications. However, a major challenge is the degradation of the semiconductor materials that cause a reduction in device performance. Here, we present our investigations of Organic Thin Film Transistors (OTFT) based...... that correlates with the local charge density indicates a pronounced exciton quenching by the injected charges. Subsequent FLIM measurements on previously biased OTFT devices show a general decrease in fluorescence lifetime suggesting degradation of the organic semiconductor. This is correlated with the results...... on the material 5,5-bis(naphthyl)-2,20-bithiophene (NaT2). These types of OTFT have previously been shown to have light emitting properties. Fluorescence Lifetime Imaging Microscopy (FLIM) has been used to investigate the exciton-polaron quenching in biased OTFTs. A clear reduction in fluorescence lifetime...

  18. A 72 × 60 Angle-Sensitive SPAD Imaging Array for Lens-less FLIM

    Directory of Open Access Journals (Sweden)

    Changhyuk Lee

    2016-09-01

    Full Text Available We present a 72 × 60, angle-sensitive single photon avalanche diode (A-SPAD array for lens-less 3D fluorescence lifetime imaging. An A-SPAD pixel consists of (1 a SPAD to provide precise photon arrival time where a time-resolved operation is utilized to avoid stimulus-induced saturation, and (2 integrated diffraction gratings on top of the SPAD to extract incident angles of the incoming light. The combination enables mapping of fluorescent sources with different lifetimes in 3D space down to micrometer scale. Futhermore, the chip presented herein integrates pixel-level counters to reduce output data-rate and to enable a precise timing control. The array is implemented in standard 180 nm complementary metal-oxide-semiconductor (CMOS technology and characterized without any post-processing.

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

    Science.gov (United States)

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

    2008-04-15

    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 oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91(phox) are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91(phox). By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91(phox) are approximately 1.38 and approximately 1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane.

  20. A dual-modality optical coherence tomography and fluorescence lifetime imaging microscopy system for simultaneous morphological and biochemical tissue characterization.

    Science.gov (United States)

    Park, Jesung; Jo, Javier A; Shrestha, Sebina; Pande, Paritosh; Wan, Qiujie; Applegate, Brian E

    2010-07-16

    Most pathological conditions elicit changes in the tissue optical response that may be interrogated by one or more optical imaging modalities. Any single modality typically only furnishes an incomplete picture of the tissue optical response, hence an approach that integrates complementary optical imaging modalities is needed for a more comprehensive non-destructive and minimally-invasive tissue characterization. We have developed a dual-modality system, incorporating optical coherence tomography (OCT) and fluorescence lifetime imaging microscopy (FLIM), that is capable of simultaneously characterizing the 3-D tissue morphology and its biochemical composition. The Fourier domain OCT subsystem, at an 830 nm center wavelength, provided high-resolution morphological volumetric tissue images with an axial and lateral resolution of 7.3 and 13.4 µm, respectively. The multispectral FLIM subsystem, based on a direct pulse-recording approach (upon 355 nm laser excitation), provided two-dimensional superficial maps of the tissue autofluorescence intensity and lifetime at three customizable emission bands with 100 µm lateral resolution. Both subsystems share the same excitation/illumination optical path and are simultaneously raster scanned on the sample to generate coregistered OCT volumes and FLIM images. The developed OCT/FLIM system was capable of a maximum A-line rate of 59 KHz for OCT and a pixel rate of up to 30 KHz for FLIM. The dual-modality system was validated with standard fluorophore solutions and subsequently applied to the characterization of two biological tissue types: postmortem human coronary atherosclerotic plaques, and in vivo normal and cancerous hamster cheek pouch epithelial tissue.

  1. 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.

  2. Cellular resolution multiplexed FLIM tomography with dual-color Bessel beam.

    Science.gov (United States)

    Xu, Dongli; Zhou, Weibin; Peng, Leilei

    2017-02-01

    Fourier multiplexed FLIM (FmFLIM) tomography enables multiplexed 3D lifetime imaging of whole embryos. In our previous FmFLIM system, the spatial resolution was limited to 25 μm because of the trade-off between the spatial resolution and the imaging depth. In order to achieve cellular resolution imaging of thick specimens, we built a tomography system with dual-color Bessel beam. In combination with FmFLIM, the Bessel FmFLIM tomography system can perform parallel 3D lifetime imaging on multiple excitation-emission channels at a cellular resolution of 2.8 μm. The image capability of the Bessel FmFLIM tomography system was demonstrated by 3D lifetime imaging of dual-labeled transgenic zebrafish embryos.

  3. Studying Biological Tissue with Fluorescence Lifetime Imaging: Microscopy, Endoscopy, and Complex Decay Profiles

    Science.gov (United States)

    Siegel, Jan; Elson, Daniel S.; Webb, Stephen E. D.; Lee, K. C. Benny; Vlandas, Alexis; Gambaruto, Giovanni L.; Léveque-Fort, Sandrine; Lever, M. John; Tadrous, Paul J.; Stamp, Gordon W. H.; Wallace, Andrew L.; Sandison, Ann; Watson, Tim F.; Alvarez, Fernando; French, Paul M. W.

    2003-06-01

    We have applied fluorescence lifetime imaging (FLIM) to the autofluorescence of different kinds of biological tissue in vitro , including animal tissue sections and knee joints as well as human teeth, obtaining two-dimensional maps with functional contrast. We find that fluorescence decay profiles of biological tissue are well described by the stretched exponential function (StrEF), which can represent the complex nature of tissue. The StrEF yields a continuous distribution of fluorescence lifetimes, which can be extracted with an inverse Laplace transformation, and additional information is provided by the width of the distribution. Our experimental results from FLIM microscopy in combination with the StrEF analysis indicate that this technique is ready for clinical deployment, including portability that is through the use of a compact picosecond diode laser as the excitation source. The results obtained with our FLIM endoscope successfully demonstrated the viability of this modality, though they need further optimization. We expect a custom-designed endoscope with optimized illumination and detection efficiencies to provide significantly improved performance.

  4. Advanced Imaging Approaches to Characterize Stromal and Metabolic Changes in In Vivo Mammary Tumor Models

    Science.gov (United States)

    2013-03-01

    have been using Fluorescence Lifetime Imaging Microscopy (FLIM) (7) to examine Nicotinamide adenine dinucleotide (NADH) and Flavin adenine...an optical imaging window into the skin of these mice above the tumor. We will then collect SHG from collagen and FLIM data for NADH using an MPM

  5. Multimodal optical setup for nonlinear and fluorescence lifetime imaging microscopies: improvement on a commercial confocal inverted microscope

    Science.gov (United States)

    Pelegati, V. B.; Adur, J.; de Thomaz, A. A.; Almeida, D. B.; Baratti, M. O.; Carvalho, H. F.; Cesar, C. L.

    2012-03-01

    In this work we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus FV300) to include nonlinear optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). The NLO microscopies included two-photon fluorescence (TPFE), Second Harmonic Generation (SHG) and Third Harmonic Generation (THG). The whole system, including FLIM, used only one laser source composed of an 80 MHz femtosecond laser. The commercial Ti:sapphire lasers can be tuned up to 690-1040 nm bringing the THG signal to the 350 nm region where most microscope optics do not work. However, the third harmonic is only generated at the sample, meaning that we only have to take care of the collection optics. To do that we used a remote photomultiplier to acquire the THG signal at the 310-350 nm wavelength window. After performing the tests to guarantee that we are observing actually SHG/THG signals we than used this system to acquire multimodal images of several biological samples, from epithelial cancer to vegetables. The ability to see the collagen network together with the cell nuclei proved to be important for cancer tissues diagnosis. Moreover, FLIM provides information about the cell metabolism, also very important for cancer cell processes.

  6. 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.

  7. Effects of anti-cancer drug doxorubicin on endogenous biomarkers NAD(P)H, FAD and Trp in prostate cancer cells: a FLIM Study

    Science.gov (United States)

    Rehman Alam, Shagufta; Wallrabe, Horst; Svindrych, Zdenek; Christopher, Kathryn G.; Chandra, Dhyan; Periasamy, Ammasi

    2017-02-01

    Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to identify changes in metabolic activity during cancer progression and upon anti-cancer drug treatment. Prostate cancer (PCa) is one of the leading cancers in men in the USA. This research focusses on understanding the lifetime changes of endogenous biomarkers: NAD(P)H, FAD and Trp in LNCaP cells upon treatment with doxorubicin using our 3-channel FLIM approach. The LNCaP cells were treated with doxorubicin for 24hr. Images using FLIM of LNCaP control and treated cells were acquired on Zeiss 780 multiphoton confocal microscope coupled with B and H TCSPC FLIM board. After FLIM data fitting and processing we observed increase in the mean fluorescence lifetime of Trp, NAD(P)H and FAD with doxorubicin treatment. Additionally, we saw reduction in the NAD(P)H/FAD redox ratio with doxorubicin treatment. Our results identify the changes in the lifetime of these endogenous biomarkers and in the cellular redox state as a metabolic response with doxorubicin treatment in prostate cancer cells.

  8. ImageJ for microscopy.

    Science.gov (United States)

    Collins, Tony J

    2007-07-01

    ImageJ is an essential tool for us that fulfills most of our routine image processing and analysis requirements. The near-comprehensive range of import filters that allow easy access to image and meta-data, a broad suite processing and analysis routine, and enthusiastic support from a friendly mailing list are invaluable for all microscopy labs and facilities-not just those on a budget.

  9. A comparative study of metabolic state of stem cells during osteogenic and adipogenic differentiations via fluorescence lifetime imaging microscopy

    Science.gov (United States)

    Chakraborty, Sandeep; Ou, Meng-Hsin; Kuo, Jean-Cheng; Chiou, Arthur

    2016-10-01

    Cellular metabolic state can serve as a biomarker to indicate the differentiation potential of stem cells into other specialized cell lineages. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was applied to determine the fluorescence lifetime and the amounts of the auto-fluorescent metabolic co-factor reduced nicotinamide adenine dinucleotide (NADH) to elucidate the cellular metabolism of human mesenchymal stem cells (hMSCs) in osteogenic and adipogenic differentiation processes. 2P-FLIM provides the free to protein-bound NADH ratio which can serve as the indicator of cellular metabolic state. We measured NADH fluorescence lifetime at 0, 7, and 14 days after hMSCs were induced for either osteogenesis or adipogenesis. In both cases, the average fluorescence lifetime increased significantly at day 14 (P stem cells into other specialized cell lineages.

  10. 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.

  11. Increasing precision of lifetime determination in fluorescence lifetime imaging

    Science.gov (United States)

    Chang, Ching-Wei; Mycek, Mary-Ann

    2010-02-01

    The interest in fluorescence lifetime imaging microscopy (FLIM) is increasing, as commercial FLIM modules become available for confocal and multi-photon microscopy. In biological FLIM applications, low fluorescence signals from samples can be a challenge, and this causes poor precision in lifetime. In this study, for the first time, we applied wavelet-based denoising methods in time-domain FLIM, and compared them with our previously developed total variation (TV) denoising methods. They were first tested using artificial FLIM images. We then applied them to lowlight live-cell images. The results demonstrated that our TV methods could improve lifetime precision multi-fold in FLIM images and preserve the overall lifetime and pre-exponential term values when improving local lifetime fitting, while wavelet-based methods were faster. The results here can enhance the precision of FLIM, especially for low-light and / or fast video-rate imaging, to improve current and rapidly emerging new applications of FLIM such as live-cell, in vivo whole-animal, or endoscopic imaging.

  12. Frequency domain phosphorescence lifetime Imaging measurements and applications by ISS FastFLIM and multi pulse excitation

    Science.gov (United States)

    Coskun, Ulas C.; Lam, Sandra; Sun, Yuansheng; Liao, Shih-Chu Jeff; George, Steven C.; Barbieri, Beniamino

    2017-02-01

    Phosphorescence probes can have significantly long lifetimes, on the order of micro- to milli-seconds or longer. In addition, environmental changes can affect the lifetimes of these phosphorescence probes. Thus, Phosphorescence Lifetime Imaging Microscopy (PLIM) is a very useful tool to localize the phosphorescence probes based on their lifetimes to study the variance in the lifetimes due to the micro environmental changes. Since the probes respond to the biologically relevant parameters like oxygen concentration, they can be used to study various biologically relevant processes like cellular metabolism, protein interaction etc. In this case, we study the effects of oxygen on Oxyphor G4 with PLIM. Since The Oxyphor G4 can be quenched by O2, it is a good example of such a probe and has a lifetime around 250us. Here we present the digital frequency domain PLIM technique and study the lifetime of the Oxyphor G4 as a function of the O2 concentration. The lifetime data are successfully presented in a phasor plot for various O2 concentrations and are consistent with the time domain data. Overall, we can analyze the oxygen consumption of varying cells using this technique.

  13. Increased metabolic activity detected by FLIM in human breast cancer cells with desmoplastic reaction: a pilot study

    Science.gov (United States)

    Natal, Rodrigo de Andrade; Pelegati, Vitor B.; Bondarik, Caroline; Mendonça, Guilherme R.; Derchain, Sophie F.; Lima, Carmen P.; Cesar, Carlos L.; Sarian, Luís. O.; Vassallo, José

    2015-07-01

    Introduction: In breast cancer (BC), desmoplastic reaction, assembled primarily by fibroblasts, is associated with unfavorable prognosis, but the reason of this fact remains still unclear. In this context, nonlinear optics microscopy, including Fluorescence Lifetime Imaging Microscopy (FLIM), has provided advancement in cellular metabolism research. In this paper, our purpose is to differentiate BC cells metabolism with or without contact to desmoplastic reaction. Formalin fixed, paraffin embedded samples were used at different points of hematoxylin stained sections. Methodology: Sections from 14 patients with invasive ductal breast carcinoma were analyzed with FLIM methodology to NAD(P)H and FAD fluorescence lifetime on a Confocal Upright LSM780 NLO device (Carl Zeiss AG, Germany). Quantification of the fluorescence lifetime and fluorescence intensity was evaluated by SPC Image software (Becker &Hickl) and ImageJ (NIH), respectively. Optical redox ratio was calculated by dividing the FAD fluorescence intensity by NAD(P)H fluorescence intensity. Data value for FLIM measurements and fluorescence intensities were calculated using Wilcoxon test; p< 0.05 was considered significant. Results: BC cells in contact with desmoplastic reaction presented a significantly lower NAD(P)H and FAD fluorescence lifetime. Furthermore, optical redox ratio was also lower in these tumor cells. Conclusion: Our results suggest that contact of BC cells with desmoplastic reaction increase their metabolic activity, which might explain the adverse prognosis of cases associated with higher peritumoral desmoplastic reaction.

  14. Image scanning microscopy with radially polarized light

    Science.gov (United States)

    Xiao, Yun; Zhang, Yunhai; Wei, Tongda; Huang, Wei; Shi, Yaqin

    2017-03-01

    In order to improve the resolution of image scanning microscopy, we present a method based on image scanning microscopy and radially polarized light. According to the theory of image scanning microscopy, we get the effective point spread function of image scanning microscopy with the longitudinal component of radially polarized light and a 1 AU detection area, and obtain imaging results of the analyzed samples using this method. Results show that the resolution can be enhanced by 7% compared with that in image scanning microscopy with circularly polarized light, and is 1.54-fold higher than that in confocal microscopy with a pinhole of 1 AU. Additionally, the peak intensity of ISM is 1.54-fold higher than that of a confocal microscopy with a pinhole of 1 AU. In conclusion, the combination of the image scanning microscopy and the radially polarized light could improve the resolution, and it could realize high-resolution and high SNR imaging at the same time.

  15. 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.

  16. 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.

  17. Fluorescence lifetime imaging microscopy analysis of defects in multi-tube physical vapor transport grown Cd{sub 1-x}Zn{sub x}Te

    Energy Technology Data Exchange (ETDEWEB)

    Schneider, Andreas; Veale, Matthew C.; Wilson, Matthew D.; Seller, Paul; Botchway, Stanley W. [Science and Technology Facility Council, Rutherford Appleton Laboratory, Detector Development Group and Central Laser Facility, Harwell Oxford, Didcot, OX11 0QX (United Kingdom); Bell, Steven J. [Faculty of Engineering and Physical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH (United Kingdom); Duarte, Diana D. [Science and Technology Facility Council, Rutherford Appleton Laboratory, Detector Development Group and Central Laser Facility, Harwell Oxford, Didcot, OX11 0QX (United Kingdom); Faculty of Engineering and Physical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH (United Kingdom); Choubey, Ashutosh; Halliday, Douglas [Department of Physics, Durham University, Rochester Building, South Road, Durham, DH1 3LE (United Kingdom)

    2014-09-15

    Cadmium zinc telluride (CZT) is the material of choice for high-energy room-temperature X-ray and γ-ray detectors. However, the performance of pixelated detectors is greatly influenced by the quality of CZT. Crystal defects and impurities are one source of shallow and deep level traps for charge carriers. Fluorescence lifetime of the recombination of optically excited charges may indicate the presence and type of defects and impurities in CZT. Fluorescence lifetime imaging microscopy (FLIM) is used to examine the excited-state lifetime in CZT fabricated by different growth methods and conditions. The FLIM set-up analyzes luminescence emitted from the sample following photo excitation. Samples were optically excited above band gap with a pulsed laser (590 nm) for raster scanning a 220 x 165 μm{sup 2} sample area. In-situ room-temperature photoluminescence (PL) and FLIM were recorded simultaneously. In order to analyze the FLIM data, two dominant charge carrier decay processes (τ{sub 1}, τ{sub 2}) were identified. The luminescence signal decays with a rapid lifetime of τ{sub 1} ∼ 50-200 ps, and a large variety of long-lifetime components τ{sub 2} were found in the range of 225-900 ps. CZT grown by multi-tube physical vapor transport (MTPVT) showed extremely long-lived recombination decay times up to 3.5 ns in the vicinity of the interface at growth start. Further away from this interface, the recombination lifetime was in the typical range of fast transitions similar to those found in detector-grade CZT fabricated by travelling heater method. Crystalline material quality strongly influences FLIM lifetime. Time-resolved transients of MTPVT-grown CZT compared with industry-leading detector grade CZT (dots: measured data; lines: fitted exponential decay curves). (copyright 2014 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  18. Spectral and lifetime fluorescence imaging microscopies: new modalities of multiphoton microscopy applied to tissue or cell engineering.

    Science.gov (United States)

    Dumas, D; Gaborit, N; Grossin, L; Riquelme, B; Gigant-Huselstein, C; De Isla, N; Gillet, P; Netter, P; Stoltz, J F

    2004-01-01

    Spectral and multiphoton imaging is the preferred approach for non-invasive study allowing deeper penetration to image molecular processes in living cells. But currently available fluorescence microscopic techniques based on fluorescence intensity, such as confocal or multiphoton excitation, cannot provide detailed quantitative information about the dynamic of complex cellular structure (molecular interaction). Due to the variation of the probe concentration, photostability, cross-talking, its effects cannot be distinguished in simple intensity images. Therefore, Time Resolved fluorescence image is required to investigate molecular interactions in biological systems. Fluorescence lifetimes are generally absolute, sensitive to environment, independent of the concentration of the probe and allow the use of probes with overlapping spectra but that not have the same fluorescence lifetime. In this work, we present the possibilities that are opened up by Fluorescence Lifetime Imaging Microscopy, firstly to collect images based on fluorescence lifetime contrast of GFP variants used as a reporter of gene expression in chondrocytes and secondly, to measure molecular proximity in erythrocyte (glycophorin/membrane) by Fluorescence Resonance Energy Transfer (FLIM-FRET).

  19. Visualizing heterogeneity of photosynthetic properties of plant leaves with two-photon fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Iermak, Ievgeniia; Vink, Jochem; Bader, Arjen N; Wientjes, Emilie; van Amerongen, Herbert

    2016-09-01

    Two-photon fluorescence lifetime imaging microscopy (FLIM) was used to analyse the distribution and properties of Photosystem I (PSI) and Photosystem II (PSII) in palisade and spongy chloroplasts of leaves from the C3 plant Arabidopsis thaliana and the C4 plant Miscanthus x giganteus. This was achieved by separating the time-resolved fluorescence of PSI and PSII in the leaf. It is found that the PSII antenna size is larger on the abaxial side of A. thaliana leaves, presumably because chloroplasts in the spongy mesophyll are "shaded" by the palisade cells. The number of chlorophylls in PSI on the adaxial side of the A. thaliana leaf is slightly higher. The C4 plant M. x giganteus contains both mesophyll and bundle sheath cells, which have a different PSI/PSII ratio. It is shown that the time-resolved fluorescence of bundle sheath and mesophyll cells can be analysed separately. The relative number of chlorophylls, which belong to PSI (as compared to PSII) in the bundle sheath cells is at least 2.5 times higher than in mesophyll cells. FLIM is thus demonstrated to be a useful technique to study the PSI/PSII ratio and PSII antenna size in well-defined regions of plant leaves without having to isolate pigment-protein complexes.

  20. Innovating lifetime microscopy: a compact and simple tool for life sciences, screening, and diagnostics.

    Science.gov (United States)

    Esposito, Alessandro; Gerritsen, Hans C; Oggier, Thierry; Lustenberger, Felix; Wouters, Fred S

    2006-01-01

    Fluorescence lifetime imaging microscopy (FLIM) allows the investigation of the physicochemical environment of fluorochromes and protein-protein interaction mapping by Forster resonance energy transfer (FRET) in living cells. However, simpler and cheaper solutions are required before this powerful analytical technique finds a broader application in the life sciences. Wide-field frequency-domain FLIM represents a solution whose application is currently limited by the need for multichannel-plate image intensifiers. We recently showed the feasibility of using a charge-coupled device/complementory metal-oxide semiconductor (CCD/CMOS) hybrid lock-in imager, originally developed for 3-D vision, as an add-on device for lifetime measurements on existing wide-field microscopes. In the present work, the performance of the setup is validated by comparison with well-established wide-field frequency-domain FLIM measurements. Furthermore, we combine the lock-in imager with solid-state light sources. This results in a simple, inexpensive, and compact FLIM system, operating at a video rate and capable of single-shot acquisition by virtue of the unique parallel retrieval of two phase-dependent images. This novel FLIM setup is used for cellular and FRET imaging, and for high-throughput and fast imaging applications. The all-solid-state design bridges the technological gap that limits the use of FLIM in areas such as drug discovery and medical diagnostics.

  1. 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...

  2. Applying phasor approach analysis of multiphoton FLIM measurements to probe the metabolic activity of three-dimensional in vitro cell culture models.

    Science.gov (United States)

    Lakner, Pirmin H; Monaghan, Michael G; Möller, Yvonne; Olayioye, Monilola A; Schenke-Layland, Katja

    2017-02-13

    Fluorescence lifetime imaging microscopy (FLIM) can measure and discriminate endogenous fluorophores present in biological samples. This study seeks to identify FLIM as a suitable method to non-invasively detect a shift in cellular metabolic activity towards glycolysis or oxidative phosphorylation in 3D Caco-2 models of colorectal carcinoma. These models were treated with potassium cyanide or hydrogen peroxide as controls, and epidermal growth factor (EGF) as a physiologically-relevant influencer of cell metabolic behaviour. Autofluorescence, attributed to nicotinamide adenine dinucleotide (NADH), was induced by two-photon laser excitation and its lifetime decay was analysed using a standard multi-exponential decay approach and also a novel custom-written code for phasor-based analysis. While both methods enabled detection of a statistically significant shift of metabolic activity towards glycolysis using potassium cyanide, and oxidative phosphorylation using hydrogen peroxide, employing the phasor approach required fewer initial assumptions to quantify the lifetimes of contributing fluorophores. 3D Caco-2 models treated with EGF had increased glucose consumption, production of lactate, and presence of ATP. FLIM analyses of these cultures revealed a significant shift in the contribution of protein-bound NADH towards free NADH, indicating increased glycolysis-mediated metabolic activity. This data demonstrate that FLIM is suitable to interpret metabolic changes in 3D in vitro models.

  3. 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.

  4. Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy.

    Science.gov (United States)

    Görlitz, Frederik; Kelly, Douglas J; Warren, Sean C; Alibhai, Dominic; West, Lucien; Kumar, Sunil; Alexandrov, Yuriy; Munro, Ian; Garcia, Edwin; McGinty, James; Talbot, Clifford; Serwa, Remigiusz A; Thinon, Emmanuelle; da Paola, Vincenzo; Murray, Edward J; Stuhmeier, Frank; Neil, Mark A A; Tate, Edward W; Dunsby, Christopher; French, Paul M W

    2017-01-18

    We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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...

  10. 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.

  11. Edge detection in microscopy images using curvelets

    OpenAIRE

    Koumoutsakos Petros; Gebäck Tobias

    2009-01-01

    Abstract Background Despite significant progress in imaging technologies, the efficient detection of edges and elongated features in images of intracellular and multicellular structures acquired using light or electron microscopy is a challenging and time consuming task in many laboratories. Results We present a novel method, based on the discrete curvelet transform, to extract a directional field from the image that indicates the location and direction of the edges. This directional field is...

  12. Detection of the interaction between SNAP25 and rabphilin in neuroendocrine PC12 cells using the FLIM/FRET technique.

    Science.gov (United States)

    Lee, Jiung-De; Chang, Yu-Fen; Kao, Fu-Jen; Kao, Lung-Sen; Lin, Chung-Chih; Lu, Ai-Chu; Shyu, Bai-Chuang; Chiou, Shih-Hwa; Yang, De-Ming

    2008-01-01

    Exocytosis has been proposed to contain four sequential steps, namely docking, priming, fusion, and recycling, and to be regulated by various proteins-protein interactions. Synaptosomal-associated protein of 25 kDa (SNAP25) has recently been found to bind rabphilin, the Rab3A specific binding protein, in vitro. However, it is still unclear whether SNAP25 and rabphilin interact during exocytosis within cells in vivo. This problem was addressed by the integration of fluorescence resonance energy transfer (FRET) with high sensitivity fluorescence lifetime imaging microscopy (FLIM) to observe this protein-protein interaction. Enhanced green fluorescence protein-labeled SNAP25 (donor) and red fluorescence protein-labeled rabphilin (acceptor) were expressed in neuroendocrine PC12 cells as a FRET pair and ATP stimulation was carried out for various durations. With 10 s stimulation, a 0.17-ns left shift of the lifetime peak was found when compared with donor only. Analysis of the lifetime image further suggested that the lifetime recovered to a similar level as the donor only in a time dependent manner. Four-dimensional (4D) images by FLIM provided useful information indicating that the interaction of SNAP25 and rabphilin occurred particularly within optical sections near cell membrane. Together the results suggest that SNAP25 bound rabphilin loosely at docking step before exocytosis and the binding became tighter at the very start of exocytosis. Finally, these two proteins dissociated after stimulation. To our knowledge, this is the first report to demonstrate the interaction of SNAP25 and rabphilin in situ using the FLIM-FRET technique within neuroendocrine cells.

  13. Correlated FLIM and PLIM for cell metabolism

    Science.gov (United States)

    Rück, A.; Breymayer, J.; Kalinina, S.

    2016-03-01

    Correlated imaging of phosphorescence and fluorescence lifetime parameters of metabolic markers is a challenge for direct investigating mechanisms related to cell metabolism and oxygen tension. A large variety of clinical phenotypes is associated with mitochondrial defects accomplished with changes in cell metabolism. In many cases the hypoxic microenvironment of cancer cells shifts metabolism from oxidative phosphorylation (OXPHOS) to anaerobic or aerobic glycolysis, a process known as "Warburg" effect. Also during stem cell differentiation a switch in cell metabolism is observed. A defective mitochondrial function associated with hypoxia has been invoked in many complex disorders such as type 2 diabetes, Alzheimers disease, cardiac ischemia/reperfusion injury, tissue inflammation and cancer. Cellular responses to oxygen tension have been studied extensively, optical imaging techniques based on time correlated single photon counting (TCSPC) to detect the underlying metabolic mechanisms are therefore of prominent interest. They offer the possibility by inspecting fluorescence decay characteristics of intrinsic coenzymes to directly image metabolic pathways. Moreover oxygen tension can be determined by considering the phosphorescence lifetime of a phosphorescent probe. The combination of both fluorescence lifetime imaging (FLIM) of coenzymes like NADH and FAD and phosphorescence lifetime (PLIM) of phosphorescent dyes could provide valuable information about correlation of metabolic pathways and oxygen tension.

  14. 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.

  15. 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.

  16. 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

    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.

  17. 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.

  18. Quantitative imaging of bilirubin by photoacoustic microscopy

    Science.gov (United States)

    Zhou, Yong; Zhang, Chi; Yao, Da-Kang; Wang, Lihong V.

    2013-03-01

    Noninvasive detection of both bilirubin concentration and its distribution is important for disease diagnosis. Here we implemented photoacoustic microscopy (PAM) to detect bilirubin distribution. We first demonstrate that our PAM system can measure the absorption spectra of bilirubin and blood. We also image bilirubin distributions in tissuemimicking samples, both without and with blood mixed. Our results show that PAM has the potential to quantitatively image bilirubin in vivo for clinical applications.

  19. Image Correlation Microscopy for Uniform Illumination

    Science.gov (United States)

    Gaborski, Thomas R.; Sealander, Michael N.; Ehrenberg, Morton; Waugh, Richard E.; McGrath, James L.

    2011-01-01

    Image cross-correlation microscopy (ICM) is a technique that quantifies the motion of fluorescent features in an image by measuring the temporal autocorrelation function decay in a time-lapse image sequence. ICM has traditionally employed laser-scanning microscopes because the technique emerged as an extension of laser-based fluorescence correlation spectroscopy (FCS). In this work, we show that image correlation can also be used to measure fluorescence dynamics in uniform illumination or wide-field imaging systems and we call our new approach uniform illumination image correlation microscopy (UI-ICM). Wide-field microscopy is not only a simpler, less expensive imaging modality, but it offers the capability of greater temporal resolution over laser-scanning systems. In traditional laser-scanning ICM, lateral mobility is calculated from the temporal de-correlation of an image, where the characteristic length is the illuminating laser beam width. In wide-field microscopy, the diffusion length is defined by the feature size using the spatial autocorrelation function (SACF). Correlation function decay in time occurs as an object diffuses from its original position. We show that theoretical and simulated comparisons between Gaussian and uniform features indicate the temporal autocorrelation function (TACF) depends strongly on particle size and not particle shape. In this report, we establish the relationships between the SACF feature size, TACF characteristic time and the diffusion coefficient for UI-ICM using analytical, Monte-Carlo and experimental validation with particle tracking algorithms. Additionally, we demonstrate UI-ICM analysis of adhesion molecule domain aggregation and diffusion on the surface of human neutrophils. PMID:20055917

  20. Confocal microscopy imaging of solid tissue

    Science.gov (United States)

    Confocal laser scanning microscopy (CLSM) is a technique that is capable of generating serial sections of whole-mount tissue and then reassembling the computer acquired images as a virtual 3-dimensional structure. In many ways CLSM offers an alternative to traditional sectioning ...

  1. microlith : Image Simulation for Biological Phase Microscopy

    CERN Document Server

    Mehta, Shalin B

    2013-01-01

    Accurate simulation of image formation remains under-exploited for biological phase microscopy methods that employ partially coherent illumination, despite being important for the design of imaging systems and the reconstruction algorithms. We present an open-source MATLAB toolbox, microlith (https://code.google.com/p/microlith), that provides accurate simulation of the 3D image of a thin specimen under any partially coherent imaging system, including coherent or incoherent systems. We demonstrate the accuracy of the microlith toolbox by comparing simulated images and experimental images of a phase-only Siemens star test target using dark field and differential interference contrast microscopes. The comparison leads to intriguing insights about the sensitivity of the dark-field microscope to sub-resolution features and effects of specimen birefringence on differential interference contrast.

  2. Spiral phase contrast imaging in microscopy.

    Science.gov (United States)

    Fürhapter, Severin; Jesacher, Alexander; Bernet, Stefan; Ritsch-Marte, Monika

    2005-02-07

    We demonstrate an optical method for edge contrast enhancement in light microscopy. The method is based on holographic Fourier plane filtering of the microscopic image with a spiral phase element (also called vortex phase or helical phase filter) displayed as an off-axis hologram at a computer controlled high resolution spatial light modulator (SLM) in the optical imaging pathway. The phase hologram imprints a helical phase term of the form exp(i phi) on the diffracted light field in its Fourier plane. In the image plane, this results in a strong and isotropic edge contrast enhancement for both amplitude and phase objects.

  3. Imaging acute thermal burns by photoacoustic microscopy

    OpenAIRE

    Zhang, Hao F.; Maslov, Konstantin; Stoica, George; Wang, Lihong V.

    2006-01-01

    The clinical significance of a burn depends on the percentage of total body involved and the depth of the burn. Hence a noninvasive method that is able to evaluate burn depth would be of great help in clinical evaluation. To this end, photoacoustic microscopy is used to determine the depth of acute thermal burns by imaging the total hemoglobin concentration in the blood that accumulates along the boundaries of injuries as a result of thermal damage to the vasculature. We induce acute thermal ...

  4. Study of intracellular delivery of doxorubicin from poly(lactide-co-glycolide) nanoparticles by means of fluorescence lifetime imaging and confocal Raman microscopy.

    Science.gov (United States)

    Romero, Gabriela; Qiu, Yuan; Murray, Richard A; Moya, Sergio E

    2013-02-01

    The intracellular delivery of Doxorubicin (Dox) from poly(lactide-co-glycolide) (PLGA) nanoparticles stabilised with bovine serum albumin, in HepG2 cells, is studied via flow cytometry, fluorescence lifetime imaging microscopy (FLIM), confocal Raman microscopy (CRM) and cell viability studies. Flow cytometry shows that the initial uptake of PLGA and Dox follow the same kinetics. However, following 8 h of incubation, the fluorescence intensity and cellular uptake of Dox decreases, while in the case of PLGA both parameters remain constant. FLIM shows the presence of a single-lifetime species, with a lifetime of 1.15 ns when measured inside the cells. Cell viability decreases by approximately 20% when incubated for 24 h with PLGA loaded with Dox, with a particle concentration of 100 µg · mL(-1). At the single-cell level, CRM shows changes in the bands from DNA and proteins in the cell nucleus when incubated with PLGA loaded with Dox. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Edge detection in microscopy images using curvelets

    Directory of Open Access Journals (Sweden)

    Koumoutsakos Petros

    2009-03-01

    Full Text Available Abstract Background Despite significant progress in imaging technologies, the efficient detection of edges and elongated features in images of intracellular and multicellular structures acquired using light or electron microscopy is a challenging and time consuming task in many laboratories. Results We present a novel method, based on the discrete curvelet transform, to extract a directional field from the image that indicates the location and direction of the edges. This directional field is then processed using the non-maximal suppression and thresholding steps of the Canny algorithm to trace along the edges and mark them. Optionally, the edges may then be extended along the directions given by the curvelets to provide a more connected edge map. We compare our scheme to the Canny edge detector and an edge detector based on Gabor filters, and show that our scheme performs better in detecting larger, elongated structures possibly composed of several step or ridge edges. Conclusion The proposed curvelet based edge detection is a novel and competitive approach for imaging problems. We expect that the methodology and the accompanying software will facilitate and improve edge detection in images available using light or electron microscopy.

  6. Classification of microscopy images of Langerhans islets

    Science.gov (United States)

    Å vihlík, Jan; Kybic, Jan; Habart, David; Berková, Zuzana; Girman, Peter; Kříž, Jan; Zacharovová, Klára

    2014-03-01

    Evaluation of images of Langerhans islets is a crucial procedure for planning an islet transplantation, which is a promising diabetes treatment. This paper deals with segmentation of microscopy images of Langerhans islets and evaluation of islet parameters such as area, diameter, or volume (IE). For all the available images, the ground truth and the islet parameters were independently evaluated by four medical experts. We use a pixelwise linear classifier (perceptron algorithm) and SVM (support vector machine) for image segmentation. The volume is estimated based on circle or ellipse fitting to individual islets. The segmentations were compared with the corresponding ground truth. Quantitative islet parameters were also evaluated and compared with parameters given by medical experts. We can conclude that accuracy of the presented fully automatic algorithm is fully comparable with medical experts.

  7. Dynamic force microscopy imaging of native membranes

    Energy Technology Data Exchange (ETDEWEB)

    Kienberger, Ferry; Stroh, Cordula; Kada, Gerald; Moser, Rosita; Baumgartner, Werner; Pastushenko, Vassili; Rankl, Christian; Schmidt, Ute; Mueller, Harald; Orlova, Elena; LeGrimellec, Christian; Drenckhahn, Detlev; Blaas, Dieter; Hinterdorfer, Peter

    2003-10-15

    We employed magnetic ACmode atomic force microscopy (MACmode AFM) as a novel dynamic force microscopy method to image surfaces of biological membranes in their native environments. The lateral resolution achieved under optimized imaging conditions was in the nanometer range, even when the sample was only weakly attached to the support. Purple membranes (PM) from Halobacterium salinarum were used as a test standard for topographical imaging. The hexagonal arrangement of the bacteriorhodopsin trimers on the cytoplasmic side of PM was resolved with 1.5 nm lateral accuracy, a resolution similar to images obtained in contact and tapping-mode AFM. Human rhinovirus 2 (HRV2) particles were attached to mica surfaces via nonspecific interactions. The capsid structure and 2 nm sized protein loops of HRV2 were routinely obtained without any displacement of the virus. Globular and filamentous structures on living and fixed endothelial cells were observed with a resolution of 5-20 nm. These examples show that MACmode AFM is a favorable method in studying the topography of soft and weakly attached biological samples with high resolution under physiological conditions.

  8. Nanoscale imaging of RNA with expansion microscopy.

    Science.gov (United States)

    Chen, Fei; Wassie, Asmamaw T; Cote, Allison J; Sinha, Anubhav; Alon, Shahar; Asano, Shoh; Daugharthy, Evan R; Chang, Jae-Byum; Marblestone, Adam; Church, George M; Raj, Arjun; Boyden, Edward S

    2016-08-01

    The ability to image RNA identity and location with nanoscale precision in intact tissues is of great interest for defining cell types and states in normal and pathological biological settings. Here, we present a strategy for expansion microscopy of RNA. We developed a small-molecule linker that enables RNA to be covalently attached to a swellable polyelectrolyte gel synthesized throughout a biological specimen. Then, postexpansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity as well as single-molecule precision in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) separates RNAs and supports amplification of single-molecule signals (i.e., via hybridization chain reaction) as well as multiplexed RNA FISH readout. ExFISH thus enables super-resolution imaging of RNA structure and location with diffraction-limited microscopes in thick specimens, such as intact brain tissue and other tissues of importance to biology and medicine.

  9. Fast image analysis in polarization SHG microscopy.

    Science.gov (United States)

    Amat-Roldan, Ivan; Psilodimitrakopoulos, Sotiris; Loza-Alvarez, Pablo; Artigas, David

    2010-08-02

    Pixel resolution polarization-sensitive second harmonic generation (PSHG) imaging has been recently shown as a promising imaging modality, by largely enhancing the capabilities of conventional intensity-based SHG microscopy. PSHG is able to obtain structural information from the elementary SHG active structures, which play an important role in many biological processes. Although the technique is of major interest, acquiring such information requires long offline processing, even with current computers. In this paper, we present an approach based on Fourier analysis of the anisotropy signature that allows processing the PSHG images in less than a second in standard single core computers. This represents a temporal improvement of several orders of magnitude compared to conventional fitting algorithms. This opens up the possibility for fast PSHG information with the subsequent benefit of potential use in medical applications.

  10. A flexible wide-field FLIM endoscope utilising blue excitation light for label-free contrast of tissue.

    Science.gov (United States)

    Sparks, Hugh; Warren, Sean; Guedes, Joana; Yoshida, Nagisa; Charn, Tze Choong; Guerra, Nadia; Tatla, Taranjit; Dunsby, Christopher; French, Paul

    2015-01-01

    Fluorescence lifetime imaging (FLIM) has previously been shown to provide contrast between normal and diseased tissue. Here we present progress towards clinical and preclinical FLIM endoscopy of tissue autofluorescence, demonstrating a flexible wide-field endoscope that utilised a low average power blue picosecond laser diode excitation source and was able to acquire ∼mm-scale spatial maps of autofluorescence lifetimes from fresh ex vivo diseased human larynx biopsies in ∼8 seconds using an average excitation power of ∼0.5 mW at the specimen. To illustrate its potential for FLIM at higher acquisition rates, a higher power mode-locked frequency doubled Ti:Sapphire laser was used to demonstrate FLIM of ex vivo mouse bowel at up to 2.5 Hz using 10 mW of average excitation power at the specimen.

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

    NARCIS (Netherlands)

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

    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 des

  12. [Mobile phone based wireless microscopy imaging technology].

    Science.gov (United States)

    Yuan, Yucheng; Liu, Jing

    2011-03-01

    This article proposes a new device named "Wireless Cellscope" that combining mobile phone and optical microscope together. The established wireless microscope platform consists of mobile phone, network monitor, miniaturized microscope or high resolution microscope etc. A series of conceptual experiments were performed on microscopic observation of ordinary objects and mice tumor tissue slices. It was demonstrated that, the new method could acquire microscopy images via a wireless way, which is spatially independent. With small size and low cost, the device thus developed has rather wide applicability in non-disturbing investigation of cell/tissue culture and long distance observation of dangerous biological sample etc.

  13. Adaptive nonlinear microscopy for whole tissue imaging

    Science.gov (United States)

    Müllenbroich, M. Caroline; McGhee, Ewan J.; Wright, Amanda J.; Anderson, Kurt I.; Mathieson, Keith

    2013-02-01

    Nonlinear microscopy is capable of imaging biological tissue non-invasively with sub-cellular resolution in three dimensions. For efficient multiphoton signal generation, it is necessary to focus high power, ultra-fast laser pulses into a volume of femtolitres. Aberrations introduced either by the system's optical setup or the sample under investigation cause a broadening of the diffraction limited focal spot which leads to loss of image intensity and resolution. Adaptive optics provides a means to compensate for these aberrations and is capable of restoring resolution and signal strength when imaging at depth. We describe the use of a micro-electro-mechanical systems (MEMS) deformable membrane mirror in a multiphoton adaptive microscope. The aberration correction is determined in a wavefront sensorless approach by rapidly altering the mirror shape with a random search algorithm until the fluorescence or second harmonic signal intensity is improved. We demonstrate the benefits of wavefront correction in a wide-variety of samples, including urea crystals, convallaria and organotypic tissue cultures. We show how the optimization algorithm can be adjusted, for example by including a bleaching compensation, to allow the user to switch between different imaging modalities, producing a versatile approach to aberration correction.

  14. 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 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.

  15. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Pennycook, S.J.; Jesson, D.E.; Chisholm, M.F. (Oak Ridge National Lab., TN (United States)); Ferridge, A.G.; Seddon, M.J. (Wellcome Research Lab., Beckenham (United Kingdom))

    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.

  17. Sub-{Angstrom} microscopy through incoherent imaging and image reconstruction

    Energy Technology Data Exchange (ETDEWEB)

    Pennycook, S.J.; Jesson, D.E.; Chisholm, M.F. [Oak Ridge National Lab., TN (United States); Ferridge, A.G.; Seddon, M.J. [Wellcome Research Lab., Beckenham (United Kingdom)

    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.

  18. Imaging white adipose tissue with confocal microscopy.

    Science.gov (United States)

    Martinez-Santibañez, Gabriel; Cho, Kae Won; Lumeng, Carey N

    2014-01-01

    Adipose tissue is composed of a variety of cell types that include mature adipocytes, endothelial cells, fibroblasts, adipocyte progenitors, and a range of inflammatory leukocytes. These cells work in concert to promote nutrient storage in adipose tissue depots and vary widely based on location. In addition, overnutrition and obesity impart significant changes in the architecture of adipose tissue that are strongly associated with metabolic dysfunction. Recent studies have called attention to the importance of adipose tissue microenvironments in regulating adipocyte function and therefore require techniques that preserve cellular interactions and permit detailed analysis of three-dimensional structures in fat. This chapter summarizes our experience with the use of laser scanning confocal microscopy for imaging adipose tissue in rodents.

  19. Confocal microscopy imaging of the biofilm matrix.

    Science.gov (United States)

    Schlafer, Sebastian; Meyer, Rikke L

    2017-07-01

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  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. Nanoscale chemical imaging by photoinduced force microscopy

    Science.gov (United States)

    Nowak, Derek; Morrison, William; Wickramasinghe, H. Kumar; Jahng, Junghoon; Potma, Eric; Wan, Lei; Ruiz, Ricardo; Albrecht, Thomas R.; Schmidt, Kristin; Frommer, Jane; Sanders, Daniel P.; Park, Sung

    2016-01-01

    Correlating spatial chemical information with the morphology of closely packed nanostructures remains a challenge for the scientific community. For example, supramolecular self-assembly, which provides a powerful and low-cost way to create nanoscale patterns and engineered nanostructures, is not easily interrogated in real space via existing nondestructive techniques based on optics or electrons. A novel scanning probe technique called infrared photoinduced force microscopy (IR PiFM) directly measures the photoinduced polarizability of the sample in the near field by detecting the time-integrated force between the tip and the sample. By imaging at multiple IR wavelengths corresponding to absorption peaks of different chemical species, PiFM has demonstrated the ability to spatially map nm-scale patterns of the individual chemical components of two different types of self-assembled block copolymer films. With chemical-specific nanometer-scale imaging, PiFM provides a powerful new analytical method for deepening our understanding of nanomaterials. PMID:27051870

  2. 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. PMID:26498781

  3. A phosphorescent silver(I)-gold (I) cluster complex that specifically lights up the nucleolus of living cells with FLIM imaging.

    Science.gov (United States)

    Chen, Min; Lei, Zhen; Feng, Wei; Li, Chunyan; Wang, Quan-Ming; Li, Fuyou

    2013-06-01

    The phosphorescent silver(I)-gold(I) cluster complex [CAu6Ag2(dppy)6](BF4)4 (N1) selectively stains the nucleolus, with a much lower uptake in the nucleus and cytoplasm, and exhibits excellent photostability. This Ag-Au cluster, which has a photoluminescent lifetime of microseconds, is particularly attractive as a probe in applications of time-gated microscopy. Investigation of the pathway of cellular entry indicated that N1 permeates the outer membrane and nuclear membrane of living cells through an energy-dependent and non-endocytic route within 10 min. High concentrations of N1 in the nucleolus have been quantified by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and transmission electron microscopy coupled with an energy dispersive X-ray analysis (TEM-EDXA), which also helped to elucidate the mechanism of the specific staining. Intracellular selective staining may be correlated with the microenvironment of the nucleolus, which is consistent with experiments conducted at different phases of the cell cycle. These results prove that N1 is a very attractive phosphorescent staining reagent for visualizing the nucleolus of living cells.

  4. Multi-modal registration for correlative microscopy using image analogies.

    Science.gov (United States)

    Cao, Tian; Zach, Christopher; Modla, Shannon; Powell, Debbie; Czymmek, Kirk; Niethammer, Marc

    2014-08-01

    Correlative microscopy is a methodology combining the functionality of light microscopy with the high resolution of electron microscopy and other microscopy technologies for the same biological specimen. In this paper, we propose an image registration method for correlative microscopy, which is challenging due to the distinct appearance of biological structures when imaged with different modalities. Our method is based on image analogies and allows to transform images of a given modality into the appearance-space of another modality. Hence, the registration between two different types of microscopy images can be transformed to a mono-modality image registration. We use a sparse representation model to obtain image analogies. The method makes use of corresponding image training patches of two different imaging modalities to learn a dictionary capturing appearance relations. We test our approach on backscattered electron (BSE) scanning electron microscopy (SEM)/confocal and transmission electron microscopy (TEM)/confocal images. We perform rigid, affine, and deformable registration via B-splines and show improvements over direct registration using both mutual information and sum of squared differences similarity measures to account for differences in image appearance. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. 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.

  6. 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.

  7. Fluorescence lifetime FRET imaging of receptor-ligand complexes in tumor cells in vitro and in vivo

    Science.gov (United States)

    Rudkouskaya, Alena; Sinsuebphon, Nattawut; Intes, Xavier; Mazurkiewicz, Joseph E.; Barroso, Margarida

    2017-02-01

    To guide the development of targeted therapies with improved efficacy and accelerated clinical acceptance, novel imaging methodologies need to be established. Toward this goal, fluorescence lifetime Förster resonance energy transfer (FLIM-FRET) imaging assays capitalize on the ability of antibodies or protein ligands to bind dimerized membrane bound receptors to measure their target engagement levels in cancer cells. Conventional FLIM FRET microscopy has been widely applied at visible wavelengths to detect protein-protein interactions in vitro. However, operation at these wavelengths restricts imaging quality and ability to quantitate lifetime changes in in vivo small animal optical imaging due to high auto-fluorescence and light scattering. Here, we have analyzed the uptake of iron-bound transferrin (Tf) probes into human breast cancer cells using FLIM-FRET microscopy in the visible and near-infrared (NIR) range. The development of NIR FLIM FRET microscopy allows for the use of quantitative lifetime-based molecular assays to measure drug-target engagement levels at multiple scales: from in vitro microscopy to in vivo small animal optical imaging (macroscopy). This novel approach can be extended to other receptors, currently targeted in oncology. Hence, lifetime-based molecular imaging can find numerous applications in drug delivery and targeted therapy assessment and optimization.

  8. Bioluminescence microscopy using a short focal-length imaging lens.

    Science.gov (United States)

    Ogoh, K; Akiyoshi, R; May-Maw-Thet; Sugiyama, T; Dosaka, S; Hatta-Ohashi, Y; Suzuki, H

    2014-03-01

    Bioluminescence from cells is so dim that bioluminescence microscopy is performed using an ultra low-light imaging camera. Although the image sensor of such cameras has been greatly improved over time, such improvements have not been made commercially available for microscopes until now. Here, we customized the optical system of a microscope for bioluminescence imaging. As a result, bioluminescence images of cells could be captured with a conventional objective lens and colour imaging camera. As bioluminescence microscopy requires no excitation light, it lacks the photo-toxicity associated with fluorescence imaging and permits the long-term, nonlethal observation of living cells. Thus, bioluminescence microscopy would be a powerful tool in cellular biology that complements fluorescence microscopy.

  9. Imaging DNA Structure by Atomic Force Microscopy.

    Science.gov (United States)

    Pyne, Alice L B; Hoogenboom, Bart W

    2016-01-01

    Atomic force microscopy (AFM) is a microscopy technique that uses a sharp probe to trace a sample surface at nanometre resolution. For biological applications, one of its key advantages is its ability to visualize substructure of single molecules and molecular complexes in an aqueous environment. Here, we describe the application of AFM to determine superstructure and secondary structure of surface-bound DNA. The method is also readily applicable to probe DNA-DNA interactions and DNA-protein complexes.

  10. Biological imaging with coherent Raman scattering microscopy: a tutorial

    Science.gov (United States)

    Alfonso-García, Alba; Mittal, Richa; Lee, Eun Seong; Potma, Eric O.

    2014-01-01

    Abstract. Coherent Raman scattering (CRS) microscopy is gaining acceptance as a valuable addition to the imaging toolset of biological researchers. Optimal use of this label-free imaging technique benefits from a basic understanding of the physical principles and technical merits of the CRS microscope. This tutorial offers qualitative explanations of the principles behind CRS microscopy and provides information about the applicability of this nonlinear optical imaging approach for biological research. PMID:24615671

  11. Alpha hemolysin induces an increase of erythrocytes calcium: a FLIM 2-photon phasor analysis approach.

    Directory of Open Access Journals (Sweden)

    Susana Sanchez

    Full Text Available α-Hemolysin (HlyA from Escherichia coli is considered as the prototype of a family of toxins called RTX (repeat in toxin, a group of proteins that share genetic and structural features. HlyA is an important virulence factor in E. coli extraintestinal infections, such as meningitis, septicemia and urinary infections. High concentrations of the toxin cause the lysis of several cells such as erythrocytes, granulocytes, monocytes, endothelial and renal epithelial cells of different species. At low concentrations it induces the production of cytokines and apoptosis. Since many of the subcytolytic effects in other cells have been reported to be triggered by the increase of intracellular calcium, we followed the calcium concentration inside the erythrocytes while incubating with sublytic concentrations of HlyA. Calcium concentration was monitored using the calcium indicator Green 1, 2-photon excitation, and fluorescence lifetime imaging microscopy (FLIM. Data were analyzed using the phasor representation. In this report, we present evidence that, at sublytic concentrations, HlyA induces an increase of calcium concentration in rabbit erythrocytes in the first 10 s. Results are discussed in relation to the difficulties of measuring calcium concentrations in erythrocytes where hemoglobin is present, the contribution of the background and the heterogeneity of the response observed in individual cells.

  12. ns-time resolution for multispecies STED-FLIM and artifact free STED-FCS

    Science.gov (United States)

    Koenig, Marcelle; Reisch, Paja; Dowler, Rhys; Kraemer, Benedikt; Tannert, Sebastian; Patting, Matthias; Clausen, Mathias P.; Galiani, Silvia; Eggeling, Christian; Koberling, Felix; Erdmann, Rainer

    2016-03-01

    Stimulated Emission Depletion (STED) Microscopy has evolved into a well established method offering optical superresolution below 50 nm. Running both excitation and depletion lasers in picosecond pulsed modes allows for highest optical resolution as well as fully exploiting the photon arrival time information using time-resolved single photon counting (TCSPC). Non-superresolved contributions can be easily dismissed through time-gated detection (gated STED) or a more detailed fluorescence decay analysis (FLIM-STED), both leading to an even further improved imaging resolution. Furthermore, these methods allow for accurate separation of different fluorescent species, especially if subtle differences in the excitation and emission spectra as well as the fluorescence decay are taken into account in parallel. STED can also be used to shrink the observation volume while studying the dynamics of diffusing species in Fluorescence Correlation Spectroscopy (FCS) to overcome averaging issues along long transit paths. A further unique advantage of STED-FCS is that the observation spot diameter can be tuned in a gradual manner enabling, for example, determining the type of hindered diffusion in lipid membrane studies. Our completely pulsed illumination scheme allows realizing an improved STED-FCS data acquisition using pulsed interleaved excitation (PIE). PIE-STED-FCS allows for a straightforward online check whether the STED laser has an influence on the investigated diffusion dynamics.

  13. 3D super-resolution imaging by localization microscopy.

    Science.gov (United States)

    Magenau, Astrid; Gaus, Katharina

    2015-01-01

    Fluorescence microscopy is an important tool in all fields of biology to visualize structures and monitor dynamic processes and distributions. Contrary to conventional microscopy techniques such as confocal microscopy, which are limited by their spatial resolution, super-resolution techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) have made it possible to observe and quantify structure and processes on the single molecule level. Here, we describe a method to image and quantify the molecular distribution of membrane-associated proteins in two and three dimensions with nanometer resolution.

  14. 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.

  15. Reconstruction of Undersampled Atomic Force Microscopy Images

    DEFF Research Database (Denmark)

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

    2013-01-01

    . Moreover, it is often required to take several images before a relevant observation region is identified. In this paper we show how to significantly reduce the image acquisition time by undersampling. The reconstruction of an undersampled AFM image can be viewed as an inpainting, interpolating problem...

  16. 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.

  17. Label-free fluorescence lifetime and second harmonic generation imaging microscopy improves quantification of experimental renal fibrosis.

    Science.gov (United States)

    Ranjit, Suman; Dobrinskikh, Evgenia; Montford, John; Dvornikov, Alexander; Lehman, Allison; Orlicky, David J; Nemenoff, Raphael; Gratton, Enrico; Levi, Moshe; Furgeson, Seth

    2016-11-01

    All forms of progressive renal diseases develop a final pathway of tubulointerstitial fibrosis and glomerulosclerosis. Renal fibrosis is usually quantified using histological staining, a process that is time-consuming and pathologist dependent. Here we develop a fast and operator-independent method to measure fibrosis utilizing the murine unilateral ureteral obstruction model which manifests a time-dependent fibrotic increase in obstructed kidneys while the contralateral kidneys are used as controls. After ureteral obstruction, kidneys were analyzed at 7, 14, and 21 days. Fibrosis was quantified using fluorescence lifetime imaging (FLIM) and second harmonic generation (SHG) in a Deep Imaging via Enhanced photon Recovery deep tissue imaging microscope. This microscope was developed for deep tissue along with second and third harmonic generation imaging and has extraordinary sensitivity toward harmonic generation. SHG data suggest the presence of more fibrillar collagen in the obstructed kidneys. The combination of short-wavelength FLIM and SHG analysis results in a robust assessment procedure independent of observer interpretation and let us create criteria to quantify the extent of fibrosis directly from the image. Thus, the FLIM-SHG technique shows remarkable improvement in quantification of renal fibrosis compared to standard histological techniques. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  18. Bioluminescence microscopy using a short focal-length imaging lens

    OpenAIRE

    Ogoh, K; Akiyoshi, R; May-Maw-Thet,; Sugiyama, T; Dosaka, S; Hatta-Ohashi, Y; Suzuki, H.

    2014-01-01

    Bioluminescence from cells is so dim that bioluminescence microscopy is performed using an ultra low-light imaging camera. Although the image sensor of such cameras has been greatly improved over time, such improvements have not been made commercially available for microscopes until now. Here, we customized the optical system of a microscope for bioluminescence imaging. As a result, bioluminescence images of cells could be captured with a conventional objective lens and colour imaging camera....

  19. Surgical implantation of an abdominal imaging window for intravital microscopy

    NARCIS (Netherlands)

    Ritsma, L.; Steller, E.J.; Ellenbroek, S.I.; Kranenburg, O.; Rinkes, I.H.; van Rheenen, J.

    2013-01-01

    High-resolution intravital microscopy through imaging windows has become an indispensable technique for the long-term visualization of dynamic processes in living animals. Easily accessible sites such as the skin, the breast and the skull can be imaged using various different imaging windows;

  20. Surgical implantation of an abdominal imaging window for intravital microscopy

    NARCIS (Netherlands)

    Ritsma, L.; Steller, E.J.; Ellenbroek, S.I.; Kranenburg, O.; Rinkes, I.H.; van Rheenen, J.

    2013-01-01

    High-resolution intravital microscopy through imaging windows has become an indispensable technique for the long-term visualization of dynamic processes in living animals. Easily accessible sites such as the skin, the breast and the skull can be imaged using various different imaging windows; howeve

  1. Confocal Microscopy Imaging of the Biofilm Matrix

    DEFF Research Database (Denmark)

    Schlafer, Sebastian; Meyer, Rikke Louise

    2016-01-01

    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...... the concentration of solutes and the diffusive properties of the biofilm matrix....

  2. 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...

  3. 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 in the indi......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...... in the indicator film and unevenness of light, as well as additive signals from detector bias, external light sources, etc. When properly corrected a better measurement of the local magnetic field can be made, even in the case of heavily damaged films. For superconductors the magnetic field distributions may...

  4. Low energy electron microscopy imaging using Medipix2 detector

    NARCIS (Netherlands)

    Sikharulidze, I.; Gastel, van R.; Schramm, S.; Abrahams, J.P.; Poelsema, B.; Tromp, R.M.; Molen, van der S.J.

    2011-01-01

    Low Energy Electron Microscopy (LEEM) and Photo-Emission Electron Microscopy (PEEM) predominantly use a combination of microchannel plate (MCP), phosphor screen and optical camera to record images formed by 10–20 keV electrons. We have tested the performance of a LEEM/PEEM instrument with a Medipix2

  5. In Vivo Confocal Microscopy expanding horizons in corneal imaging

    NARCIS (Netherlands)

    T. Hillenaar (Toine)

    2012-01-01

    textabstractConfocal microscopy is an emerging optical technique that allows the living human cornea to be imaged on a cellular level. As such, confocal microscopy enables morphologic and quantitative analysis of corneal resident cells in health and disease and provides an exciting bridge between in

  6. In Vivo Confocal Microscopy expanding horizons in corneal imaging

    NARCIS (Netherlands)

    T. Hillenaar (Toine)

    2012-01-01

    textabstractConfocal microscopy is an emerging optical technique that allows the living human cornea to be imaged on a cellular level. As such, confocal microscopy enables morphologic and quantitative analysis of corneal resident cells in health and disease and provides an exciting bridge between in

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

    CSIR Research Space (South Africa)

    Mabaso, M

    2016-02-01

    Full Text Available of the 9th International Joint Conference on Biomedical Engineering Systems and Technologies, Rome, Italy. 21-23 February, 2016 A Framework for Creating Realistic Synthetic Fluorescence Microscopy Image Sequences Matsilele Mabaso1, Daniel Withey1...

  8. Correlative atomic force microscopy and localization-based super-resolution microscopy: revealing labelling and image reconstruction artefacts.

    Science.gov (United States)

    Monserrate, Aitor; Casado, Santiago; Flors, Cristina

    2014-03-17

    Hybrid microscopy: A correlative microscopy tool that combines in situ super-resolution fluorescence microscopy based on single-molecule localization and atomic force microscopy is presented. Direct comparison with high- resolution topography allows the authors to improve fluorescence labeling and image analysis in super-resolution imaging.

  9. Nonlinear optical microscopy for imaging thin films and surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Smilowitz, L.B.; McBranch, D.W.; Robinson, J.M.

    1995-03-01

    We have used the inherent surface sensitivity of second harmonic generation to develop an instrument for nonlinear optical microscopy of surfaces and interfaces. We have demonstrated the use of several nonlinear optical responses for imaging thin films. The second harmonic response of a thin film of C{sub 60} has been used to image patterned films. Two photon absorption light induced fluorescence has been used to image patterned thin films of Rhodamine 6G. Applications of nonlinear optical microscopy include the imaging of charge injection and photoinduced charge transfer between layers in semiconductor heterojunction devices as well as across membranes in biological systems.

  10. 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.

  11. Simulating Realistic Imaging Conditions For In-Situ Liquid Microscopy

    Science.gov (United States)

    Welch, David A.; Faller, Roland; Evans, James E.; Browning, Nigel D.

    2013-01-01

    In situ transmission electron microscopy enables the imaging of biological cells, macromolecular protein complexes, nanoparticles, and other systems in a near-native environment. In order to improve interpretation of image contrast features and also predict ideal imaging conditions ahead of time, new virtual electron microscopic techniques are needed. A technique for virtual fluid-stage high-angle annular dark-field scanning transmission electron microscopy with the multislice method is presented that enables the virtual imaging of model fluid-stage systems composed of millions of atoms. The virtual technique is exemplified by simulating images of PbS nanoparticles under different imaging conditions and the results agree with previous experimental findings. General insight is obtained on the influence of the effects of fluid path length, membrane thickness, nanoparticle position, defocus and other microscope parameters on attainable image quality. PMID:23872040

  12. Simulating realistic imaging conditions for in situ liquid microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Welch, David A.; Faller, Roland; Evans, James E.; Browning, Nigel D.

    2013-12-01

    In situ transmission electron microscopy enables the imaging of biological cells, macromolecular protein complexes, nanoparticles, and other systems in a near-native environment. In order to improve interpretation of image contrast features and also predict ideal imaging conditions ahead of time, new virtual electron microscopic techniques are needed. A technique for virtual fluid-stage high-angle annular dark-field scanning transmission electron microscopy with the multislice method is presented that enables the virtual imaging of model fluid-stage systems composed of millions of atoms. The virtual technique is exemplified by simulating images of PbS nanoparticles under different imaging conditions and the results agree with previous experimental findings. General insight is obtained on the influence of the effects of fluid path length, membrane thickness, nanoparticle position, defocus and other microscope parameters on attainable image quality.

  13. Photobleaching correction in fluorescence microscopy images

    Energy Technology Data Exchange (ETDEWEB)

    Vicente, Nathalie B; Diaz Zamboni, Javier E; Adur, Javier F; Paravani, Enrique V; Casco, Victor H [Microscopy Laboratory, School of Engineering - Bioengineering, National University of Entre Rios (UNER), Ruta 11, Km 10 (3101), Oro Verde, Entre Rios (Argentina)

    2007-11-15

    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.

  14. 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

  15. Simulating realistic imaging conditions for in situ liquid microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Welch, David A., E-mail: dawelch@ucdavis.edu [Department of Chemical Engineering and Materials Science, University of California, Davis, CA (United States); Faller, Roland [Department of Chemical Engineering and Materials Science, University of California, Davis, CA (United States); Evans, James E. [Pacific Northwest National Laboratory, Environmental Molecular Sciences Laboratory, Richland, WA (United States); Browning, Nigel D. [Pacific Northwest National Laboratory, Fundamental Computational Sciences Directorate, Richland, WA (United States)

    2013-12-15

    In situ transmission electron microscopy enables the imaging of biological cells, macromolecular protein complexes, nanoparticles, and other systems in a near-native environment. In order to improve interpretation of image contrast features and also predict ideal imaging conditions ahead of time, new virtual electron microscopic techniques are needed. A technique for virtual fluid-stage high-angle annular dark-field scanning transmission electron microscopy with the multislice method is presented that enables the virtual imaging of model fluid-stage systems composed of millions of atoms. The virtual technique is exemplified by simulating images of PbS nanoparticles under different imaging conditions and the results agree with previous experimental findings. General insight is obtained on the influence of the effects of fluid path length, membrane thickness, nanoparticle position, defocus and other microscope parameters on attainable image quality. - Highlights: • Image simulation has been performed to understand in situ electron microscopy experiments. • Experimentally observed resolution of in situ grown PbS nanoparticles has been virtually reproduced. • General relationships between image resolution and in situ holder design, defocus, and particle size have been determined. • The presented image simulation technique can predict the obtainable resolution of future experiments.

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

    Directory of Open Access Journals (Sweden)

    Strandh Christer

    2008-07-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusion 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.

  17. Simultaneous morphological and biochemical endogenous optical imaging of atherosclerosis.

    Science.gov (United States)

    Jo, Javier A; Park, Jesung; Pande, Paritosh; Shrestha, Sebina; Serafino, Michael J; Rico Jimenez, J de Jesus; Clubb, Fred; Walton, Brian; Buja, L Maximilian; Phipps, Jennifer E; Feldman, Marc D; Adame, Jessie; Applegate, Brian E

    2015-08-01

    The aim of this study was to validate novel imaging technology for simultaneous morphological and biochemical endogenous optical imaging of coronary atherosclerotic plaque. Optical coherence tomography (OCT) generates high-resolution 3D images of plaque morphology and endogenous fluorescence lifetime imaging microscopy (FLIM) characterizes biochemical composition. Both imaging modalities rely on plaque's intrinsic optical characteristics, making contrast agents unnecessary. A multimodal OCT/FLIM system was utilized to generate luminal biochemical maps superimposed on high-resolution (7 µm axial and 13 µm lateral) structural volumetric images. Forty-seven fresh postmortem human coronary segments were imaged: pathological intimal thickening (PIT, n = 26), fibroatheroma (FA, n = 12), thin-cap FA (TCFA, n = 2), and fibrocalcific plaque (CA, n = 7), determined by histopathology. Multimodal images were evaluated, and each plaque identified as PIT, FA, TCFA, or CA based on expert OCT readers, and as having high-lipid (HL), high-collagen (HC), or low-collagen/low-lipid (LCL) luminal composition based on linear discriminant analysis of FLIM. Of 47 plaques, 89.4% (42/47) of the plaques were correctly identified based on OCT/FLIM evaluation using tissue histopathology and immunohistochemistry as the gold standard. Four of the misclassifications corresponded to confusing PIT with HL luminal composition for FA with HL cap. The other corresponded to confusing FA with a HC cap for FA with an LCL cap. We have demonstrated the feasibility of accurate simultaneous OCT/FLIM morphological and biochemical characterization of coronary plaques at spatial resolutions and acquisition speeds compatible with catheter-based intravascular imaging. The success of this pilot study sets up future development of a multimodal intravascular imaging system that will enable studies that could help improve our understanding of plaque pathogenesis. Published on behalf of the European Society of

  18. Modulated CMOS camera for fluorescence lifetime microscopy.

    Science.gov (United States)

    Chen, Hongtao; Holst, Gerhard; Gratton, Enrico

    2015-12-01

    Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate method to measure the fluorescence lifetime of entire images. However, the complexity and high costs involved in construction of such a system limit the extensive use of this technique. PCO AG recently released the first luminescence lifetime imaging camera based on a high frequency modulated CMOS image sensor, QMFLIM2. Here we tested and provide operational procedures to calibrate the camera and to improve the accuracy using corrections necessary for image analysis. With its flexible input/output options, we are able to use a modulated laser diode or a 20 MHz pulsed white supercontinuum laser as the light source. The output of the camera consists of a stack of modulated images that can be analyzed by the SimFCS software using the phasor approach. The nonuniform system response across the image sensor must be calibrated at the pixel level. This pixel calibration is crucial and needed for every camera settings, e.g. modulation frequency and exposure time. A significant dependency of the modulation signal on the intensity was also observed and hence an additional calibration is needed for each pixel depending on the pixel intensity level. These corrections are important not only for the fundamental frequency, but also for the higher harmonics when using the pulsed supercontinuum laser. With these post data acquisition corrections, the PCO CMOS-FLIM camera can be used for various biomedical applications requiring a large frame and high speed acquisition.

  19. 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.

  20. Imaging photothermal microscopy for absorption measurements of optical coatings

    Institute of Scientific and Technical Information of China (English)

    Chunxian Tao; Yuanan Zhao; Hongbo He; Dawei Li; Jianda Shao; Zhengxiu Fan

    2009-01-01

    @@ For absorption measurement of large-aperture optical coatings, a novel method of imaging photothermal microscopy based on image lock-in technique is presented.Detailed theoretical analysis and numerical calculation are made based on the image photothermal technique.The feasibility of this imaging method is proved through the coincidence between the theoretical results of single spot method and multi-channel method.The measuring speed of this imaging method can be increased hundreds of times compared with that of the raster scanning.This technique can expand the applications of photothermal technique.

  1. Image analysis and microscopy: a useful combination

    Directory of Open Access Journals (Sweden)

    Pinotti L.

    2009-01-01

    Full Text Available The TSE Roadmap published in 2005 (DG for Health and Consumer Protection, 2005 suggests that short and medium term (2005-2009 amendments to control BSE policy should include “a relaxation of certain measures of the current total feed ban when certain conditions are met”. The same document noted “the starting point when revising the current feed ban provisions should be risk-based but at the same time taking into account the control tools in place to evaluate and ensure the proper implementation of this feed ban”. The clear implication is that adequate analytical methods to detect constituents of animal origin in feedstuffs are required. The official analytical method for the detection of constituents of animal origin in feedstuffs is the microscopic examination technique as described in Commission Directive 2003/126/EC of 23 December 2003 [OJ L 339, 24.12.2003, 78]. Although the microscopic method is usually able to distinguish fish from land animal material, it is often unable to distinguish between different terrestrial animals. Fulfillments of the requirements of Regulation 1774/2002/EC laying down health rules concerning animal by-products not intended for human consumption, clearly implies that it must be possible to identify the origin animal materials, at higher taxonomic levels than in the past. Thus improvements in all methods of detecting constituents of animal origin are required, including the microscopic method. This article will examine the problem of meat and bone meal in animal feeds, and the use of microscopic methods in association with computer image analysis to identify the source species of these feedstuff contaminants. Image processing, integrated with morphometric measurements can provide accurate and reliable results and can be a very useful aid to the analyst in the characterization, analysis and control of feedstuffs.

  2. Real-time fluorescence lifetime imaging system with a 32 × 32 0.13μm 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 0.13μm CMOS singlephoton avalanche diodes (SPADs) is demonstrated. Fast background-insensitive fluorescence lifetime determination is achieved by use of a recently proposed algorithm called

  3. 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.

  4. 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.

  5. Non-linear image scanning microscopy (Conference Presentation)

    Science.gov (United States)

    Gregor, Ingo; Ros, Robert; Enderlein, Jörg

    2017-02-01

    Nowadays, multiphoton microscopy can be considered as a routine method for the observation of living cells, organs, up to whole organisms. Second-harmonics generation (SHG) imaging has evolved to a powerful qualitative and label-free method for studying fibrillar structures, like collagen networks. However, examples of super-resolution non-linear microscopy are rare. So far, such approaches require complex setups and advanced synchronization of scanning elements limiting the image acquisition rates. We describe theory and realization of a super-resolution image scanning microscope [1, 2] using two-photon excited fluorescence as well as second-harmonic generation. It requires only minor modifications compared to a classical two-photon laser-scanning microscope and allows image acquisition at the high frame rates of a resonant galvo-scanner. We achieve excellent sensitivity and high frame-rate in combination with two-times improved lateral resolution. We applied this method to fixed cells, collagen hydrogels, as well as living fly embryos. Further, we proofed the excellent image quality of our setup for deep tissue imaging. 1. Müller C.B. and Enderlein J. (2010) Image scanning microscopy. Phys. Rev. Lett. 104(19), 198101. 2. Sheppard C.J.R. (1988) Super-resolution in confocal imaging. Optik (Stuttg) 80 53-54.

  6. Segmentation and learning in the quantitative analysis of microscopy images

    Science.gov (United States)

    Ruggiero, Christy; Ross, Amy; Porter, Reid

    2015-02-01

    In material science and bio-medical domains the quantity and quality of microscopy images is rapidly increasing and there is a great need to automatically detect, delineate and quantify particles, grains, cells, neurons and other functional "objects" within these images. These are challenging problems for image processing because of the variability in object appearance that inevitably arises in real world image acquisition and analysis. One of the most promising (and practical) ways to address these challenges is interactive image segmentation. These algorithms are designed to incorporate input from a human operator to tailor the segmentation method to the image at hand. Interactive image segmentation is now a key tool in a wide range of applications in microscopy and elsewhere. Historically, interactive image segmentation algorithms have tailored segmentation on an image-by-image basis, and information derived from operator input is not transferred between images. But recently there has been increasing interest to use machine learning in segmentation to provide interactive tools that accumulate and learn from the operator input over longer periods of time. These new learning algorithms reduce the need for operator input over time, and can potentially provide a more dynamic balance between customization and automation for different applications. This paper reviews the state of the art in this area, provides a unified view of these algorithms, and compares the segmentation performance of various design choices.

  7. Imaging diffusion in a microfluidic device by third harmonic microscopy

    Science.gov (United States)

    Petzold, Uwe; Büchel, Andreas; Hardt, Steffen; Halfmann, Thomas

    2012-09-01

    We monitor and characterize near-surface diffusion of miscible, transparent liquids in a microfluidic device by third harmonic microscopy. The technique enables observations even of transparent or index-matched media without perturbation of the sample. In particular, we image concentrations of ethanol diffusing in water and estimate the diffusion coefficient from the third harmonic images. We obtain a diffusion coefficient D = (460 ± 30) μm2/s, which is consistent with theoretical predictions. The investigations clearly demonstrate the potential of harmonic microscopy also under the challenging conditions of transparent fluids.

  8. 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 contributi......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...

  9. 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

  10. Detecting overlapping instances in microscopy images using extremal region trees.

    Science.gov (United States)

    Arteta, Carlos; Lempitsky, Victor; Noble, J Alison; Zisserman, Andrew

    2016-01-01

    In many microscopy applications the images may contain both regions of low and high cell densities corresponding to different tissues or colonies at different stages of growth. This poses a challenge to most previously developed automated cell detection and counting methods, which are designed to handle either the low-density scenario (through cell detection) or the high-density scenario (through density estimation or texture analysis). The objective of this work is to detect all the instances of an object of interest in microscopy images. The instances may be partially overlapping and clustered. To this end we introduce a tree-structured discrete graphical model that is used to select and label a set of non-overlapping regions in the image by a global optimization of a classification score. Each region is labeled with the number of instances it contains - for example regions can be selected that contain two or three object instances, by defining separate classes for tuples of objects in the detection process. We show that this formulation can be learned within the structured output SVM framework and that the inference in such a model can be accomplished using dynamic programming on a tree structured region graph. Furthermore, the learning only requires weak annotations - a dot on each instance. The candidate regions for the selection are obtained as extremal region of a surface computed from the microscopy image, and we show that the performance of the model can be improved by considering a proxy problem for learning the surface that allows better selection of the extremal regions. Furthermore, we consider a number of variations for the loss function used in the structured output learning. The model is applied and evaluated over six quite disparate data sets of images covering: fluorescence microscopy, weak-fluorescence molecular images, phase contrast microscopy and histopathology images, and is shown to exceed the state of the art in performance.

  11. Three-dimensional volume imaging with electron microscopy toward connectome.

    Science.gov (United States)

    Ohno, Nobuhiko; Katoh, Mitsuhiko; Saitoh, Yurika; Saitoh, Sei; Ohno, Shinichi

    2015-02-01

    Ultrastructural analyses with electron microscopy have provided indispensable information to understand physiology and pathology of the nervous system. Recent advancement in imaging methodology paved the way for complete reconstruction of the neuronal connection map in the central nervous system, which is termed 'connectome' and would provide key insights to understand the functions of the brain. The critical advancement includes serial ultrastructural observation with scanning electron microscopy (SEM) instead of conventional serial sectioning transmission electron microscopy along with specific tissue preparation methods to increase heavy metal deposition for efficient SEM imaging. The advanced imaging methods using SEM have distinct advantages and disadvantages in multiple aspects, such as resolution and imaging speed, and should be selected depending on the observation conditions, such as target tissue sizes, required spatial resolution and necessity for re-observation. Dealing with the huge dataset remained to be a major obstacle, and automation in segmentation and 3D reconstruction would be critical to understand neuronal circuits in a larger volume of the brain. Future improvement in acquisition and analyses of the morphological data obtained with the advanced SEM imaging is awaited to elucidate the significance of whole connectome as the structural basis of the consciousness, intelligence and memory of a subject. © The Author 2014. 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.

  12. Reconstruction of complementary images in second harmonic generation microscopy

    Science.gov (United States)

    Gao, Liang; Jin, Lei; Xue, Ping; Xu, Jun; Wang, Yi; Ma, Hui; Chen, Dieyan

    2006-05-01

    Second harmonic generation microscopy(SHGM) has become widely used to image biological samples. Due to the complexity of biological samples, more and more effort has been put on polarization imaging in SHGM technology to uncover their structures. In this work, we put forward a novel stitching method based on careful mathematical calculation, and accomplish it by rotating laser polarization. We first show its validity in imaging a perfectly synthesized bio-origin polymer poly (3-hyroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Then, we test its power by getting a true image of fibrillar collagen structure of rat-tail tendon.

  13. A wide-field TCSPC FLIM system based on an MCP PMT with a delay-line anode

    Science.gov (United States)

    Becker, Wolfgang; Hirvonen, Liisa M.; Milnes, James; Conneely, Thomas; Jagutzki, Ottmar; Netz, Holger; Smietana, Stefan; Suhling, Klaus

    2016-09-01

    We report on the implementation of a wide-field time-correlated single photon counting (TCSPC) method for fluorescence lifetime imaging (FLIM). It is based on a 40 mm diameter crossed delay line anode detector, where the readout is performed by three standard TCSPC boards. Excitation is performed by a picosecond diode laser with 50 MHz repetition rate. The photon arrival timing is obtained directly from the microchannel plates, with an instrumental response of ˜190 to 230 ps full width at half maximum depending on the position on the photocathode. The position of the photon event is obtained from the pulse propagation time along the two delay lines, one in x and one in y. One end of a delay line is fed into the "start" input of the corresponding TCSPC board, and the other end is delayed by 40 ns and fed into the "stop" input. The time between start and stop is directly converted into position, with a resolution of 200-250 μm. The data acquisition software builds up the distribution of the photons over their spatial coordinates, x and y, and their times after the excitation pulses, typically into 512 × 512 pixels and 1024 time channels per pixel. We apply the system to fluorescence lifetime imaging of cells labelled with Alexa 488 phalloidin in an epi-fluorescence microscope and discuss the application of our approach to other fluorescence microscopy methods.

  14. 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.

  15. 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.

  16. 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.

  17. In vivo simultaneous morphological and biochemical optical imaging of oral epithelial cancer.

    Science.gov (United States)

    Jo, Javier A; Applegate, Brian E; Park, Jesung; Shrestha, Sebina; Pande, Paritosh; Gimenez-Conti, Irma B; Brandon, Jimi L

    2010-10-01

    Early detection of cancer is key to reducing morbidity and mortality. Morphological and chemical biomarkers presage the transition from normal to cancerous tissue. We have developed a noninvasive imaging system incorporating optical coherence tomography (OCT) and fluorescence lifetime imaging microscopy (FLIM) into a single optical system for the first time, in order to acquire both sets of biomarkers. OCT can provide morphological images of tissue with high resolution, while FLIM can provide biochemical tissue maps. Coregistered OCT volumes and FLIM images have been acquired simultaneously in an in vivo hamster cheek pouch model of oral cancer. The OCT images indicate morphological biomarkers for cancer including thickening of the epithelial layer and loss of the layered structure. The FLIM images indicate chemical biomarkers including increased nicotinamide adenine dinucleotide and reduced collagen emission. While both sets of biomarkers can differentiate normal and cancerous tissue, we believe their combination will enable the discrimination of benign lesions possessing some of the indicated biomarkers, e.g., scarring or inflammation.

  18. Imaging of RNA in situ hybridization by atomic force microscopy

    NARCIS (Netherlands)

    Kalle, W.H.J.; Macville, M.V.E.; van de Corput, M.P.C.; de Grooth, B.G.; Tanke, H.J.; Raap, A.K.

    In this study we investigated the possibility of imaging internal cellular molecules after cytochemical detection with atomic force microscopy (AFM). To this end, rat 9G and HeLa cells were hybridized with haptenized probes for 28S ribosomal RNA, human elongation factor mRNA and cytomegalovirus

  19. X-ray holographic microscopy: Improved images of zymogen granules

    Energy Technology Data Exchange (ETDEWEB)

    Jacobsen, C.; Howells, M.; Kirz, J.; McQuaid, K.; Rothman, S.

    1988-10-01

    Soft x-ray holography has long been considered as a technique for x-ray microscopy. It has been only recently, however, that sub-micron resolution has been obtained in x-ray holography. This paper will concentrate on recent progress we have made in obtaining reconstructed images of improved quality. 15 refs., 6 figs.

  20. Intermolecular Contrast in Atomic Force Microscopy Images without Intermolecular Bonds

    NARCIS (Netherlands)

    Hämäläinen, Sampsa K.; van der Heijden, N.J. (Nadine); van der Lit, Joost; den Hartog, Stephan; Liljeroth, Peter; Swart, Ingmar

    2014-01-01

    Intermolecular features in atomic force microscopy images of organic molecules have been ascribed to intermolecular bonds. A recent theoretical study [P. Hapala et al., Phys. Rev. B 90, 085421 (2014)] showed that these features can also be explained by the flexibility of molecule-terminated tips. We

  1. 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

  2. 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 requirem

  3. 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 requirem

  4. 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

  5. Scanning transmission electron microscopy imaging dynamics at low accelerating voltages

    Energy Technology Data Exchange (ETDEWEB)

    Lugg, N.R. [School of Physics, University of Melbourne, Parkville, Victoria 3010 (Australia); Findlay, S.D. [Institute of Engineering Innovation, The University of Tokyo, Tokyo 116-0013 (Japan); Shibata, N. [Institute of Engineering Innovation, The University of Tokyo, Tokyo 116-0013 (Japan); PRESTO, Japan Science and Technology Agency, Saitama 332-0012 (Japan); Mizoguchi, T. [Institute of Industrial Science, The University of Tokyo, Tokyo 153-8505 (Japan); D' Alfonso, A.J. [School of Physics, University of Melbourne, Parkville, Victoria 3010 (Australia); Allen, L.J., E-mail: lja@unimelb.edu.au [School of Physics, University of Melbourne, Parkville, Victoria 3010 (Australia); Ikuhara, Y. [Institute of Engineering Innovation, The University of Tokyo, Tokyo 116-0013 (Japan); Nanostructures Research Laboratory, Japan Fine Ceramic Center, Nagoya 456-8587 (Japan); WPI Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577 (Japan)

    2011-07-15

    Motivated by the desire to minimize specimen damage in beam sensitive specimens, there has been a recent push toward using relatively low accelerating voltages (<100kV) in scanning transmission electron microscopy. To complement experimental efforts on this front, this paper seeks to explore the variations with accelerating voltage of the imaging dynamics, both of the channelling of the fast electron and of the inelastic interactions. High-angle annular-dark field, electron energy loss spectroscopic imaging and annular bright field imaging are all considered. -- Highlights: {yields} Both elastic and inelastic scattering in STEM are acceleration voltage dependent. {yields} HAADF, EELS and ABF imaging are assessed with a view to optimum imaging. {yields} Lower accelerating voltages improve STEM EELS contrast in very thin crystals. {yields} Higher accelerating voltages give better STEM EELS contrast in thicker crystals. {yields} At fixed resolution, higher accelerating voltage aids ABF imaging of light elements.

  6. Synergizing superresolution optical fluctuation imaging with single molecule localization microscopy

    CERN Document Server

    Schidorsky, Shachar; Razvag, Yair; Golan, Yonatan; Weiss, Shimon; Sherman, Eilon

    2016-01-01

    Single molecule localization microscopy (SMLM) techniques enable imaging biological samples well beyond the diffraction limit of light, but they vary significantly in their spatial and temporal resolutions. High-order statistical analysis of temporal fluctuations as in superresolution optical fluctuation imaging (SOFI) also enable imaging beyond diffraction limit, but usually at a lower resolution as compared to SMLM. Since the same data format is acquired for both methods, their algorithms can be applied to the same data set, and thus may be combined synergistically to improve overall imaging performance. Here, we find that SOFI converges much faster than SMLM, provides additive information to SMLM, and can efficiently reject background. We then show how SOFI-assisted SMLM imaging can improve SMLM image reconstruction by rejecting common sources of background, especially under low signal-to-background conditions. The performance of our approach was evaluated using a realistic simulation of fluorescence imagi...

  7. Quantification of photoacoustic microscopy images for ovarian cancer detection

    Science.gov (United States)

    Wang, Tianheng; Yang, Yi; Alqasemi, Umar; Kumavor, Patrick D.; Wang, Xiaohong; Sanders, Melinda; Brewer, Molly; Zhu, Quing

    2014-03-01

    In this paper, human ovarian tissues with malignant and benign features were imaged ex vivo by using an opticalresolution photoacoustic microscopy (OR-PAM) system. Several features were quantitatively extracted from PAM images to describe photoacoustic signal distributions and fluctuations. 106 PAM images from 18 human ovaries were classified by applying those extracted features to a logistic prediction model. 57 images from 9 ovaries were used as a training set to train the logistic model, and 49 images from another 9 ovaries were used to test our prediction model. We assumed that if one image from one malignant ovary was classified as malignant, it is sufficient to classify this ovary as malignant. For the training set, we achieved 100% sensitivity and 83.3% specificity; for testing set, we achieved 100% sensitivity and 66.7% specificity. These preliminary results demonstrate that PAM could be extremely valuable in assisting and guiding surgeons for in vivo evaluation of ovarian tissue.

  8. 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.

  9. Divided-aperture differential confocal fast-imaging microscopy

    Science.gov (United States)

    Wang, Yun; Qiu, Lirong; Zhao, Xiangye; Zhao, Weiqian

    2017-03-01

    A new method, laser divided-aperture differential confocal microscopy (DDCM), is proposed to achieve high-resolution 3D imaging of microstructures of large-scale sample surfaces. This method uses a divided-aperture confocal structure to significantly improve the axial resolution of confocal microscopy and keep a long working distance simultaneously; uses two radically offset point detectors to achieve differential detection to further improve the axial response sensitivity and realize fast imaging of a large-scale sample surface with a big axial scan-step interval. Theoretical analyses and experimental results show that the DDCM can reach an axial resolution of 5 nm with a 3.1 mm working distance with a 3 times imaging speed of a confocal system with the same resolution.

  10. High-speed atomic force microscopy: imaging and force spectroscopy.

    Science.gov (United States)

    Eghiaian, Frédéric; Rico, Felix; Colom, Adai; Casuso, Ignacio; Scheuring, Simon

    2014-10-01

    Atomic force microscopy (AFM) is the type of scanning probe microscopy that is probably best adapted for imaging biological samples in physiological conditions with submolecular lateral and vertical resolution. In addition, AFM is a method of choice to study the mechanical unfolding of proteins or for cellular force spectroscopy. In spite of 28 years of successful use in biological sciences, AFM is far from enjoying the same popularity as electron and fluorescence microscopy. The advent of high-speed atomic force microscopy (HS-AFM), about 10 years ago, has provided unprecedented insights into the dynamics of membrane proteins and molecular machines from the single-molecule to the cellular level. HS-AFM imaging at nanometer-resolution and sub-second frame rate may open novel research fields depicting dynamic events at the single bio-molecule level. As such, HS-AFM is complementary to other structural and cellular biology techniques, and hopefully will gain acceptance from researchers from various fields. In this review we describe some of the most recent reports of dynamic bio-molecular imaging by HS-AFM, as well as the advent of high-speed force spectroscopy (HS-FS) for single protein unfolding.

  11. 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.

  12. TCSPC FLIM in the wavelength range from 800 nm to 1700 nm (Conference Presentation)

    Science.gov (United States)

    Becker, Wolfgang; Shcheslavsky, Vladislav

    2016-03-01

    Excitation and detection in the wavelength range above 800nm is a convenient and relatively inexpensive way to increase the penetration depth in optical microscopy. Moreover, detection at long wavelength avoids the problem that tissue autofluorescence contaminates the signals from endogenous fluorescence probes. FLIM at NIR wavelength may therefore be complementary to multiphoton microscopy, especially if the lifetimes of NIR fluorophores report biological parameters of the tissue structures they are bound to. Unfortunately, neither the excitation sources nor the detectors of standard confocal and multiphoton laser scanning systems are directly suitable for excitation and detection of NIR fluorescence. Most of these problems can be solved, however, by using ps diode lasers or Ti:Sapphire lasers at their fundamental wavelength, and NIR-sensitive detectors. With NIR-sensitive PMTs the detection wavelength range can be extended up to 900 nm, with InGaAs SPAD detectors up to 1700 nm. Here, we demonstrate the use of a combination of laser scanning, multi-dimensional TCSPC, and advanced excitation sources and detectors for FLIM at up to 1700 nm. The performance was tested at tissue samples incubated with NIR dyes. The fluorescence lifetimes generally get shorter with increasing absorption and emission wavelengths of the dyes. For the cyanine dye IR1061, absorbing around 1060 nm, the lifetime was found to be as short as 70 ps. Nevertheless the fluorescence decay could still be clearly detected. Almost all dyes showed clear lifetime changes depending on the binding to different tissue constituents.

  13. 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.

  14. Fast rasterscanning enables FLIM in macroscopic samples up to several centimeters

    Science.gov (United States)

    Koberling, F.; Buschmann, V.; Hille, C.; Patting, M.; Dosche, C.; Sandberg, A.; Wheelock, A.; Erdmann, R.

    2010-02-01

    Fluorescence Lifetime Imaging (FLIM) based on Time-Correlated Single Photon Counting (TCSPC) is nowadays a well established technique that is very often realised as an add-on for confocal laser scanning microscopes. However, the standard laser scanning technique limits the maximum scan range in these setups to a few millimetre, making it therefore unsuited for e.g. fluorescence multiplexing in multi well plate based assays or for macroscopic material science studies on solar cells, wafers and similar material. In order to also realize larger scanning ranges, we have developed a sample scanning approach based on a xy-cross stage equipped with piezo linear motors. Using online position monitoring, this approach permits fast acceleration and scanning as well as precise positioning and features scan ranges from 100×100 microns up to 80×80 mm with submicron positioning accuracy. Standard upright and inverse microscope bodies can easily be equipped with this scanning device. Along with the necessary excitation and detection components "largearea" FLIM thus becomes possible. We will show new results obtained with a modified MicroTime 100 (PicoQuant GmbH) illustrating the system capabilities for lifetime based imaging in macroscopic samples such as the improvement of the fluorescence sensitivity in 2D gel electrophoresis or the possibility to perform lifetime based fluorescence multiplexing in μ-well plate based assays. Even Two Photon Excitation (TPE) imaging is possible with this widerange sample scanning approach and first FLIM results on cockroach salivary glands, loaded with a chloride sensitive dye (MQAE) will be presented.

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

    CERN Document Server

    Kurvits, Jonathan A; Zia, Rashid

    2015-01-01

    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, which 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 ide...

  16. Scanning electron microscopy: preparation and imaging for SEM.

    Science.gov (United States)

    Jones, Chris G

    2012-01-01

    Scanning electron microscopy (SEM) has been almost universally applied for the surface examination and characterization of both natural and man-made objects. Although an invasive technique, developments in electron microscopy over the years has given the microscopist a much clearer choice in how invasive the technique will be. With the advent of low vacuum SEM in the 1970s (The environmental cold stage, 1970) and environmental SEM in the late 1980s (J Microsc 160(pt. 1):9-19, 1989), it is now possible in some circumstances to examine samples without preparation. However, for the examination of biological tissue and cells it is still advisable to chemically fix, dehydrate, and coat samples for SEM imaging and analysis. This chapter aims to provide an overview of SEM as an imaging tool, and a general introduction to some of the methods applied for the preparation of samples.

  17. 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.

  18. 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.

  19. 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...... specific segmentation problem using supervised learning. This approach was tested on five different segmentation problems in bright field, differential interference contrast, fluorescence and laser confocal scanning microscopy. In all instance good results were achieved with the segmentation quality...

  20. Confocal supercritical angle fluorescence microscopy for cell membrane imaging

    CERN Document Server

    Sivankutty, Siddharth; Mayet, Céline; Dupuis, Guillaume; Fort, Emmanuel; Lévêque-Fort, Sandrine

    2013-01-01

    We demonstrate sub-wavelength sectioning on biological samples with a conventional confocal microscope. This optical sectioning is achieved by the phenomenon of supercritical angle fuorescence, wherein only a fluorophore next to the interface of a refractive index discontinuity can emit propagating components of radiation into the so-called forbidden angles. The simplicity of this technique allows it to be integrated with a high numerical aperture confocal scanning microscope by only a simple modi?cation on the detection channel. Confocal-SAF microscopy would be a powerful tool to achieve high resolution surface imaging, especially for membrane imaging in biological samples

  1. Neuron Segmentation in Electron Microscopy Images Using Partial Differential Equations.

    Science.gov (United States)

    Jones, Cory; Sayedhosseini, Mojtaba; Ellisman, Mark; Tasdizen, Tolga

    2013-01-01

    In connectomics, neuroscientists seek to identify the synaptic connections between neurons. Segmentation of cell membranes using supervised learning algorithms on electron microscopy images of brain tissue is often done to assist in this effort. Here we present a partial differential equation with a novel growth term to improve the results of a supervised learning algorithm. We also introduce a new method for representing the resulting image that allows for a more dynamic thresholding to further improve the result. Using these two processes we are able to close small to medium sized gaps in the cell membrane detection and improve the Rand error by as much as 9% over the initial supervised segmentation.

  2. 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.

  3. Imaging carious dental tissues with multiphoton fluorescence lifetime imaging microscopy

    Science.gov (United States)

    Lin, Po-Yen; Lyu, Hong-Chou; Hsu, Chin-Ying Stephen; Chang, Chia-Seng; Kao, Fu-Jen

    2011-01-01

    In this study, multiphoton excitation was utilized to image normal and carious dental tissues noninvasively. Unique structures in dental tissues were identified using the available multimodality (second harmonic, autofluorescence, and fluorescence lifetime analysis) without labeling. The collagen in dentin exhibits a strong second harmonic response. Both dentin and enamel emit strong autofluorescence that reveals in detail morphological features (such as dentinal tubules and enamel rods) and, despite their very similar spectral profiles, can be differentiated by lifetime analysis. Specifically, the carious dental tissue exhibits a greatly reduced autofluorescence lifetime, which result is consistent with the degree of demineralization, determined by micro-computed tomography. Our findings suggest that two-photon excited fluorescence lifetime imaging may be a promising tool for diagnosing and monitoring dental caries. PMID:21326645

  4. 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.

  5. Canning plasmonic microscopy by image reconstruction from the Fourier space

    CERN Document Server

    Mollet, O; Drezet, A

    2014-01-01

    We demonstrate a simple scheme for high-resolution imaging of nanoplasmonic structures that basically removes most of the resolution limiting allowed light usually transmitted to the far field. This is achieved by implementing a Fourier lens in a near-field scanning optical microscope (NSOM) operating in the leakage-radiation microscopy (LRM) mode. The method consists of reconstructing optical images solely from the plasmonic `forbidden' light collected in the Fourier space. It is demonstrated by using a point-like nanodiamond-based tip that illuminates a thin gold film patterned with a sub-wavelength annular slit. The reconstructed image of the slit shows a spatial resolution enhanced by a factor $\\simeq 4$ compared to NSOM images acquired directly in the real space.

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

    Science.gov (United States)

    Wang, Feifei; Li, Yi; Jia, Boliang; Liu, Lianqing; Li, Wen Jung

    2016-01-01

    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. PMID:27768774

  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. High resolution surface plasmon microscopy for cell imaging

    Science.gov (United States)

    Argoul, F.; Monier, K.; Roland, T.; Elezgaray, J.; Berguiga, L.

    2010-04-01

    We introduce a new non-labeling high resolution microscopy method for cellular imaging. This method called SSPM (Scanning Surface Plasmon Microscopy) pushes down the resolution limit of surface plasmon resonance imaging (SPRi) to sub-micronic scales. High resolution SPRi is obtained by the surface plasmon lauching with a high numerical aperture objective lens. The advantages of SPPM compared to other high resolution SPRi's rely on three aspects; (i) the interferometric detection of the back reflected light after plasmon excitation, (ii) the twodimensional scanning of the sample for image reconstruction, (iii) the radial polarization of light, enhancing both resolution and sensitivity. This microscope can afford a lateral resolution of - 150 nm in liquid environment and - 200 nm in air. We present in this paper images of IMR90 fibroblasts obtained with SSPM in dried environment. Internal compartments such as nucleus, nucleolus, mitochondria, cellular and nuclear membrane can be recognized without labelling. We propose an interpretation of the ability of SSPM to reveal high index contrast zones by a local decomposition of the V (Z) function describing the response of the SSPM.

  9. Registration and 3D visualization of large microscopy images

    Science.gov (United States)

    Mosaliganti, Kishore; Pan, Tony; Sharp, Richard; Ridgway, Randall; Iyengar, Srivathsan; Gulacy, Alexandra; Wenzel, Pamela; de Bruin, Alain; Machiraju, Raghu; Huang, Kun; Leone, Gustavo; Saltz, Joel

    2006-03-01

    Inactivation of the retinoblastoma gene in mouse embryos causes tissue infiltrations into critical sections of the placenta, which has been shown to affect fetal survivability. Our collaborators in cancer genetics are extremely interested in examining the three dimensional nature of these infiltrations given a stack of two dimensional light microscopy images. Three sets of wildtype and mutant placentas was sectioned serially and digitized using a commercial light microscopy scanner. Each individual placenta dataset consisted of approximately 1000 images totaling 700 GB in size, which were registered into a volumetric dataset using National Library of Medicine's (NIH/NLM) Insight Segmentation and Registration Toolkit (ITK). This paper describes our method for image registration to aid in volume visualization of tissue level intermixing for both wildtype and Rb - specimens. The registration process faces many challenges arising from the large image sizes, damages during sectioning, staining gradients both within and across sections, and background noise. These issues limit the direct application of standard registration techniques due to frequent convergence to local solutions. In this work, we develop a mixture of automated and semi-automated enhancements with ground-truth validation for the mutual information-based registration algorithm. Our final volume renderings clearly show tissue intermixing differences between both wildtype and Rb - specimens which are not obvious prior to registration.

  10. Coherent imaging with incoherent light in digital holographic microscopy

    Science.gov (United States)

    Chmelik, Radim

    2012-01-01

    Digital holographic microscope (DHM) allows for imaging with a quantitative phase contrast. In this way it becomes an important instrument, a completely non-invasive tool for a contrast intravital observation of living cells and a cell drymass density distribution measurement. A serious drawback of current DHMs is highly coherent illumination which makes the lateral resolution worse and impairs the image quality by a coherence noise and a parasitic interference. An uncompromising solution to this problem can be found in the Leith concept of incoherent holography. An off-axis hologram can be formed with arbitrary degree of light coherence in systems equipped with an achromatic interferometer and thus the resolution and the image quality typical for an incoherent-light wide-field microscopy can be achieved. In addition, advanced imaging modes based on limited coherence can be utilized. The typical example is a coherence-gating effect which provides a finite axial resolution and makes DHM image similar to that of a confocal microscope. These possibilities were described theoretically using the formalism of three-dimensional coherent transfer functions and proved experimentally by the coherence-controlled holographic microscope which is DHM based on the Leith achromatic interferometer. Quantitative-phase-contrast imaging is demonstrated with incoherent light by the living cancer cells observation and their motility evaluation. The coherence-gating effect was proved by imaging of model samples through a scattering layer and living cells inside an opalescent medium.

  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. Confocal laser scanning microscopy image correlation for nanoparticle flow velocimetry

    CERN Document Server

    Jun, Brian; Yang, Haisheng; Main, Russell; Vlachos, Pavlos

    2016-01-01

    We present a new particle image correlation technique for resolving nanoparticle flow velocity using confocal laser scanning microscopy (CLSM). The two primary issues that complicate nanoparticle scanning laser image correlation (SLIC) based velocimetry are (1) the use of diffusion dominated nanoparticles as flow tracers, which introduce a random decorrelating error into the velocity estimate, and (2) the effects of the scanning laser image acquisition, which introduces a bias error. To date, no study has quantified these errors or demonstrated a means to deal with them in SLIC velocimetry. In this work, we build upon the robust phase correlation (RPC) and existing methods of SLIC to quantify and mitigate these errors. First, we implement an ensemble RPC instead of using an ensemble standard cross correlation, and develop an SLIC optimal filter that maximizes the correlation strength in order to reliably and accurately detect the correlation peak representing the most probable average displacement of the nano...

  13. Compressive Fluorescence Microscopy for Biological and Hyperspectral Imaging

    CERN Document Server

    Studer, Vincent; Chahid, Makhlad; Moussavi, Hamed; Candes, Emmanuel; Dahan, Maxime

    2012-01-01

    The mathematical theory of compressed sensing (CS) asserts that one can acquire signals from measurements whose rate is much lower than the total bandwidth. Whereas the CS theory is now well developed, challenges concerning hardware implementations of CS-based acquisition devices---especially in optics---have only started being addressed. This paper presents an implementation of compressive sensing in fluorescence microscopy and its applications to biomedical imaging. Our CS microscope combines a dynamic structured wide-field illumination and a fast and sensitive single-point fluorescence detection to enable reconstructions of images of fluorescent beads, cells and tissues with undersampling ratios (between the number of pixels and number of measurements) up to 32. We further demonstrate a hyperspectral mode and record images with 128 spectral channels and undersampling ratios up to 64, illustrating the potential benefits of CS acquisition for higher dimensional signals which typically exhibits extreme redund...

  14. CARS and non-linear microscopy imaging of brain tumors

    Science.gov (United States)

    Galli, Roberta; Uckermann, Ortrud; Tamosaityte, Sandra; Geiger, Kathrin; Schackert, Gabriele; Steiner, Gerald; Koch, Edmund; Kirsch, Matthias

    2013-06-01

    Nonlinear optical microscopy offers a series of techniques that have the potential to be applied in vivo, for intraoperative identification of tumor border and in situ pathology. By addressing the different content of lipids that characterize the tumors with respect to the normal brain tissue, CARS microscopy enables to discern primary and secondary brain tumors from healthy tissue. A study performed in mouse models shows that the reduction of the CARS signal is a reliable quantity to identify brain tumors, irrespective from the tumor type. Moreover it enables to identify tumor borders and infiltrations at a cellular resolution. Integration of CARS with autogenous TPEF and SHG adds morphological and compositional details about the tissue. Examples of multimodal CARS imaging of different human tumor biopsies demonstrate the ability of the technique to retrieve information useful for histopathological diagnosis.

  15. Directional bilateral filters for smoothing fluorescence microscopy images

    Directory of Open Access Journals (Sweden)

    Manasij Venkatesh

    2015-08-01

    Full Text Available Images obtained through fluorescence microscopy at low numerical aperture (NA are noisy and have poor resolution. Images of specimens such as F-actin filaments obtained using confocal or widefield fluorescence microscopes contain directional information and it is important that an image smoothing or filtering technique preserve the directionality. F-actin filaments are widely studied in pathology because the abnormalities in actin dynamics play a key role in diagnosis of cancer, cardiac diseases, vascular diseases, myofibrillar myopathies, neurological disorders, etc. We develop the directional bilateral filter as a means of filtering out the noise in the image without significantly altering the directionality of the F-actin filaments. The bilateral filter is anisotropic to start with, but we add an additional degree of anisotropy by employing an oriented domain kernel for smoothing. The orientation is locally adapted using a structure tensor and the parameters of the bilateral filter are optimized for within the framework of statistical risk minimization. We show that the directional bilateral filter has better denoising performance than the traditional Gaussian bilateral filter and other denoising techniques such as SURE-LET, non-local means, and guided image filtering at various noise levels in terms of peak signal-to-noise ratio (PSNR. We also show quantitative improvements in low NA images of F-actin filaments.

  16. Nonlinear optical microscopy and ultrasound imaging of human cervical structure

    Science.gov (United States)

    Reusch, Lisa M.; Feltovich, Helen; Carlson, Lindsey C.; Hall, Gunnsteinn; Campagnola, Paul J.; Eliceiri, Kevin W.; Hall, Timothy J.

    2013-03-01

    The cervix softens and shortens as its collagen microstructure rearranges in preparation for birth, but premature change may lead to premature birth. The global preterm birth rate has not decreased despite decades of research, likely because cervical microstructure is poorly understood. Our group has developed a multilevel approach to evaluating the human cervix. We are developing quantitative ultrasound (QUS) techniques for noninvasive interrogation of cervical microstructure and corroborating those results with high-resolution images of microstructure from second harmonic generation imaging (SHG) microscopy. We obtain ultrasound measurements from hysterectomy specimens, prepare the tissue for SHG, and stitch together several hundred images to create a comprehensive view of large areas of cervix. The images are analyzed for collagen orientation and alignment with curvelet transform, and registered with QUS data, facilitating multiscale analysis in which the micron-scale SHG images and millimeter-scale ultrasound data interpretation inform each other. This novel combination of modalities allows comprehensive characterization of cervical microstructure in high resolution. Through a detailed comparative study, we demonstrate that SHG imaging both corroborates the quantitative ultrasound measurements and provides further insight. Ultimately, a comprehensive understanding of specific microstructural cervical change in pregnancy should lead to novel approaches to the prevention of preterm birth.

  17. Quantitative analysis of in vivo confocal microscopy images: a review.

    Science.gov (United States)

    Patel, Dipika V; McGhee, Charles N

    2013-01-01

    In vivo confocal microscopy (IVCM) is a non-invasive method of examining the living human cornea. The recent trend towards quantitative studies using IVCM has led to the development of a variety of methods for quantifying image parameters. When selecting IVCM images for quantitative analysis, it is important to be consistent regarding the location, depth, and quality of images. All images should be de-identified, randomized, and calibrated prior to analysis. Numerous image analysis software are available, each with their own advantages and disadvantages. Criteria for analyzing corneal epithelium, sub-basal nerves, keratocytes, endothelium, and immune/inflammatory cells have been developed, although there is inconsistency among research groups regarding parameter definition. The quantification of stromal nerve parameters, however, remains a challenge. Most studies report lower inter-observer repeatability compared with intra-observer repeatability, and observer experience is known to be an important factor. Standardization of IVCM image analysis through the use of a reading center would be crucial for any future large, multi-centre clinical trials using IVCM.

  18. High resolution magnetic imaging: MicroSQUID Force Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Hasselbach, K; Ladam, C; Dolocan, V O; Hykel, D; Crozes, T [Institut Neel, CNRS et Universite Joseph Fourier, BP 166, F-38042 Grenoble Cedex 9 (France); Schuster, K [Institut de RadioAstronomie Millimetrique 300 rue de la Piscine, Domaine Universitaire F-38406 Saint Martin d' Heres (France); Mailly, D [Laboratoire de Photonique et de Nanostructures, CNRS, Site Alcatel de Marcoussis Route de Nozay F-91460 Marcoussis (France)], E-mail: klaus.hasselbach@grenoble.cnrs.fr

    2008-02-01

    Magnetic imaging at the micrometer scale with high sensitivity is a challenge difficult to be met. Magnetic force microscopy has a very high spatial resolution but is limited in magnetic resolution. Hall probe microscopy is very powerful but sensor fabrication at the one micron scale is difficult and effects due to discreteness of charge appear in the form of significant 1/f noise. SQUID microscopy is very powerful, having high magnetic resolution, but spatial resolution is usually of the order of 10 {mu}m. The difficulties lay mostly in an efficient way to couple flux to the sensor. The only way to improve spatial resolution is to place the probe close to the very edge of the support, thus maximising coupling and spatial resolution. If there has been found a way to bring close the tip, there must be also found a reliable a way to maintain distance during scanning. We want to present recent improvements on scanning microsquid microscopy: Namely the improved fabrication of microSQUID tips using silicon micro machining and the precise positioning of the micrometer diameter microSQUID loop by electron beam lithography. The microSQUID is a microbridge DC SQUID, with two opposite microbridges. The constrictions are patterned by high-resolution e-beam lithography and have a width of 20 nm and a length of about 100 nm. The distance control during scanning is obtained by integrating the microSQUID sensor with a piezoelectric tuning fork acting as a force sensor allowing to control height and even topographic imaging. The detector is placed in a custom built near field microscope and the sample temperature can be varied between 0.1 Kelvin and 10 K. The microscope is used to study magnetic flux structures in unconventional superconductors and will be used to observe thermal domains in superconducting detectors in the voltage state.

  19. 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...

  20. Mueller matrix signature in advanced fluorescence microscopy imaging

    Science.gov (United States)

    Mazumder, Nirmal; Qiu, Jianjun; Kao, Fu-Jen; Diaspro, Alberto

    2017-02-01

    We have demonstrated the measurement and characterization of the polarization properties of a fluorescence signal using four-channel photon counting based Stokes-Mueller polarization microscopy. Thus, Lu-Chipman decomposition was applied to extract the critical polarization properties such as depolarization, linear retardance and the optical rotation of collagen type I fiber. We observed the spatial distribution of anisotropic and helical molecules of collagen from the reconstructed 2D Mueller images based on the fluorescence signal in a pixel-by-pixel manner.

  1. High-Speed Fluorescence Microscopy: Lifetime Imaging in the Biomedical Sciences

    Science.gov (United States)

    Periasamy, Ammasi; Wang, Xue F.; Wodnick, Pawel; Gordon, Gerald W.; Kwon, Seongwook; Diliberto, Pamela A.; Herman, Brian

    1995-02-01

    The ability to observe the behavior of living cells and tissues provides unparalleled access to information regarding the organization and dynamics of complex cellular structures. While great strides have been made over the past 30 to 40 years in the design and application of a variety of novel optical microscopic techniques, until recently, it has not been possible to image biological phenomena that occur over very short time periods (nanosecond to millisecond) or over short distances (10 to 1000 [Angstrom capital A, ring]). However, the recent combination of (1) very rapidly gated and sensitive image intensifiers and (2) the ability to deliver fluorescence excitation energy to intact living biological specimens in a pulsed or sinusoidally modulated fashion has allowed such measurements to become a reality through the imaging of the lifetimes of fluorescent molecules. This capability has resulted in the ability to observe the dynamic organization and interaction of cellular components on a spatial and temporal scale previously not possible using other microscopic techniques. This paper discusses the implementation of a fluorescence lifetime imaging microscope (FLIM) and provides a review of some of the applications of such an instrument. These include measurements of receptor topography and subunit interactions using fluorescence resonance energy transfer (FRET), fluorescence anisotropy of phospholipids in cell membranes, cytosolic free calcium (Ca2+)i and the detection of human papillomavirus (HPV) infection in clinical cervicovaginal smears.

  2. Image recombination transform algorithm for superresolution structured illumination microscopy

    Science.gov (United States)

    Zhou, Xing; Lei, Ming; Dan, Dan; Yao, Baoli; Yang, Yanlong; Qian, Jia; Chen, Guangde; Bianco, Piero R.

    2016-09-01

    Structured illumination microscopy (SIM) is an attractive choice for fast superresolution imaging. The generation of structured illumination patterns made by interference of laser beams is broadly employed to obtain high modulation depth of patterns, while the polarizations of the laser beams must be elaborately controlled to guarantee the high contrast of interference intensity, which brings a more complex configuration for the polarization control. The emerging pattern projection strategy is much more compact, but the modulation depth of patterns is deteriorated by the optical transfer function of the optical system, especially in high spatial frequency near the diffraction limit. Therefore, the traditional superresolution reconstruction algorithm for interference-based SIM will suffer from many artifacts in the case of projection-based SIM that possesses a low modulation depth. Here, we propose an alternative reconstruction algorithm based on image recombination transform, which provides an alternative solution to address this problem even in a weak modulation depth. We demonstrated the effectiveness of this algorithm in the multicolor superresolution imaging of bovine pulmonary arterial endothelial cells in our developed projection-based SIM system, which applies a computer controlled digital micromirror device for fast fringe generation and multicolor light-emitting diodes for illumination. The merit of the system incorporated with the proposed algorithm allows for a low excitation intensity fluorescence imaging even less than 1 W/cm2, which is beneficial for the long-term, in vivo superresolved imaging of live cells and tissues.

  3. Multiphoton microscopy as a diagnostic imaging modality for lung cancer

    Science.gov (United States)

    Pavlova, Ina; Hume, Kelly R.; Yazinski, Stephanie A.; Peters, Rachel M.; Weiss, Robert S.; Webb, Watt W.

    2010-02-01

    Lung cancer is the leading killer among all cancers for both men and women in the US, and is associated with one of the lowest 5-year survival rates. Current diagnostic techniques, such as histopathological assessment of tissue obtained by computed tomography guided biopsies, have limited accuracy, especially for small lesions. Early diagnosis of lung cancer can be improved by introducing a real-time, optical guidance method based on the in vivo application of multiphoton microscopy (MPM). In particular, we hypothesize that MPM imaging of living lung tissue based on twophoton excited intrinsic fluorescence and second harmonic generation can provide sufficient morphologic and spectroscopic information to distinguish between normal and diseased lung tissue. Here, we used an experimental approach based on MPM with multichannel fluorescence detection for initial discovery that MPM spectral imaging could differentiate between normal and neoplastic lung in ex vivo samples from a murine model of lung cancer. Current results indicate that MPM imaging can directly distinguish normal and neoplastic lung tissues based on their distinct morphologies and fluorescence emission properties in non-processed lung tissue. Moreover, we found initial indication that MPM imaging differentiates between normal alveolar tissue, inflammatory foci, and lung neoplasms. Our long-term goal is to apply results from ex vivo lung specimens to aid in the development of multiphoton endoscopy for in vivo imaging of lung abnormalities in various animal models, and ultimately for the diagnosis of human lung cancer.

  4. Imaging theory of nonlinear second harmonic and third harmonic generations in confocal microscopy

    Institute of Scientific and Technical Information of China (English)

    TANG; Zhilie; XING; Da; LIU; Songhao

    2004-01-01

    The imaging theory of nonlinear second harmonic generation (SHG) and third harmonic generation (THG) in confocal microscopy is presented in this paper. The nonlinear effect of SHG and THG on the imaging properties of confocal microscopy has been analyzed in detail by the imaging theory. It is proved that the imaging process of SHG and THG in confocal microscopy, which is different from conventional coherent imaging or incoherent imaging, can be divided into two different processes of coherent imaging. The three-dimensional point spread functions (3D-PSF) of SHG and THG confocal microscopy are derived based on the nonlinear principles of SHG and THG. The imaging properties of SHG and THG confocal microscopy are discussed in detail according to its 3D-PSF. It is shown that the resolution of SHG and THG confocal microscopy is higher than that of single-and two-photon confocal microscopy.

  5. Image Restoration Phase-Filtering Lateral Superresolution Confocal Microscopy

    Institute of Scientific and Technical Information of China (English)

    ZHAO Wei-Qian; QIU Li-Rong; CHEN Shan-Shan; FENG Zheng-De

    2006-01-01

    @@ Image restoration phase-filtering lateral superresolution confocal microscopy, a new approach, is proposed to achieve lateral superresolution using a confocal microscope. This approach uses a lateral superresolution pupil filter to preliminarily improve its lateral resolution and uses a single-image superresolution restoration technique based on a maximum likelihood estimate to further improve its lateral resolution. The new approach has the advantages of a low cost and the remarkable superresolution effect without excessive system complexity. Experiments indicate that the proposed approach can improve the lateral resolution of a confocal microscope from 0.3μm to less than 0.1 μm when λ = 632.8 nm and NA =0.85.

  6. Dynamic force microscopy for imaging of viruses under physiological conditions

    Directory of Open Access Journals (Sweden)

    Kienberger Ferry

    2004-01-01

    Full Text Available Dynamic force microscopy (DFM allows imaging of the structure and the assessment of the function of biological specimens in their physiological environment. In DFM, the cantilever is oscillated at a given frequency and touches the sample only at the end of its downward movement. Accordingly, the problem of lateral forces displacing or even destroying bio-molecules is virtually inexistent as the contact time and friction forces are reduced. Here, we describe the use of DFM in studies of human rhinovirus serotype 2 (HRV2 weakly adhering to mica surfaces. The capsid of HRV2 was reproducibly imaged without any displacement of the virus. Release of the genomic RNA from the virions was initiated by exposure to low pH buffer and snapshots of the extrusion process were obtained. In the following, the technical details of previous DFM investigations of HRV2 are summarized.

  7. Segmentation of fluorescence microscopy cell images using unsupervised mining.

    Science.gov (United States)

    Du, Xian; Dua, Sumeet

    2010-05-28

    The accurate measurement of cell and nuclei contours are critical for the sensitive and specific detection of changes in normal cells in several medical informatics disciplines. Within microscopy, this task is facilitated using fluorescence cell stains, and segmentation is often the first step in such approaches. Due to the complex nature of cell issues and problems inherent to microscopy, unsupervised mining approaches of clustering can be incorporated in the segmentation of cells. In this study, we have developed and evaluated the performance of multiple unsupervised data mining techniques in cell image segmentation. We adapt four distinctive, yet complementary, methods for unsupervised learning, including those based on k-means clustering, EM, Otsu's threshold, and GMAC. Validation measures are defined, and the performance of the techniques is evaluated both quantitatively and qualitatively using synthetic and recently published real data. Experimental results demonstrate that k-means, Otsu's threshold, and GMAC perform similarly, and have more precise segmentation results than EM. We report that EM has higher recall values and lower precision results from under-segmentation due to its Gaussian model assumption. We also demonstrate that these methods need spatial information to segment complex real cell images with a high degree of efficacy, as expected in many medical informatics applications.

  8. Imaging domains in transmission electron microscopy (invited) (abstract)

    Science.gov (United States)

    Mishra, R. K.

    1987-04-01

    Magnetic domain walls and domains inside thin electron transparent specimens of ferromagnetic materials can be imaged using the Fresnel and Focault techniques in a transmission electron microscope. Combined with the diffraction, microstructural and microchemical capabilities of modern microscopes, Lorentz microscopy offers one of the most powerful tools to study structure-property relationships in magnetic materials. In addition, using this technique, it is possible to deduce the local magnetization distribution around inhomogeneities and complex Bloch and Néel walls. Lorentz images can be used to quantitatively measure domain wall thickness and estimate domain wall energy. With modified sample holders and pole pieces, one can study in situ domain wall motion and the interaction of domains with microstructural features such as second phases, grain boundaries, structural defects, etc. All these will be illustrated with examples of Lorentz images from soft and hard magnets with special emphasis on the Nd-Fe-B hard magnets. Finally, the limitations of the Lorentz imaging technique utilizing the deflected electron intensities will be outlined and a new technique which utilizes the phase changes in the electron beam as it passes through the material in a scanning transmission microscope will be reviewed.

  9. 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

  10. A mTurquoise-based cAMP sensor for both FLIM and ratiometric read-out has improved dynamic range.

    Directory of Open Access Journals (Sweden)

    Jeffrey B Klarenbeek

    Full Text Available FRET-based sensors for cyclic Adenosine Mono Phosphate (cAMP have revolutionized the way in which this important intracellular messenger is studied. The currently prevailing sensors consist of the cAMP-binding protein Epac1, sandwiched between suitable donor- and acceptor fluorescent proteins (FPs. Through a conformational change in Epac1, alterations in cellular cAMP levels lead to a change in FRET that is most commonly detected by either Fluorescence Lifetime Imaging (FLIM or by Sensitized Emission (SE, e.g., by simple ratio-imaging. We recently reported a range of different Epac-based cAMP sensors with high dynamic range and signal-to-noise ratio. We showed that constructs with cyan FP as donor are optimal for readout by SE, whereas other constructs with green FP donors appeared much more suited for FLIM detection. In this study, we present a new cAMP sensor, termed (TEpac(VV, which employs mTurquoise as donor. Spectrally very similar to CFP, mTurquoise has about doubled quantum efficiency and unlike CFP, its fluorescence decay is strictly single-exponential. We show that (TEpac(VV appears optimal for detection both by FLIM and SE, that it has outstanding FRET span and signal-to-noise ratio, and improved photostability. Hence, (TEpac(VV should become the cAMP sensor of choice for new experiments, both for FLIM and ratiometric detection.

  11. 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

    2017-05-26

    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

  12. 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.

  13. Enhanced live cell imaging via photonic crystal enhanced fluorescence microscopy.

    Science.gov (United States)

    Chen, Weili; Long, Kenneth D; Yu, Hojeong; Tan, Yafang; Choi, Ji Sun; Harley, Brendan A; Cunningham, Brian T

    2014-11-21

    We demonstrate photonic crystal enhanced fluorescence (PCEF) microscopy as a surface-specific fluorescence imaging technique to study the adhesion of live cells by visualizing variations in cell-substrate gap distance. This approach utilizes a photonic crystal surface incorporated into a standard microscope slide as the substrate for cell adhesion, and a microscope integrated with a custom illumination source as the detection instrument. When illuminated with a monochromatic light source, angle-specific optical resonances supported by the photonic crystal enable efficient excitation of surface-confined and amplified electromagnetic fields when excited at an on-resonance condition, while no field enhancement occurs when the same photonic crystal is illuminated in an off-resonance state. By mapping the fluorescence enhancement factor for fluorophore-tagged cellular components between on- and off-resonance states and comparing the results to numerical calculations, the vertical distance of labelled cellular components from the photonic crystal substrate can be estimated, providing critical and quantitative information regarding the spatial distribution of the specific components of cells attaching to a surface. As an initial demonstration of the concept, 3T3 fibroblast cells were grown on fibronectin-coated photonic crystals with fluorophore-labelled plasma membrane or nucleus. We demonstrate that PCEF microscopy is capable of providing information about the spatial distribution of cell-surface interactions at the single-cell level that is not available from other existing forms of microscopy, and that the approach is amenable to large fields of view, without the need for coupling prisms, coupling fluids, or special microscope objectives.

  14. Multiphoton fluorescence and second harmonic generation microscopy for imaging keratoconus

    Science.gov (United States)

    Sun, Yen; Lo, Wen; Lin, Sung-Jan; Lin, Wei-Chou; Jee, Shiou-Hwa; Tan, Hsin-Yuan; Dong, Chen-Yuan

    2006-02-01

    The purpose of this study is to assess the possible application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for imaging the structural features of keratoconus cornea and to evaluate its potential as being a clinical in vivo monitoring technique. Using the near-infrared excitation source from a titanium-sapphire laser pumped by a diode-pumped, solid state (DPSS) laser system, we can induce and simultaneously acquire multiphoton autofluorescence and SHG signals from the cornea specimens with keratoconus. A home-modified commercial microscope system with specified optical components is used for optimal signal detection. Keratoconus cornea button from patient with typical clinical presentation of keratoconus was obtained at the time of penetrating keratoplasty. The specimen was also sent for the histological examination as comparison. In all samples of keratoconus, destruction of lamellar structure with altered collagen fiber orientation was observed within whole layer of the diseased stromal area. In addition, the orientation of the altered collagen fibers within the cone area shows a trend directing toward the apex of the cone, which might implicate the biomechanical response of the keratoconus stroma to the intraocular pressure. Moreover, increased autofluorescent cells were also found in the cone area, with increased density as one approaches the apical area. In conclusion, multiphoton autofluorescence and SHG microscopy non-invasively demonstrated the morphological features of keratoconus cornea, especially the structural alternations of the stromal lamellae. We believe that in the future the multiphoton microscopy can be applied in vivo as an effective, non-invasive diagnostic and monitoring technique for keratoconus.

  15. Char porosity characterisation by scanning electron microscopy and image analysis

    Energy Technology Data Exchange (ETDEWEB)

    Soerensen, H.S.; Rosenberg, P.; Petersen, H.I.; Soerensen, L.H. [Danfoss A/S, Nordborg (Denmark)

    2000-09-01

    No significant change in either the morphotype composition or the macroporosity (pores {gt}5 {mu}m) in the 0-30 wt.% char burnout interval were revealed by reflected light microscopy or image analysis. Two high temperature char series from a Tertiary South American coal (C1) and a Permian Gondwana coal (C2) were therefore examined by scanning electron microscopy to provide information on the combustion process up to {approximately} 60 wt% char burnout. This study documents a significant mesopore ({approximately} 0.1-5 {mu}m) development on the fused chars in the burnout interval studied. A method to quantify the size and amount of the mesopores is described and both the parameters increased with increasing char burnout. Above a char burnout of {approximately} 30 wt% an increase in macroporosity was detected and ascribed to coalescence of mesopores to form large pores. Although the measurement of mesoporosity is restricted to fused chars, i.e. pores in fragments and the char morphotypes inertoid, fusinoid and solid could not be measured, the consideration of mesoporosity seems to be fundamental in understanding, evaluating and modelling combustion processes in the char burnout interval studied. 7 refs., 9 figs., 4 tabs.

  16. Imaging Photon Lattice States by Scanning Defect Microscopy

    Science.gov (United States)

    Underwood, D. L.; Shanks, W. E.; Li, Andy C. Y.; Ateshian, Lamia; Koch, Jens; Houck, A. A.

    2016-04-01

    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.

  17. 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.

  18. 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...

  19. 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 BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

  20. All-optically integrated multimodality imaging system: combined photoacoustic microscopy, optical coherence tomography, and fluorescence imaging

    Science.gov (United States)

    Chen, Zhongjiang; Yang, Sihua; Xing, Da

    2016-10-01

    We have developed a multimodality imaging system by optically integrating all-optical photoacoustic microscopy (AOPAM), optical coherence tomography (OCT) and fluorescence microscopy (FLM) to provide complementary information including optical absorption, optical back-scattering and fluorescence contrast of biological tissue. By sharing the same low-coherence Michelson interferometer, AOPAM and OCT could be organically optically combined to obtain the absorption and scattering information of the biological tissues. Also, owing to using the same laser source and objective lens, intrinsically registered photoacoustic and fluorescence signals are obtained to present the radiative and nonradiative transition process of absorption. Simultaneously photoacoustic angiography, tissue structure and fluorescence molecular in vivo images of mouse ear were acquired to demonstrate the capabilities of the optically integrated trimodality imaging system, which can present more information to study tumor angiogenesis, vasculature, anatomical structure and microenvironments in vivo.

  1. Comparison of mouse mammary gland imaging techniques and applications: Reflectance confocal microscopy, GFP Imaging, and ultrasound

    Directory of Open Access Journals (Sweden)

    Cotarla Ion

    2008-01-01

    Full Text Available Abstract Background Genetically engineered mouse models of mammary gland cancer enable the in vivo study of molecular mechanisms and signaling during development and cancer pathophysiology. However, traditional whole mount and histological imaging modalities are only applicable to non-viable tissue. Methods We evaluated three techniques that can be quickly applied to living tissue for imaging normal and cancerous mammary gland: reflectance confocal microscopy, green fluorescent protein imaging, and ultrasound imaging. Results In the current study, reflectance confocal imaging offered the highest resolution and was used to optically section mammary ductal structures in the whole mammary gland. Glands remained viable in mammary gland whole organ culture when 1% acetic acid was used as a contrast agent. Our application of using green fluorescent protein expressing transgenic mice in our study allowed for whole mammary gland ductal structures imaging and enabled straightforward serial imaging of mammary gland ducts in whole organ culture to visualize the growth and differentiation process. Ultrasound imaging showed the lowest resolution. However, ultrasound was able to detect mammary preneoplastic lesions 0.2 mm in size and was used to follow cancer growth with serial imaging in living mice. Conclusion In conclusion, each technique enabled serial imaging of living mammary tissue and visualization of growth and development, quickly and with minimal tissue preparation. The use of the higher resolution reflectance confocal and green fluorescent protein imaging techniques and lower resolution ultrasound were complementary.

  2. 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.

  3. 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.

  4. Hybrid wide-field and scanning microscopy for high-speed 3D imaging.

    Science.gov (United States)

    Duan, Yubo; Chen, Nanguang

    2015-11-15

    Wide-field optical microscopy is efficient and robust in biological imaging, but it lacks depth sectioning. In contrast, scanning microscopic techniques, such as confocal microscopy and multiphoton microscopy, have been successfully used for three-dimensional (3D) imaging with optical sectioning capability. However, these microscopic techniques are not very suitable for dynamic real-time imaging because they usually take a long time for temporal and spatial scanning. Here, a hybrid imaging technique combining wide-field microscopy and scanning microscopy is proposed to accelerate the image acquisition process while maintaining the 3D optical sectioning capability. The performance was demonstrated by proof-of-concept imaging experiments with fluorescent beads and zebrafish liver.

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

    OpenAIRE

    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 multiphoton 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 progr...

  6. A wide-field TCSPC FLIM system based on an MCP PMT with a delay-line anode

    OpenAIRE

    Becker, Wolfgang, 1962-; Hirvonen, Liisa; Milnes, James; Conneely, Thomas; Jagutzki, Ottmar; Netz, Holger; Smietana, Stefan; Suhling, Klaus

    2016-01-01

    We report on the implementation of a wide-field time-correlated single photon counting (TCSPC) method for fluorescence lifetime imaging (FLIM). It is based on a 40 mm diameter crossed delay line anode detector, where the readout is performed by three standard TCSPC boards. Excitation is performed by a picosecond diode laser with 50 MHz repetition rate. The photon arrival timing is obtained directly from the microchannel plates, with an instrumental response of ∼190 to 230 ps full width at hal...

  7. Nanoscale fluorescence lifetime imaging with a single diamond NV center

    CERN Document Server

    Beams, Ryan; Johnson, Timothy W; Oh, Sang-Hyun; Novotny, Lukas; Vamivakas, Nick

    2013-01-01

    Solid-state quantum emitters, such as artificially engineered quantum dots or naturally occurring defects in solids, are being investigated for applications ranging from quantum information science and optoelectronics to biomedical imaging. Recently, these same systems have also been studied from the perspective of nanoscale metrology. In this letter we study the near-field optical properties of a diamond nanocrystal hosting a single nitrogen vacancy center. We find that the nitrogen vacancy center is a sensitive probe of the surrounding electromagnetic mode structure. We exploit this sensitivity to demonstrate nanoscale fluorescence lifetime imaging microscopy (FLIM) with a single nitrogen vacancy center by imaging the local density of states of an optical antenna.

  8. Efficient parallel Levenberg-Marquardt model fitting towards real-time automated parametric imaging microscopy.

    Science.gov (United States)

    Zhu, Xiang; Zhang, Dianwen

    2013-01-01

    We present a fast, accurate and robust parallel Levenberg-Marquardt minimization optimizer, GPU-LMFit, which is implemented on graphics processing unit for high performance scalable parallel model fitting processing. GPU-LMFit can provide a dramatic speed-up in massive model fitting analyses to enable real-time automated pixel-wise parametric imaging microscopy. We demonstrate the performance of GPU-LMFit for the applications in superresolution localization microscopy and fluorescence lifetime imaging microscopy.

  9. Immobilization Techniques of Bacteria for Live Super-resolution Imaging Using Structured Illumination Microscopy.

    Science.gov (United States)

    Bottomley, Amy L; Turnbull, Lynne; Whitchurch, Cynthia B; Harry, Elizabeth J

    2017-01-01

    Advancements in optical microscopy technology have allowed huge progression in the ability to understand protein structure and dynamics in live bacterial cells using fluorescence microscopy. Paramount to high-quality microscopy is good sample preparation to avoid bacterial cell movement that can result in motion blur during image acquisition. Here, we describe two techniques of sample preparation that reduce unwanted cell movement and are suitable for application to a number of bacterial species and imaging methods.

  10. Segmentation of scanning electron microscopy images from natural rubber samples with gold nanoparticles using starlet wavelets

    OpenAIRE

    de Siqueira, Alexandre Fioravante; Cabrera, Flavio Camargo [UNESP; Pagamisse, Aylton; Job,Aldo Eloizo

    2016-01-01

    Electronic microscopy has been used for morphology evaluation of different materials structures. However, microscopy results may be affected by several factors. Image processing methods can be used to correct and improve the quality of these results. In this article, we propose an algorithm based on starlets to perform the segmentation of scanning electron microscopy images. An application is presented in order to locate gold nanoparticles in natural rubber membranes. In this application, our...

  11. A fast image registration approach of neural activities in light-sheet fluorescence microscopy images

    Science.gov (United States)

    Meng, Hui; Hui, Hui; Hu, Chaoen; Yang, Xin; Tian, Jie

    2017-03-01

    The ability of fast and single-neuron resolution imaging of neural activities enables light-sheet fluorescence microscopy (LSFM) as a powerful imaging technique in functional neural connection applications. The state-of-art LSFM imaging system can record the neuronal activities of entire brain for small animal, such as zebrafish or C. elegans at single-neuron resolution. However, the stimulated and spontaneous movements in animal brain result in inconsistent neuron positions during recording process. It is time consuming to register the acquired large-scale images with conventional method. In this work, we address the problem of fast registration of neural positions in stacks of LSFM images. This is necessary to register brain structures and activities. To achieve fast registration of neural activities, we present a rigid registration architecture by implementation of Graphics Processing Unit (GPU). In this approach, the image stacks were preprocessed on GPU by mean stretching to reduce the computation effort. The present image was registered to the previous image stack that considered as reference. A fast Fourier transform (FFT) algorithm was used for calculating the shift of the image stack. The calculations for image registration were performed in different threads while the preparation functionality was refactored and called only once by the master thread. We implemented our registration algorithm on NVIDIA Quadro K4200 GPU under Compute Unified Device Architecture (CUDA) programming environment. The experimental results showed that the registration computation can speed-up to 550ms for a full high-resolution brain image. Our approach also has potential to be used for other dynamic image registrations in biomedical applications.

  12. An epifluorescence microscopy method for generalized polarization imaging

    DEFF Research Database (Denmark)

    Hansen, Jesper Søndergaard; Helix Nielsen, Claus

    2011-01-01

    Generalized polarization (GP) microscopy represents an excellent tool to study lipid–lipid and lipid–protein interactions in situ and in vitro. Here, we present an efficient and cost effective method to perform GP microscopy using a standard light-emitting diode (LED) epifluorescence microscope...

  13. Imaging photonic crystals using Fourier plane imaging and Fourier ptychographic microscopy techniques implemented with a computer controlled hemispherical digital condenser

    Science.gov (United States)

    Sen, Sanchari; Desai, Darshan B.; Alsubaie, Meznh H.; Zhelyeznyakov, Maksym V.; Molina, L.; Sarraf, Hamed Sari; Bernussi, Ayrton A.; Peralta, Luis Grave de

    2017-01-01

    Fourier plane imaging (FPIM) and Fourier ptychographic (FPM) microscopy techniques were used to image photonic crystals. A computer-controlled hemispherical digital condenser provided required sample illumination with variable inclination. Notable improvement in image resolution was obtained with both methods. However, it was determined that the FPM technique cannot surpass the Rayleigh resolution limit when imaging photonic crystals.

  14. 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.

  15. 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.

  16. Multiwavelength FLIM: new applications and algorithms

    Science.gov (United States)

    Rück, A.; Strat, D.; Dolp, F.; von Einem, B.; von Arnim, C. A. F.

    2011-03-01

    The combination of time-resolved and spectral resolved techniques as achieved by SLIM (spectrally resolved fluorescence lifetime imaging) improves the analysis of complex situations, when different fluorophores have to be distinguished. This could be the case when endogenous fluorophores of living cells and tissues are observed to identify the redox state and oxidative metabolic changes of the mitochondria. Other examples are FRET (resonant energy transfer) measurements, when different donor/acceptor pairs are observed simultaneously. SLIM is working in the time domain employing excitation with short light pulses and detection of the fluorescence intensity decay in many cases with time-correlated single photon counting (TCSPC). Spectral resolved detection is achieved by a polychromator in the detection path and a 16-channel multianode photomultiplier tube with the appropriate routing electronics. Within this paper special attention will be focused on FRET measurements with respect to protein interactions in Alzheimers disease. Using global analysis as the phasor plot approach or integration of the kinetic equations taking into account the multidimensional datasets in every spectral channel we could demonstrate considerable improvement of our calculations.

  17. Integral imaging microscopy with enhanced depth-of-field using a spatial multiplexing.

    Science.gov (United States)

    Kwon, Ki-Chul; Erdenebat, Munkh-Uchral; Alam, Md Ashraful; Lim, Young-Tae; Kim, Kwang Gi; Kim, Nam

    2016-02-08

    A depth-of-field enhancement method for integral imaging microscopy system using a spatial multiplexing structure consisting of a beamsplitter with dual video channels and micro lens arrays is proposed. A computational integral imaging reconstruction algorithm generates two sets of depth-sliced images for the acquired depth information of the captured elemental image arrays and the well-focused depth-slices of both image sets are combined where each is focused on a different depth plane of the specimen. A prototype is implemented, and the experimental results demonstrate that the depth-of-field of the reconstructed images in the proposed integral imaging microscopy is significantly increased compared with conventional integral imaging microscopy systems.

  18. 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.

  19. Spectrally resolved fluorescence lifetime imaging of Nile red for measurements of intracellular polarity

    Science.gov (United States)

    Levitt, James A.; Chung, Pei-Hua; Suhling, Klaus

    2015-09-01

    Spectrally resolved confocal microscopy and fluorescence lifetime imaging have been used to measure the polarity of lipid-rich regions in living HeLa cells stained with Nile red. The emission peak from the solvatochromic dye in lipid droplets is at a shorter wavelength than other, more polar, stained internal membranes, and this is indicative of a low polarity environment. We estimate that the dielectric constant, ɛ, is around 5 in lipid droplets and 25<ɛ<40 in other lipid-rich regions. Our spectrally resolved fluorescence lifetime imaging microscopy (FLIM) data show that intracellular Nile red exhibits complex, multiexponential fluorescence decays due to emission from a short lifetime locally excited state and a longer lifetime intramolecular charge transfer state. We measure an increase in the average fluorescence lifetime of the dye with increasing emission wavelength, as shown using phasor plots of the FLIM data. We also show using these phasor plots that the shortest lifetime decay components arise from lipid droplets. Thus, fluorescence lifetime is a viable contrast parameter for distinguishing lipid droplets from other stained lipid-rich regions. Finally, we discuss the FLIM of Nile red as a method for simultaneously mapping both polarity and relative viscosity based on fluorescence lifetime measurements.

  20. Hardware-friendly bi-exponential fluorescence lifetime imaging algorithms and phasor approaches

    Science.gov (United States)

    Li, David; Chen, Yu

    2015-07-01

    A newly developed hardware-friendly non-iterative fluorescence lifetime imaging (FLIM) analysis method was verified in an FPGA chip. Its performances were also demonstrated on two-photon FLIM images of gold nanorods (GNRs)-Cy5 labelled Hela cells. The results obtained by the proposed method can be presented in a polor plot to be compared to the widely used phasor (Phasor) approach. Combining our method with Phasor will be very useful in FLIM analysis.

  1. 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.

  2. Super-resolution deep imaging with hollow Bessel beam STED microscopy

    CERN Document Server

    Yu, Wentao; Dong, Dashan; Yang, Xusan; Xiao, Yunfeng; Gong, Qihuang; Xi, Peng; Shi, Kebin

    2015-01-01

    Stimulated emission depletion (STED) microscopy has become a powerful imaging and localized excitation method beating the diffraction barrier for improved lateral spatial resolution in cellular imaging, lithography, etc. Due to specimen-induced aberrations and scattering distortion, it has been a great challenge for STED to maintain consistent lateral resolution deeply inside the specimens. Here we report on a deep imaging STED microscopy by using Gaussian beam for excitation and hollow Bessel beam for depletion (GB-STED). The proposed scheme shows the improved imaging depth up to ~155{\\mu}m in solid agarose sample, ~115{\\mu}m in PDMS and ~100{\\mu}m in phantom of gray matter in brain tissue with consistent super resolution, while the standard STED microscopy shown a significantly reduced lateral resolution at the same imaging depth. The results indicate the excellent imaging penetration capability of GB-STED, making it a promising tool for deep 3D imaging optical nanoscopy and laser fabrication.

  3. 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.

  4. Automatic detection of cell divisions (mitosis) in live-imaging microscopy images using Convolutional Neural Networks.

    Science.gov (United States)

    Shkolyar, Anat; Gefen, Amit; Benayahu, Dafna; Greenspan, Hayit

    2015-08-01

    We propose a semi-automated pipeline for the detection of possible cell divisions in live-imaging microscopy and the classification of these mitosis candidates using a Convolutional Neural Network (CNN). We use time-lapse images of NIH3T3 scratch assay cultures, extract patches around bright candidate regions that then undergo segmentation and binarization, followed by a classification of the binary patches into either containing or not containing cell division. The classification is performed by training a Convolutional Neural Network on a specially constructed database. We show strong results of AUC = 0.91 and F-score = 0.89, competitive with state-of-the-art methods in this field.

  5. Nanoscale Imaging Technology for THz Frequency Transmission Microscopy

    Science.gov (United States)

    2014-12-16

    Microscopy, Analytical Chemistry , (06 2013): 0. doi: 10.1021/ac4010088 Yung Yu Wang, Peter J. Burke. Polyelectrolyte multilayer electrostatic gating of...C. G. Densmore, S. K. Doorn, P. J. Burke. Effect of source, surfactant , and deposition process on electronic properties of nanotube arrays, (09

  6. Laser differential fitting confocal microscopy with high imaging efficiency.

    Science.gov (United States)

    Sheng, Zhong; Wang, Yun; Zhao, Weiqian; Qiu, Lirong; Sun, Yingbin

    2016-09-01

    Based on the optical arrangement of a bipolar differential confocal microscopy (BDCM), laser differential fitting confocal microscopy (DFCM) is proposed in this paper using the feature of BDCM that a zero-crossing point (ZCP) of the axial response curve precisely corresponds to the focus of the system objective. A linear segment of the DFCM axial response around the ZCP is used to fit a straight line. Focus can be determined by solving the equations of the fitting lines, and then, the sample surface could be measured and reconstructed with a high resolution. Compared with the curve-fitting peak detection, which is an algorithm for focus detection widely used in conventional confocal microscopy, the line-fitting zero solution method used in DFCM has several advantages, such as high precision and sensitivity. Most importantly, precise focus detection can be realized using less data, i.e., DFCM has a high measurement efficiency. Furthermore, DFCM can effectively eliminate common-mode noise in a confocal microscopy system and has good noise suppression and disturbance resistance capability.

  7. Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging.

    NARCIS (Netherlands)

    Andresen, V.; Alexander, S.; Heupel, W.M.; Hirschberg, M.; Hoffman, R.M.; Friedl, P.H.A.

    2009-01-01

    Multiphoton microscopy (MPM) is the method of choice for investigating cells and cellular functions in deep tissue sections and organs. Here we present the setup and applications of infrared-(IR-)MPM using excitation wavelengths above 1080 nm. IR-MPM enables the use of red fluorophores and

  8. Modeling of Image Formation in Cryo-Electron Microscopy

    NARCIS (Netherlands)

    Vulovic, M.

    2013-01-01

    Knowledge of the structure of biological specimens is crucial for understanding life. Cryo-electron microscopy (cryo-EM) permits structural studies of biological specimen at their near-native state. The research performed in this thesis represents one of two subprojects of the FOM industrial partner

  9. Nonlinear Image Restoration in Confocal Microscopy : Stability under Noise

    NARCIS (Netherlands)

    Roerdink, J.B.T.M.

    1995-01-01

    In this paper we study the noise stability of iterative algorithms developed for attenuation correction in Fluorescence Confocal Microscopy using FT methods. In each iteration the convolution of the previous estimate is computed. It turns out that the estimators are robust to noise perturbation.

  10. Nonlinear Image Restoration in Confocal Microscopy : Stability under Noise

    NARCIS (Netherlands)

    Roerdink, J.B.T.M.

    1995-01-01

    In this paper we study the noise stability of iterative algorithms developed for attenuation correction in Fluorescence Confocal Microscopy using FT methods. In each iteration the convolution of the previous estimate is computed. It turns out that the estimators are robust to noise perturbation.

  11. SIMS ion microscopy as a novel, practical tool for subcellular chemical imaging in cancer research

    Energy Technology Data Exchange (ETDEWEB)

    Chandra, S

    2003-01-15

    The development of cryogenic sample preparations, subcellular image quantification schemes, and correlative confocal laser scanning microscopy and ion microscopy have made dynamic SIMS a versatile tool in biology and medicine. For example, ion microscopy can provide much needed, novel information on calcium influx and intracellular calcium stores at organelle resolution in normal and transformed cells in order to better understand the altered calcium signaling in malignant cells. 3-D SIMS imaging of cells revealed dynamic gradients of calcium in cells undergoing mitosis and cytokinesis. Studies of subcellular localization of anticancer drugs is another area of research where ion microscopy can provide novel observations in many types of cancers. Ion microscopy is already an essential tool in boron neutron capture therapy (BNCT) of brain cancer as it can be used to quantitatively image the subcellular location of boron in cells and tissues. This information is critically needed for testing the efficacy of boronated agents and for calculations of radiation dosimetry.

  12. Objective, comparative assessment of the penetration depth of temporal-focusing microscopy for imaging various organs

    Science.gov (United States)

    Rowlands, Christopher J.; Bruns, Oliver T.; Bawendi, Moungi G.; So, Peter T. C.

    2015-06-01

    Temporal focusing is a technique for performing axially resolved widefield multiphoton microscopy with a large field of view. Despite significant advantages over conventional point-scanning multiphoton microscopy in terms of imaging speed, the need to collect the whole image simultaneously means that it is expected to achieve a lower penetration depth in common biological samples compared to point-scanning. We assess the penetration depth using a rigorous objective criterion based on the modulation transfer function, comparing it to point-scanning multiphoton microscopy. Measurements are performed in a variety of mouse organs in order to provide practical guidance as to the achievable penetration depth for both imaging techniques. It is found that two-photon scanning microscopy has approximately twice the penetration depth of temporal-focusing microscopy, and that penetration depth is organ-specific; the heart has the lowest penetration depth, followed by the liver, lungs, and kidneys, then the spleen, and finally white adipose tissue.

  13. 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.

  14. 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 track

  15. 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…

  16. Spatial Modulation Microscopy for Real-Time Imaging of Plasmonic Nanoparticles and Cells

    CERN Document Server

    Fairbairn, N; Carter, R; Fernandes, R; Kanaras, A G; Elliott, T J; Somekh, M G; Pitter, M C; Muskens, O L

    2012-01-01

    Spatial modulation microscopy is a technique originally developed for quantitative spectroscopy of individual nano-objects. Here, a parallel implementation of the spatial modulation microscopy technique is demonstrated based on a line detector capable of demodulation at kHz frequencies. The capabilities of the imaging system are shown using an array of plasmonic nanoantennas and dendritic cells incubated with gold nanoparticles.

  17. Deep Imaging in Scattering Media with Single Photon Selective Plane Illumination Microscopy (SPIM)

    CERN Document Server

    Pediredla, Adithya Kumar; Avants, Ben; Ye, Fan; Nagayama, Shin; Chen, Ziying; Kemere, Caleb; Robinson, Jacob; Veeraraghavan, Ashok

    2016-01-01

    In most biological tissues, light scattering due to small differences in refractive index limits the depth of optical imaging systems. Two-photon microscopy (2PM), which significantly reduces the scattering of the excitation light, has emerged as the most common method to image deep within scattering biological tissue. This technique, however, requires high-power pulsed lasers that are both expensive and difficult to integrate into compact portable systems. In this paper, using a combination of theoretical and experimental techniques, we show that Selective Plane Illumination Microscopy (SPIM) can image nearly as deep as 2PM without the need for a high-powered pulsed laser. Compared to other single photon imaging techniques like epifluorescence and confocal microscopy, SPIM can image more than twice as deep in scattering media (approximately 10 times the mean scattering length). These results suggest that SPIM has the potential to provide deep imaging in scattering media in situations where 2PM systems would ...

  18. 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.

  19. Label-free biomedical imaging of lipids by stimulated Raman scattering microscopy.

    Science.gov (United States)

    Ramachandran, Prasanna V; Mutlu, Ayse Sena; Wang, Meng C

    2015-01-05

    Advances in modern optical microscopy have provided unparalleled tools to study intracellular structure and function, yet visualizing lipid molecules within a cell remains challenging. Stimulated Raman Scattering (SRS) microscopy is a recently developed imaging modality that addresses this challenge. By selectively imaging the vibration of chemical moieties enriched in lipids, this technique allows for rapid imaging of lipid molecules in vivo without the need for perturbative extrinsic labels. SRS microscopy has been effectively employed in the study of fat metabolism, helping uncover novel regulators of lipid storage. This unit provides a brief introduction to the principle of SRS microscopy, and describes methods for its use in imaging lipids in cells, tissues, and whole organisms.

  20. Voltammetric scanning electrochemical cell microscopy: dynamic imaging of hydrazine electro-oxidation on platinum electrodes

    NARCIS (Netherlands)

    Chen, C.-H.; Jacobse, L.; McKelvey, K.; Lai, S.C.S.; Koper, M.T.M.; Unwin, P.R.

    2015-01-01

    Voltammetric scanning electrochemical cell microscopy (SECCM) incorporates cyclic voltammetry measurements in the SECCM imaging protocol, by recording electrochemical currents in a wide potential window at each pixel in a map. This provides much more information compared to traditional fixed potenti

  1. 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.

  2. Nanoscale fluorescence lifetime imaging of an optical antenna with a single diamond NV center.

    Science.gov (United States)

    Beams, Ryan; Smith, Dallas; Johnson, Timothy W; Oh, Sang-Hyun; Novotny, Lukas; Vamivakas, A Nick

    2013-08-14

    Solid-state quantum emitters, such as artificially engineered quantum dots or naturally occurring defects in solids, are being investigated for applications ranging from quantum information science and optoelectronics to biomedical imaging. Recently, these same systems have also been studied from the perspective of nanoscale metrology. In this letter, we study the near-field optical properties of a diamond nanocrystal hosting a single nitrogen vacancy center. We find that the nitrogen vacancy center is a sensitive probe of the surrounding electromagnetic mode structure. We exploit this sensitivity to demonstrate nanoscale fluorescence lifetime imaging microscopy (FLIM) with a single nitrogen vacancy center by imaging the local density of states of an optical antenna.

  3. 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.

  4. Comparison of two detection algorithms for spot tracking in fluorescence microscopy images

    CSIR Research Space (South Africa)

    Mabaso, M

    2014-11-01

    Full Text Available for spot tracking in fluorescence microscopy images Matsilele Mabaso∗, Daniel Withey‡, Bhekisipho Twala† ∗ ‡MDS(MIAS) Council for Scientific and Industrial Research Pretoria, South Africa, Email: ∗MMabaso@csir.co.za †Department of Electrical Engineering.... The quantitative comparative results demonstrated the importance of spot detection in tracking contexts. I. INTRODUCTION In recent years, the field of fluorescence microscopy has been improved and automated, and a large volume of image data are being generated...

  5. 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.

  6. 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.

  7. Serial block face scanning electron microscopy--the future of cell ultrastructure imaging.

    Science.gov (United States)

    Hughes, Louise; Hawes, Chris; Monteith, Sandy; Vaughan, Sue

    2014-03-01

    One of the major drawbacks in transmission electron microscopy has been the production of three-dimensional views of cells and tissues. Currently, there is no one suitable 3D microscopy technique that answers all questions and serial block face scanning electron microscopy (SEM) fills the gap between 3D imaging using high-end fluorescence microscopy and the high resolution offered by electron tomography. In this review, we discuss the potential of the serial block face SEM technique for studying the three-dimensional organisation of animal, plant and microbial cells.

  8. Multicolor 3D super-resolution imaging by quantum dot stochastic optical reconstruction microscopy.

    Science.gov (United States)

    Xu, Jianquan; Tehrani, Kayvan F; Kner, Peter

    2015-03-24

    We demonstrate multicolor three-dimensional super-resolution imaging with quantum dots (QSTORM). By combining quantum dot asynchronous spectral blueing with stochastic optical reconstruction microscopy and adaptive optics, we achieve three-dimensional imaging with 24 nm lateral and 37 nm axial resolution. By pairing two short-pass filters with two appropriate quantum dots, we are able to image single blueing quantum dots on two channels simultaneously, enabling multicolor imaging with high photon counts.

  9. 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

    provides researchers in compressed sensing with a selection of algorithms for reconstructing undersampled general images, and offers a consistent and rigorous way to efficiently evaluate the researchers own developed reconstruction algorithms in terms of phase transitions. The package also serves......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...

  10. Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM

    Science.gov (United States)

    Demeautis, Claire; Sipieter, François; Roul, Julien; Chapuis, Catherine; Padilla-Parra, Sergi; Riquet, Franck B.; Tramier, Marc

    2017-01-01

    Monitoring of different signalling enzymes in a single assay using multiplex biosensing provides a multidimensional workspace to elucidate biological processes, signalling pathway crosstalk, and determine precise sequence of events at the single living cell level. In this study, we interrogate the complexity in cAMP/PKA-MAPK/ERK1&2 crosstalk by using multi-parameter biosensing experiments to correlate biochemical activities simultaneously in time and space. Using a single excitation wavelength dual colour FLIM method we are able to detect fluorescence lifetime images of two donors to simultaneously measure PKA and ERK1&2 kinase activities in the same cellular localization by using FRET biosensors. To this end, we excite two FRET donors mTFP1 and LSSmOrange with a 440 nm wavelength and we alleviate spectral bleed-through associated limitations with the very dim-fluorescent acceptor ShadowG for mTFP1 and the red-shifted mKate2 for LSSmOrange. The simultaneous recording of PKA and ERK1&2 kinase activities reveals concomitant EGF-mediated activations of both kinases in HeLa cells. Under these conditions the subsequent Forskolin-induced cAMP release reverses the transient increase of EGF-mediated ERK1&2 kinase activity while reinforcing PKA activation. Here we propose a validated methodology for multiparametric kinase biosensing in living cells using FRET-FLIM. PMID:28106114

  11. 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.

  12. Imaging zebrafish embryos by two-photon excitation time-lapse microscopy.

    Science.gov (United States)

    Carvalho, Lara; Heisenberg, Carl-Philipp

    2009-01-01

    The zebrafish is a favorite model organism to study tissue morphogenesis during development at a subcellular level. This largely results from the fact that zebrafish embryos are transparent and thus accessible to various imaging techniques, such as confocal and two-photon excitation (2PE) microscopy. In particular, 2PE microscopy has been shown to be useful for imaging deep cell layers within the embryo and following tissue morphogenesis over long periods. This chapter describes how to use 2PE microscopy to study morphogenetic movements during early zebrafish embryonic development, providing a general blueprint for its use in zebrafish.

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

    Science.gov (United States)

    Ellingsen, Pa˚L. 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.

  14. Embedding complementary imaging data in laser scanning microscopy micrographs by reversible watermarking.

    Science.gov (United States)

    Dragoi, Ioan-Catalin; Stanciu, Stefan G; Hristu, Radu; Coanda, Henri-George; Tranca, Denis E; Popescu, Marius; Coltuc, Dinu

    2016-04-01

    Complementary laser scanning microscopy micrographs are considered as pairs consisting in a master image (MI) and a slave image (SI), the latter with potential for facilitating the interpretation of the MI. We propose a strategy based on reversible watermarking for embedding a lossy compressed version of the SI into the MI. The use of reversible watermarking ensures the exact recovery of the host image. By storing and/or transmitting the watermarked MI in a single file, the information contained in both images that constitute the pair is made available to a potential end-user, which simplifies data association and transfer. Examples are presented using support images collected by two complementary techniques, confocal scanning laser microscopy and transmission laser scanning microscopy, on Hematoxylin and Eosin stained tissue fragments. A strategy for minimizing the watermarking distortions of the MI, while preserving the content of the SI, is discussed in detail.

  15. Imaging theory and resolution improvement of two-photon confocal microscopy

    Institute of Scientific and Technical Information of China (English)

    唐志列; 杨初平; 裴红津; 梁瑞生; 刘颂豪

    2002-01-01

    The nonlinear effect of two-photon excitation on the imaging property of two-photonconfocal microscopy has been analyzed by the two-photon fluorescence intensity transfer functionderived in this paper. The two-photon fluorescence intensity transfer function in a confocal micros-copy is given. Furthermore the three-dimensional point spread function (3D-PSF) and thethree-dimensional optical transfer function (3D-OTF) of two-photon confocal microscopy are de-rived based on the nonlinear effect of two-photon excitation. The imaging property of two-photonconfocal microscopy is discussed in detail based on 3D-OTF. Finally the spatial resolution limit oftwo-photon confocal microscopy is discussed according to the uncertainty principle.

  16. 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.

  17. 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.

  18. Wide-field optical sectioning for live-tissue imaging by plane-projection multiphoton microscopy

    Science.gov (United States)

    Yu, Jiun-Yann; Kuo, Chun-Hung; Holland, Daniel B.; Chen, Yenyu; Ouyang, Mingxing; Blake, Geoffrey A.; Zadoyan, Ruben; Guo, Chin-Lin

    2011-11-01

    Optical sectioning provides three-dimensional (3D) information in biological tissues. However, most imaging techniques implemented with optical sectioning are either slow or deleterious to live tissues. Here, we present a simple design for wide-field multiphoton microscopy, which provides optical sectioning at a reasonable frame rate and with a biocompatible laser dosage. The underlying mechanism of optical sectioning is diffuser-based temporal focusing. Axial resolution comparable to confocal microscopy is theoretically derived and experimentally demonstrated. To achieve a reasonable frame rate without increasing the laser power, a low-repetition-rate ultrafast laser amplifier was used in our setup. A frame rate comparable to that of epifluorescence microscopy was demonstrated in the 3D imaging of fluorescent protein expressed in live epithelial cell clusters. In this report, our design displays the potential to be widely used for video-rate live-tissue and embryo imaging with axial resolution comparable to laser scanning microscopy.

  19. High resolution imaging using scanning ion conductance microscopy with improved distance feedback control

    Institute of Scientific and Technical Information of China (English)

    Chao Li; Nicholas Johnson; Victor Ostanin; Andrew Shevchuk; Liming Ying; Yuri Korchev; David Klenerman

    2008-01-01

    Microscopy is an essential technique for observation on living cells. There is currently great interest in applying scanning probe microscopy to image-living biological cells in their natural environment at the nanometer scale. Scanning ion conductance microscopy is a new form of scanning probe microscopy, which enables non-contact high-resolution imaging of living biological cells. Based on a scanned nanopipette in physiological buffer, the distance feedback control uses the ion current to control the distance between the pipette tip and the sample surface. However, this feedback control has difficulties over slopes on convoluted cell surfaces, which limits its resolution. In this study, we present an improved form of feedback control that removes the contribution of up to the third-order slope from the ion current signal, hence providing a more accurate signal for controlling the distance. We show that this allows faster and lower noise topographic high-resolution imaging.

  20. 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.

  1. 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.

  2. Transillumination spatially modulated illumination microscopy for human chromosome imaging

    Science.gov (United States)

    Pitris, Costas; Heracleous, Peter; Patsalis, Philippos

    2005-03-01

    Human chromosome analysis is an essential task in cytogenetics, especially in prenatal screening, genetic syndrome diagnosis, cancer pathology research and mutagen dosimetry. Chromosomal analysis begins with the creation of a karyotype, which is a layout of chromosome images organized by decreasing size in pairs. Both manual and automatic classification of chromosomes are limited by the resolution of the microscope and imaging system used. One way to improve the results of classification and even detect subtleties now remaining undetected, is to enhance the resolution of the images. It is possible to achieve lateral resolution beyond the classical limit, by using spatially modulated illumination (SMI) in a wide-field, non-confocal microscope. In this case, the sample is illuminated with spatially modulated light, which makes normally inaccessible high-resolution information visible in the observed image by shifting higher frequencies within the OTF limits of the microscope. Although, SMI microscopes have been reported in the past, this manuscript reports the development of a transillumination microscope for opaque, non-fluorescent samples. The illumination path consisted of a light source illuminating a ruled grating which was subsequently imaged on the sample. The grating was mounted on a rotating and translating stage so that the magnification and rotation of the pattern could be adjusted. The imaging lens was a 1.25 NA oil immersion objective. Test samples showed resolution improvement, as judged from a comparison of the experimentally obtained FWHM. Further studies using smaller fringe distance or laser interference pattern illumination will be evaluated to further optimize the SMI results.

  3. Quantitative imaging of complex samples by spiral phase contrast microscopy.

    Science.gov (United States)

    Bernet, Stefan; Jesacher, Alexander; Fürhapter, Severin; Maurer, Christian; Ritsch-Marte, Monika

    2006-05-01

    Recently a spatial spiral phase filter in a Fourier plane of a microscopic imaging setup has been demonstrated to produce edge enhancement and relief-like shadow formation of amplitude and phase samples. Here we demonstrate that a sequence of at least 3 spatially filtered images, which are recorded with different rotational orientations of the spiral phase plate, can be used to obtain a quantitative reconstruction of both, amplitude and phase information of a complex microscopic sample, i.e. an object consisting of mixed absorptive and refractive components. The method is demonstrated using a calibrated phase sample, and an epithelial cheek cell.

  4. 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.

  5. 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...

  6. 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.

  7. Cell tracking with gadophrin-2: a bifunctional contrast agent for MR imaging, optical imaging, and fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Daldrup-Link, Heike E. [Department of Radiology, UCSF Medical Center, University of California in San Francisco, 513 Parnassus Ave, CA 94143, San Francisco (United States); Rudelius, Martina; Piontek, Guido; Schlegel, Juergen [Institute of Pathology, Technical University, Munich (Germany); Metz, Stephan; Settles, Marcus; Rummeny, Ernst J. [Department of Radiology, Technical University, Munich (Germany); Pichler, Bernd [Department of Biomedical Engineering, University of California Davis, Davis (United States); Heinzmann, Ulrich [National Research Center for Environment and Health, Technical University, Munich (Germany); Oostendorp, Robert A.J. [3. Clinic of Internal Medicine, Laboratory of Stem Cell Physiology, Technical University, Munich (Germany)

    2004-09-01

    The purpose of this study was to assess the feasibility of use of gadophrin-2 to trace intravenously injected human hematopoietic cells in athymic mice, employing magnetic resonance (MR) imaging, optical imaging (OI), and fluorescence microscopy. Mononuclear peripheral blood cells from GCSF-primed patients were labeled with gadophrin-2 (Schering AG, Berlin, Germany), a paramagnetic and fluorescent metalloporphyrin, using established transfection techniques with cationic liposomes. The labeled cells were evaluated in vitro with electron microscopy and inductively coupled plasma atomic emission spectrometry. Then, 1 x 10{sup 6}-3 x 10{sup 8} labeled cells were injected into 14 nude Balb/c mice and the in vivo cell distribution was evaluated with MR imaging and OI before and 4, 24, and 48 h after intravenous injection (p.i.). Five additional mice served as controls: three mice were untreated controls and two mice were investigated after injection of unlabeled cells. The contrast agent effect was determined quantitatively for MR imaging by calculating signal-to-noise-ratio (SNR) data. After completion of in vivo imaging studies, fluorescence microscopy of excised organs was performed. Intracellular cytoplasmatic uptake of gadophrin-2 was confirmed by electron microscopy. Spectrometry determined an uptake of 31.56 nmol Gd per 10{sup 6} cells. After intravenous injection, the distribution of gadophrin-2 labeled cells in nude mice could be visualized by MR, OI, and fluorescence microscopy. At 4 h p.i., the transplanted cells mainly distributed to lung, liver, and spleen, and 24 h p.i. they also distributed to the bone marrow. Fluorescence microscopy confirmed the distribution of gadophrin-2 labeled cells to these target organs. Gadophrin-2 is suited as a bifunctional contrast agent for MR imaging, OI, and fluorescence microscopy and may be used to combine the advantages of each individual imaging modality for in vivo tracking of intravenously injected hematopoietic cells

  8. 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

  9. 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.

  10. Intravital microscopy through an abdominal imaging window reveals a pre-micrometastasis stage during liver metastasis

    NARCIS (Netherlands)

    Ritsma, L.; Steller, E.J.; Beerling, E.; Loomans, C.J.; Zomer, A.; Gerlach, C.; Vrisekoop, N.; Seinstra, D.; van Gurp, L.; Schafer, R.; Raats, D.A.; de Graaff, A.; Schumacher, T.N.; de Koning, E.; Rinkes, I.H.; Kranenburg, O.; van Rheenen, J.

    2012-01-01

    Cell dynamics in subcutaneous and breast tumors can be studied through conventional imaging windows with intravital microscopy. By contrast, visualization of the formation of metastasis has been hampered by the lack of long-term imaging windows for metastasis-prone organs, such as the liver. We

  11. 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

  12. Submolecular Resolution Imaging of molecules by Atomic Force Microscopy:The influence of the Electrostatic Force

    NARCIS (Netherlands)

    van der Lit, J.; Cicco, F.; Hapala, P.; Jelinek, P.; Swart, Ingmar

    2016-01-01

    The forces governing the contrast in submolecular resolution imaging of molecules with atomic force microscopy (AFM) have recently become a topic of intense debate. Here, we show that the electrostatic force is essential to understand the contrast in atomically resolved AFM images of polar molecules

  13. Intensity Weighted Subtraction Microscopy Approach for Image Contrast and Resolution Enhancement

    Science.gov (United States)

    Korobchevskaya, Kseniya; Peres, Chiara; Li, Zhibin; Antipov, Alexei; Sheppard, Colin J. R.; Diaspro, Alberto; Bianchini, Paolo

    2016-05-01

    We propose and demonstrate a novel subtraction microscopy algorithm, exploiting fluorescence emission difference or switching laser mode and their derivatives for image enhancement. The key novelty of the proposed approach lies in the weighted subtraction coefficient, adjusted pixel-by-pixel with respect to the intensity distributions of initial images. This method produces significant resolution enhancement and minimizes image distortions. Our theoretical and experimental studies demonstrate that this approach can be applied to any optical microscopy techniques, including label free and non-linear methods, where common super-resolution techniques cannot be used.

  14. 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.

  15. Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Emilio J Gualda

    2014-08-01

    Full Text Available The development of three dimensional cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex three dimensional matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy is becoming an excellent tool for fast imaging of such three-dimensional biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment.

  16. Fluorescence lifetime imaging of membrane lipid order with a ratiometric fluorescent probe.

    Science.gov (United States)

    Kilin, Vasyl; Glushonkov, Oleksandr; Herdly, Lucas; Klymchenko, Andrey; Richert, Ludovic; Mely, Yves

    2015-05-19

    To monitor the lateral segregation of lipids into liquid-ordered (Lo) and -disordered (Ld) phases in lipid membranes, environment-sensitive dyes that partition in both phases but stain them differently have been developed. Of particular interest is the dual-color F2N12S probe, which can discriminate the two phases through the ratio of its two emission bands. These bands are associated with the normal (N(∗)) and tautomer (T(∗)) excited-state species that result from an excited-state intramolecular proton transfer. In this work, we investigated the potency of the time-resolved fluorescence parameters of F2N12S to discriminate lipid phases in model and cell membranes. Both the long and mean lifetime values of the T(∗) form of F2N12S were found to differ by twofold between Ld and Lo phases as a result of the restriction in the relative motions of the two aromatic moieties of F2N12S imposed by the highly packed Lo phase. This differed from the changes in the ratio of the two emission bands between the two phases, which mainly resulted from the decreased hydration of the N(∗) form in the Lo phase. Importantly, the strong difference in lifetimes between the two phases was preserved when cholesterol was added to the Ld phase. The two phases could be imaged with high contrast by fluorescence lifetime imaging microscopy (FLIM) on giant unilamellar vesicles. FLIM images of F2N12S-labeled live HeLa cells confirmed that the plasma membrane was mainly in the Lo-like phase. Furthermore, the two phases were found to be homogeneously distributed all over the plasma membrane, indicating that they are highly mixed at the spatiotemporal resolution of the FLIM setup. Finally, FLIM could also be used to sensitively monitor the change in lipid phase upon cholesterol depletion and apoptosis.

  17. 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.

  18. Imaging of magnetic and electric fields by electron microscopy.

    Science.gov (United States)

    Zweck, Josef

    2016-10-12

    Nanostructured materials become more and more a part of our daily life, partly as self-assembled particles or artificially patterned. These nanostructures often possess intrinsic magnetic and/or electric fields which determine (at least partially) their physical properties. Therefore it is important to be able to measure these fields reliably on a nanometre scale. A rather common instrument for the investigation of these fields is the transmission electron microscope as it offers high spatial resolution. The use of an electron microscope to image electric and magnetic fields on a micron down to sub-nanometre scale is treated in detail for transmission electron microscopes (TEM) and scanning transmission electron microscopes (STEM). The formation of contrast is described for the most common imaging modes, the specific advantages and disadvantages of each technique are discussed and examples are given. In addition, the experimental requirements for the use of the techniques described are listed and explained.

  19. 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.

  20. Imaging of magnetic and electric fields by electron microscopy

    Science.gov (United States)

    Zweck, Josef

    2016-10-01

    Nanostructured materials become more and more a part of our daily life, partly as self-assembled particles or artificially patterned. These nanostructures often possess intrinsic magnetic and/or electric fields which determine (at least partially) their physical properties. Therefore it is important to be able to measure these fields reliably on a nanometre scale. A rather common instrument for the investigation of these fields is the transmission electron microscope as it offers high spatial resolution. The use of an electron microscope to image electric and magnetic fields on a micron down to sub-nanometre scale is treated in detail for transmission electron microscopes (TEM) and scanning transmission electron microscopes (STEM). The formation of contrast is described for the most common imaging modes, the specific advantages and disadvantages of each technique are discussed and examples are given. In addition, the experimental requirements for the use of the techniques described are listed and explained.

  1. Tomographic imaging and scanning thermal microscopy: thermal impedance tomography

    OpenAIRE

    2002-01-01

    The application of tomographic imaging techniques developed for medical applications to the data provided by the scanning thermal microscope will give access to true three-dimensional information on the thermal properties of materials on a mm length scale. In principle, the technique involves calculating and inverting a sensitivity matrix for a uniform isotropic material, collecting ordered data at several modulation frequencies, and multiplying the inverse of the matrix with the data vector....

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

    DEFF Research Database (Denmark)

    Andersen, Jens Enevold Thaulov

    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......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...

  3. 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.

  4. 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.

  5. Live imaging of Tribolium castaneum embryonic development using light-sheet-based fluorescence microscopy.

    Science.gov (United States)

    Strobl, Frederic; Schmitz, Alexander; Stelzer, Ernst H K

    2015-10-01

    Tribolium castaneum has become an important insect model organism for evolutionary developmental biology, genetics and biotechnology. However, few protocols for live fluorescence imaging of Tribolium have been reported, and little image data is available. Here we provide a protocol for recording the development of Tribolium embryos with light-sheet-based fluorescence microscopy. The protocol can be completed in 4-7 d and provides procedural details for: embryo collection, microscope configuration, embryo preparation and mounting, noninvasive live imaging for up to 120 h along multiple directions, retrieval of the live embryo once imaging is completed, and image data processing, for which exemplary data is provided. Stringent quality control criteria for developmental biology studies are also discussed. Light-sheet-based fluorescence microscopy complements existing toolkits used to study Tribolium development, can be adapted to other insect species, and requires no advanced imaging or sample preparation skills.

  6. 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.

  7. Imaging Biological Systems using Dielectric Near-Field Microscopy

    Science.gov (United States)

    Brown, Keith; Issadore, David; Hunt, Tom; Westervelt, Robert

    2007-03-01

    We have developed a dielectric spectrometer for use on biological systems. The spectrum of dielectric response to RF electric fields is analogous to color as an optical response. Measurement of the dielectric spectrum from ˜ 10 kHz to ˜ 3 GHz will reveal information about the structure and conditions of protein solutions, protein crystals and biological tissues. We designed and built a system to test biological samples in a microfluidic chamber mounted on a circuit board. The apparatus measures the RF dielectric spectrum directly, or by analyzing the pulse response in the time domain. We have constructed several versions of the hardware for sensitive capacitive measurements, including two types of capacitive bridges, and a transmission line, incorporating precision electronics and local generation of pulses. A goal is to scale the system down and implement many dielectric spectrometers as an array of pixels on a CMOS chip for dielectric near-field microscopy of biological samples. This work made possible by NSEC NSF grant PHY-0117795 and the NCI MIT-Harvard CCNE.

  8. High-Resolution Microscopy-Coil MR Imaging of Skin Tumors: Techniques and Novel Clinical Applications.

    Science.gov (United States)

    Budak, Matthew J; Weir-McCall, Jonathan R; Yeap, Phey M; White, Richard D; Waugh, Shelley A; Sudarshan, Thiru A P; Zealley, Ian A

    2015-01-01

    High-resolution magnetic resonance (MR) imaging performed with a microscopy coil is a robust radiologic tool for the evaluation of skin lesions. Microscopy-coil MR imaging uses a small surface coil and a 1.5-T or higher MR imaging system. Simple T1- and T2-weighted imaging protocols can be implemented to yield high-quality, high-spatial-resolution images that provide an excellent depiction of dermal anatomy. The primary application of microscopy-coil MR imaging is to delineate the deep margins of skin tumors, thereby providing a preoperative road map for dermatologic surgeons. This information is particularly useful for surgeons who perform Mohs micrographic surgery and in cases of nasofacial neoplasms, where the underlying anatomy is complex. Basal cell carcinoma is the most common nonmelanocytic skin tumor and has a predilection to manifest on the face, where it can be challenging to achieve complete surgical excision while preserving the cosmetic dignity of the patient. Microscopy-coil MR imaging provides dermatologic surgeons with valuable preoperative anatomic information that is not available at conventional clinical examination. ©RSNA, 2015.

  9. Magnetic force microscopy/current contrast imaging: A new technique for internal current probing of ICs

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, A.N.; Cole, E.I. Jr.; Dodd, B.A.; Anderson, R.E.

    1993-09-01

    This invited paper describes recently reported work on the application of magnetic force microscopy (MFM) to image currents in IC conductors [1]. A computer model for MFM imaging of IC currents and experimental results demonstrating the ability to determine current direction and magnitude with a resolution of {approximately} 1 mA dc and {approximately} 1 {mu}A ac are presented. The physics of MFM signal generation and applications to current imaging and measurement are described.

  10. 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....

  11. Vibrational imaging based on stimulated Raman scattering microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Nandakumar, P; Kovalev, A; Volkmer, A [3. Physikalisches Institut, Universitaet Stuttgart, Pfaffenwaldring 57, 70550 Stuttgart (Germany)], E-mail: a.volkmer@physik.uni-stuttgart.de

    2009-03-15

    A stimulated Raman scattering microscope with near-infrared picosecond laser pulses at high repetition rates (76 MHz) and radio-frequency lock-in detection is accomplished. Based on stimulated Raman loss detection, we demonstrate noninvasive point-by-point vibrational mapping of chemical and biological samples with high sensitivity and without the requirement for labeling of the sample with natural or artificial fluorophores. We experimentally demonstrate a major benefit of this technique, which is the capability to respond exclusively to the linear Raman-resonance properties of the sample, thus allowing a direct quantitative interpretation of image contrast in terms of the number density of Raman-active modes.

  12. Vibrational imaging based on stimulated Raman scattering microscopy

    Science.gov (United States)

    Nandakumar, P.; Kovalev, A.; Volkmer, A.

    2009-03-01

    A stimulated Raman scattering microscope with near-infrared picosecond laser pulses at high repetition rates (76 MHz) and radio-frequency lock-in detection is accomplished. Based on stimulated Raman loss detection, we demonstrate noninvasive point-by-point vibrational mapping of chemical and biological samples with high sensitivity and without the requirement for labeling of the sample with natural or artificial fluorophores. We experimentally demonstrate a major benefit of this technique, which is the capability to respond exclusively to the linear Raman-resonance properties of the sample, thus allowing a direct quantitative interpretation of image contrast in terms of the number density of Raman-active modes.

  13. Photothermal Microscopy for High Sensitivity and High Resolution Absorption Contrast Imaging of Biological Tissues

    Directory of Open Access Journals (Sweden)

    Jun Miyazaki

    2017-04-01

    Full Text Available Photothermal microscopy is useful to visualize the distribution of non-fluorescence chromoproteins in biological specimens. Here, we developed a high sensitivity and high resolution photothermal microscopy with low-cost and compact laser diodes as light sources. A new detection scheme for improving signal to noise ratio more than 4-fold is presented. It is demonstrated that spatial resolution in photothermal microscopy is up to nearly twice as high as that in the conventional widefield microscopy. Furthermore, we demonstrated the ability for distinguishing or identifying biological molecules with simultaneous muti-wavelength imaging. Simultaneous photothermal and fluorescence imaging of mouse brain tissue was conducted to visualize both neurons expressing yellow fluorescent protein and endogenous non-fluorescent chromophores.

  14. Optimizing and extending light-sculpting microscopy for fast functional imaging in neuroscience

    CERN Document Server

    Rupprecht, Peter; Groessl, Florian; Haubensak, Wulf E; Vaziri, Alipasha

    2015-01-01

    A number of questions in systems biology such as understanding how dynamics of neuronal networks are related to brain function require the ability to capture the functional dynamics of large cellular populations at high speed. Recently, this has driven the development of a number of parallel and high speed imaging techniques such as light-sculpting microscopy, which has been used to capture neuronal dynamics at the whole brain and single cell level in small model organism. However, the broader applicability of light-sculpting microscopy is limited by the size of volumes for which high speed imaging can be obtained and scattering in brain tissue. Here, we present strategies for optimizing the present tradeoffs in light-sculpting microscopy. Various scanning modalities in light-sculpting microscopy are theoretically and experimentally evaluated, and strategies to maximize the obtainable volume speeds, and depth penetration in brain tissue using different laser systems are provided. Design-choices, important par...

  15. An open data mining framework for the analysis of medical images: application on obstructive nephropathy microscopy images.

    Science.gov (United States)

    Doukas, Charalampos; Goudas, Theodosis; Fischer, Simon; Mierswa, Ingo; Chatziioannou, Aristotle; Maglogiannis, Ilias

    2010-01-01

    This paper presents an open image-mining framework that provides access to tools and methods for the characterization of medical images. Several image processing and feature extraction operators have been implemented and exposed through Web Services. Rapid-Miner, an open source data mining system has been utilized for applying classification operators and creating the essential processing workflows. The proposed framework has been applied for the detection of salient objects in Obstructive Nephropathy microscopy images. Initial classification results are quite promising demonstrating the feasibility of automated characterization of kidney biopsy images.

  16. Stochastic optical reconstruction microscopy (STORM) in comparison with stimulated emission depletion (STED) and other imaging methods.

    Science.gov (United States)

    Tam, Johnny; Merino, David

    2015-11-01

    Stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED) microscopy are two super-resolution optical microscopy approaches that have rapidly gained popularity in recent years. Both modalities offer super-resolution imaging capabilities with the potential for imaging in multiple colors, three-dimensions, and the possibility to image in live cells. In this review, we focus on the specific advantages and disadvantages of each technique in the context of each other. STORM has been reported to achieve higher spatial resolution when compared to STED, but a lengthy acquisition may be required. STED utilizes relatively higher laser intensities, but is able to generate a super-resolution image immediately after acquisition without the need for any additional data processing. Ultimately, the choice between STORM and STED will depend not only on the specific application, but also on the users' ability to understand and optimize the various parameters ranging from sample preparation to image acquisition, which determine the quality of the final image. Stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED) are two super-resolution microscopy approaches that have rapidly gained popularity in recent years. STORM is based on the precise localization of a large number of individual molecules that together form a super-resolved image (bottom), whereas STED is based on the scanning of two super-imposed light sources which together allow for a super-resolved spot on the sample to be imaged (top). We discuss the specific advantages and disadvantages of each technique and explain the various parameters that affect image quality, which should be taken into consideration when planning experiments.

  17. Fluorescent Nanodiamond-Gold Hybrid Particles for Multimodal Optical and Electron Microscopy Cellular Imaging.

    Science.gov (United States)

    Liu, Weina; Naydenov, Boris; Chakrabortty, Sabyasachi; Wuensch, Bettina; Hübner, Kristina; Ritz, Sandra; Cölfen, Helmut; Barth, Holger; Koynov, Kaloian; Qi, Haoyuan; Leiter, Robert; Reuter, Rolf; Wrachtrup, Jörg; Boldt, Felix; Scheuer, Jonas; Kaiser, Ute; Sison, Miguel; Lasser, Theo; Tinnefeld, Philip; Jelezko, Fedor; Walther, Paul; Wu, Yuzhou; Weil, Tanja

    2016-10-12

    There is a continuous demand for imaging probes offering excellent performance in various microscopy techniques for comprehensive investigations of cellular processes by more than one technique. Fluorescent nanodiamond-gold nanoparticles (FND-Au) constitute a new class of "all-in-one" hybrid particles providing unique features for multimodal cellular imaging including optical imaging, electron microscopy, and, and potentially even quantum sensing. Confocal and optical coherence microscopy of the FND-Au allow fast investigations inside living cells via emission, scattering, and photothermal imaging techniques because the FND emission is not quenched by AuNPs. In electron microscopy, transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) analysis of FND-Au reveals greatly enhanced contrast due to the gold particles as well as an extraordinary flickering behavior in three-dimensional cellular environments originating from the nanodiamonds. The unique multimodal imaging characteristics of FND-Au enable detailed studies inside cells ranging from statistical distributions at the entire cellular level (micrometers) down to the tracking of individual particles in subcellular organelles (nanometers). Herein, the processes of endosomal membrane uptake and release of FNDs were elucidated for the first time by the imaging of individual FND-Au hybrid nanoparticles with single-particle resolution. Their convenient preparation, the availability of various surface groups, their flexible detection modalities, and their single-particle contrast in combination with the capability for endosomal penetration and low cytotoxicity make FND-Au unique candidates for multimodal optical-electronic imaging applications with great potential for emerging techniques, such as quantum sensing inside living cells.

  18. Atomic Force Microscopy Imaging of Filamentous Aggregates from an N-Terminal Peptide Fragment of Barnase

    Science.gov (United States)

    Shibata-Seki, Teiko; Masai, Junji; Yoshida, Kenji; Sato, Kazuki; Yanagawa, Hiroshi

    1993-06-01

    This paper reports the atomic force microscopy (AFM) imaging of filamentous aggregates derived from an N-terminal peptide fragment of barnase, a ribonuclease from Bacillus amyloliquefaciens. The sample was deposited on a freshly cleaved mica surface and observed in ambient conditions. The overall shapes of the filamentous structures imaged with two different kinds of AFMs were similar to those obtained with a transmission electron microscope (TEM), except that the filaments in AFM images were broader than those in TEM images. This broadening phenomenon characteristic of AFM images was explained in terms of the convolution-type distortion of the specimen diameter by the scanning-tip apex.

  19. Efficient Parallel Levenberg-Marquardt Model Fitting towards Real-Time Automated Parametric Imaging Microscopy

    OpenAIRE

    Xiang Zhu; Dianwen Zhang

    2013-01-01

    We present a fast, accurate and robust parallel Levenberg-Marquardt minimization optimizer, GPU-LMFit, which is implemented on graphics processing unit for high performance scalable parallel model fitting processing. GPU-LMFit can provide a dramatic speed-up in massive model fitting analyses to enable real-time automated pixel-wise parametric imaging microscopy. We demonstrate the performance of GPU-LMFit for the applications in superresolution localization microscopy and fluorescence lifetim...

  20. Deep focus; a digital image processing technique to produce improved focal depth in light microscopy:

    OpenAIRE

    Goldsmith, Noel T.

    2000-01-01

    In light microscopy, the spatial transverse resolution is a function of the wavelength and numerical aperture. The depth resolution is another function of these parameters. The factors that enable the detection of fine detail, make the sharp focusing of more than a thin slice of the depth in an object impossible. When the examination of fracture surfaces is attempted using light reflection microscopy, the roughness will often restrict the in-focus parts of an image to a small portion of the f...

  1. Deep tissue imaging by enhanced photon collection

    Directory of Open Access Journals (Sweden)

    Viera Crosignani

    2014-09-01

    Full Text Available We have developed a two-photon fluorescence microscope capable of imaging up to 4mm in turbid media with micron resolution. The key feature of this instrument is the innovative detector, capable of collecting emission photons from a wider surface area of the sample than detectors in traditional two-photon microscopes. This detection scheme is extremely efficient in the collection of emitted photons scattered by turbid media which allows eight fold increase in the imaging depth when compared with conventional two-photon microscopes. Furthermore, this system also has in-depth fluorescence lifetime imaging microscopy (FLIM imaging capability which increases image contrast. The detection scheme captures emission light in a transmission configuration, making it extremely efficient for the detection of second harmonic generation (SHG signals, which is generally forward propagating. Here we present imaging experiments of tissue phantoms and in vivo and ex vivo biological tissue performed with this microscope.

  2. Imaging of membrane proteins using antenna-based optical microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Hoeppener, Christiane; Novotny, Lukas [Institute of Optics and Department of Biomedical Engineering, University of Rochester, Rochester, NY 14627 (United States)], E-mail: novotny@optics.rochester.edu

    2008-09-24

    The localization and identification of individual proteins is of key importance for the understanding of biological processes on the molecular scale. Here, we demonstrate near-field fluorescence imaging of single proteins in their native cell membrane. Incident laser radiation is localized and enhanced with an optical antenna in the form of a spherical gold particle attached to a pointed dielectric tip. Individual proteins can be identified with a diffraction-unlimited spatial resolution of {approx}50 nm. Besides determining the concentration and distribution of specific membrane proteins, this approach makes it possible to study the colocalization of different membrane proteins. Moreover, it enables a simultaneous recording of the membrane topology. Protein distributions can be correlated with the local membrane topology, thereby providing important information on the chemical and structural organization of cellular membranes.

  3. Band Excitation in Scanning Probe Microscopy: Recognition and Functional Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Jesse, Stephen [ORNL; Vasudevan, Dr. Rama [Oak Ridge National Laboratory (ORNL); Collins, Liam [University College, Dublin; Strelcov, Evgheni [ORNL; Okatan, Mahmut B [ORNL; Belianinov, Alex [ORNL; Baddorf, Arthur P [ORNL; Proksch, Roger [Asylum Research, Santa Barbara, CA; Kalinin, Sergei V [ORNL

    2014-01-01

    Field confinement at the junction between a biased scanning probe microscope s (SPM) tip and solid surface enables local probing of various bias-induced transformations such as polarization switching, ionic motion, or electrochemical reactions to name a few. The nanoscale size of the biased region is smaller or comparable to features like grain boundaries and dislocations, potentially allows for the study of kinetics and thermodynamics at the level of a single defect. In contrast to classical statistically averaged approaches, this allows one to link structure to functionality and deterministically decipher associated mesoscopic and atomistic mechanisms. Furthermore, this type of information can serve as a fingerprint of local material functionality, allowing for local recognition imaging. Here, current progress in multidimensional SPM techniques based on band-excitation time and voltage spectroscopies is illustrated, including discussions on data acquisition, dimensionality reduction, and visualization along with future challenges and opportunities for the field.

  4. Photoelectron microscopy in the life sciences: Imaging neuron networks

    Energy Technology Data Exchange (ETDEWEB)

    Mercanti, D. (Istituto di Neurobiologia del CNR, Viale Marx 15, 00100 Roma (Italy)); De Stasio, G. (ISM-CNR, Via E. Fermi 38, 00044 Frascati, Roma (Italy)); Ciotti, M.T. (Istituto di Neurobiologia del CNR, Viale Marx 15, 00100 Roma (Italy)); Capasso, C.; Ng, W.; Ray-Chaudhuri, A.K.; Liang, S.H.; Cole, R.K.; Guo, Z.Y.; Wallace, J. (Department of Physics, University of Wisconsin, Madison, WI (USA) Electrical and Computer Engineering, University of Wisconsin, Madison, WI (USA)); Margaritondo, G. (Institut de Physique Appliquee, Ecole Polytechnique Federale de Lausanne, Ecublens (Switzerland)); Cerrina, F. (Departments of Physics, University of Wisconsin, Madison, WI (USA) Electrical and Computer Engineering, University of Wisconsin, Madison, WI (USA)); Underwood, J.; Perera, R.; Kortright, J. (Center for X-ray Optics, Lawrence Berkeley Laboratory, Berkeley, CA 94720 (USA))

    1991-05-01

    Photoemission techniques like electron spectroscopy for chemical analysis are the leading electronic probes in materials science---but their impact in the life sciences has been minimal. A critical problem is that the lateral resolution in ordinary photoemission does not exceed a few tenths of a millimeter. This space-averaged probe is nearly useless for most of the fundamental problems in biophysics and biochemistry, which deal with microstructures in the submicron range or smaller. This limit is being overcome with photoemission microscopes, such as our scanning instrument MAXIMUM. The first scanning photoelectron micrographs of a cellular system with submicron resolution are presented. Minute details of neuron networks are imaged on MAXIMUM, thereby opening the way to novel applications of photoemission in the life sciences. The details include individual neurons, axons, dendrites, and synapses, and composite large-area scanning micrographs were routinely produced with a lateral resolution of 0.5 {mu}m.

  5. 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.

  6. Nanoscopy for nanoscience: how super-resolution microscopy extends imaging for nanotechnology.

    Science.gov (United States)

    Johnson, Sam A

    2015-01-01

    Imaging methods have presented scientists with powerful means of investigation for centuries. The ability to resolve structures using light microscopes is though limited to around 200 nm. Fluorescence-based super-resolution light microscopy techniques of several principles and methods have emerged in recent years and offer great potential to extend the capabilities of microscopy. This resolution improvement is especially promising for nanoscience where the imaging of nanoscale structures is inherently restricted by the resolution limit of standard forms of light microscopy. Resolution can be improved by several distinct approaches including structured illumination microscopy, stimulated emission depletion, and single-molecule positioning methods such as photoactivated localization microscopy and stochastic optical reconstruction microscopy and several derivative variations of each of these. These methods involve substantial differences in the resolutions achievable in the different axes, speed of acquisition, compatibility with different labels, ease of use, hardware complexity, and compatibility with live biological samples. The field of super-resolution imaging and its application to nanotechnology is relatively new and still rapidly developing. An overview of how these methods may be used with nanomaterials is presented with some examples of pioneering uses of these approaches.

  7. In vivo multiphoton microscopy associated to 3D image processing for human skin characterization

    Science.gov (United States)

    Baldeweck, T.; Tancrède, E.; Dokladal, P.; Koudoro, S.; Morard, V.; Meyer, F.; Decencière, E.; Pena, A.-M.

    2012-03-01

    Multiphoton microscopy has emerged in the past decade as a promising non-invasive skin imaging technique. The aim of this study was to assess whether multiphoton microscopy coupled to specific 3D image processing tools could provide new insights into the organization of different skin components and their age-related changes. For that purpose, we performed a clinical trial on 15 young and 15 aged human female volunteers on the ventral and dorsal side of the forearm using the DermaInspectR medical imaging device. We visualized the skin by taking advantage of intrinsic multiphoton signals from cells, elastic and collagen fibers. We also developed 3D image processing algorithms adapted to in vivo multiphoton images of human skin in order to extract quantitative parameters in each layer of the skin (epidermis and superficial dermis). The results show that in vivo multiphoton microscopy is able to evidence several skin alterations due to skin aging: morphological changes in the epidermis and modifications in the quantity and organization of the collagen and elastic fibers network. In conclusion, the association of multiphoton microscopy with specific image processing allows the three-dimensional organization of skin components to be visualized and quantified thus providing a powerful tool for cosmetic and dermatological investigations.

  8. Atomic force microscopy imaging of fragments from the Martian meteorite ALH84001.

    Science.gov (United States)

    Steele, A; Goddard, D; Beech, I B; Tapper, R C; Stapleton, D; Smith, J R

    1998-01-01

    A combination of scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) techniques, as well as atomic force microscopy (AFM) methods has been used to study fragments of the Martian meteorite ALH84001. Images of the same areas on the meteorite were obtained prior to and following gold/palladium coating by mapping the surface of the fragment using ESEM coupled with energy-dispersive X-ray analysis. Viewing of the fragments demonstrated the presence of structures, previously described as nanofossils by McKay et al. (Search for past life on Mars--possible relic biogenic activity in martian meteorite ALH84001. Science, 1996, pp. 924-930) of NASA who used SEM imaging of gold-coated meteorite samples. Careful imaging of the fragments revealed that the observed structures were not an artefact introduced by the coating procedure.

  9. 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.

  10. Segmentation of scanning electron microscopy images from natural rubber samples with gold nanoparticles using starlet wavelets.

    Science.gov (United States)

    de Siqueira, Alexandre Fioravante; Cabrera, Flávio Camargo; Pagamisse, Aylton; Job, Aldo Eloizo

    2014-01-01

    Electronic microscopy has been used for morphology evaluation of different materials structures. However, microscopy results may be affected by several factors. Image processing methods can be used to correct and improve the quality of these results. In this article, we propose an algorithm based on starlets to perform the segmentation of scanning electron microscopy images. An application is presented in order to locate gold nanoparticles in natural rubber membranes. In this application, our method showed accuracy greater than 85% for all test images. Results given by this method will be used in future studies, to computationally estimate the density distribution of gold nanoparticles in natural rubber samples and to predict reduction kinetics of gold nanoparticles at different time periods.

  11. 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.

  12. Imaging rat esophagus using combination of reflectance confocal and multiphoton microscopy

    Science.gov (United States)

    Zhuo, S. M.; Chen, J. X.; Jiang, X. S.; Lu, K. C.; Xie, S. S.

    2008-08-01

    We combine reflectance confocal microscopy (RCM) with multiphoton microscopy (MPM) to image rat esophagus. The two imaging modalities allow detection of layered-resolved complementary information from esophagus. In the keratinizing layer, the keratinocytes boundaries can be characterized by RCM, while the keratinocytes cytoplasm (keratin) can be further imaged by multiphoton autofluorescence signal. In the epithelium, the epithelial cellular boundaries and nucleus can be detected by RCM, and MPM can be used for imaging epithelial cell cytoplasm and monitoring metabolic state of epithelium. In the stroma, multiphoton autofluorescence signal is used to image elastin and second harmonic generation signal is utilized to detect collagen, while RCM is used to determine the optical property of stroma. Overall, these results suggest that the combination of RCM and MPM has potential to provide more important and comprehensive information for early diagnosis of esophageal cancer.

  13. A CMOS image sensor with draining only modulation pixels for fluorescence lifetime imaging

    Science.gov (United States)

    Li, Zhuo; Yasutomi, Keita; Takasawa, Taishi; Itoh, Shinya; Kawahito, Shoji

    2011-03-01

    Fluorescence lifetime imaging is becoming a powerful tool in biology. A charge-domain CMOS Fluorescence Lifetime Imaging Microscopy (FLIM) chip using a pinned photo diode (PPD) and the pinned storage diode (PSD) with different depth of potential wells has been previously developed by the authors. However, a transfer gate between PPD and PSD causes charge transfer noise due to traps at the channel surface. This paper presents a time-resolved CMOS image sensor with draining only modulation pixels for fluorescence lifetime imaging, which removes the transfer gate between PPD and PSD. The time windowing is done by draining with a draining gate only, which is attached along the carrier path from PPD to PSD. This allows us to realize a trapping less charge transfer between PPD and PSD, leading to a very low-noise time-resolved signal detection. A video-rate CMOS FLIM chip has been fabricated using 0.18μm standard CMOS pinned diode image sensor process. The pixel consists of a PPD, a PSD, a charge draining gate (TD), a readout transfer gate (TX) between the PSD and the floating diffusion (FD), a reset transistor and a source follower amplifier transistor. The pixel array has 200(Row) x 256(Column) pixels and the pixel pitch is 7.5μm. Fundamental characteristics of the implemented CMOS FLIM chip are measured. The signal intensity of the PSD as a function of the TD gate voltage is also measured. The ratio of the signal for the TD off to the signal for the TD on is 212 : 1.

  14. 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.

  15. 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.

  16. 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.

  17. Imaging dendritic spines of rat primary hippocampal neurons using structured illumination microscopy.

    Science.gov (United States)

    Schouten, Marijn; De Luca, Giulia M R; Alatriste González, Diana K; de Jong, Babette E; Timmermans, Wendy; Xiong, Hui; Krugers, Harm; Manders, Erik M M; Fitzsimons, Carlos P

    2014-05-04

    Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm. Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the

  18. Tripling the maximum imaging depth with third-harmonic generation microscopy.

    Science.gov (United States)

    Yildirim, Murat; Durr, Nicholas; Ben-Yakar, Adela

    2015-09-01

    The growing interest in performing high-resolution, deep-tissue imaging has galvanized the use of longer excitation wavelengths and three-photon-based techniques in nonlinear imaging modalities. This study presents a threefold improvement in maximum imaging depth of ex vivo porcine vocal folds using third-harmonic generation (THG) microscopy at 1552-nm excitation wavelength compared to two-photon microscopy (TPM) at 776-nm excitation wavelength. The experimental, analytical, and Monte Carlo simulation results reveal that THG improves the maximum imaging depth observed in TPM significantly from 140 to 420 μm in a highly scattered medium, reaching the expected theoretical imaging depth of seven extinction lengths. This value almost doubles the previously reported normalized imaging depths of 3.5 to 4.5 extinction lengths using three-photon-based imaging modalities. Since tissue absorption is substantial at the excitation wavelength of 1552 nm, this study assesses the tissue thermal damage during imaging by obtaining the depth-resolved temperature distribution through a numerical simulation incorporating an experimentally obtained thermal relaxation time (τ). By shuttering the laser for a period of 2τ, the numerical algorithm estimates a maximum temperature increase of ∼2°C at the maximum imaging depth of 420 μm. The paper demonstrates that THG imaging using 1552 nm as an illumination wavelength with effective thermal management proves to be a powerful deep imaging modality for highly scattering and absorbing tissues, such as scarred vocal folds.

  19. Engineering Dark Chromoprotein Reporters for Photoacoustic Microscopy and FRET Imaging

    Science.gov (United States)

    Li, Yan; Forbrich, Alex; Wu, Jiahui; Shao, Peng; Campbell, Robert E.; Zemp, Roger

    2016-03-01

    A subset of the family of fluorescent proteins are the non-fluorescent chromoproteins which are promising probe molecules for use in photoacoustic imaging and as acceptor chromophores in Förster resonance energy transfer (FRET)-based biosensors. Typical approaches for fluorescent protein optimization by screening of large libraries of variants cannot be effectively applied to chromoproteins due to their characteristic lack of fluorescence. To address this challenge, we have developed a directed evolution method to iteratively screen large libraries of protein variants on the basis of their photoacoustic signal levels. By applying this procedure to the promising Ultramarine and cjBlue chromoprotein templates, we were able to identify improved variants with a 02-04 fold increase in photoacoustic signal-to-noise ratio after only a few evolutionary steps. These improved variants enable more accurate spectral de-mixing and localization of protein-producing bacteria in vivo and serve as effective FRET acceptors for both fluorescence- and photoacoustic-based detection of protease activity.

  20. Adult Human Neurogenesis: from Microscopy to Magnetic Resonance Imaging

    Directory of Open Access Journals (Sweden)

    Amanda eSierra

    2011-04-01

    Full Text Available Neural stem cells reside in well-defined areas of the adult human brain and are capable of gene-rating new neurons throughout the life span. In rodents, it is well established that the new born neurons are involved in olfaction as well as in certain forms of memory and learning. In humans, the functional relevance of adult human neurogenesis is being investigated, in particular its implication in the etiopathology of a variety of brain disorders. Adult neurogenesis in the human brain was discovered by utilizing methodologies directly imported from the rodent research, such as immunohistological detection of proliferation and cell-type specific biomarkers in postmortem or biopsy tissue. However, in the vast majority of cases, these methods do not support longitudinal studies; thus, the capacity of the putative stem cells to form new neurons under different disease conditions cannot be tested. More recently, new technologies have been specifically developed for the detection and quantification of neural stem cells in the living human brain. These technologies rely on the use of magnetic resonance imaging, available in hospitals worldwide. Although they require further validation in rodents and primates, these new methods hold the potential to test the contribution of adult human neurogenesis to brain function in both health and disease. This review reports on the current knowledge on adult human neurogenesis. We first review the different methods available to assess human neurogenesis, both ex vivo and in vivo and then appraise the changes of adult neurogenesis in human diseases.

  1. Sub-micron imaging of buried integrated circuit structures using scanning confocal electron microscopy.

    Energy Technology Data Exchange (ETDEWEB)

    Frigo, S. P.; Levine, Z.; Zaluzec, N. J.; Materials Science Division; Northern Arizona Univ.; NIST

    2002-09-09

    Two-dimensional images of model integrated circuit components were collected using the technique of scanning confocal electron microscopy. For structures embedded about 5 {mu}m below the surface of a silicon oxide dielectric, a lateral resolution of 76{+-}9 nm was measured. Elemental mapping via x-ray emission spectrometry is demonstrated. A parallax analysis of images taken for various tilt angles to the electron beam allowed determination of the spacing between two wiring planes. The results show that scanning confocal electron microscopy is capable of probing buried structures at resolutions that will be necessary for the inspection of next-generation integrated circuit technology.

  2. Integrated structural and functional optical imaging combining spectral-domain optical coherence and multiphoton microscopy

    CERN Document Server

    Vinegoni, C; Luo, W; Marks, D L; Ralston, T; Tan, W

    2005-01-01

    An integrated microscope that combines different optical techniques for simultaneous imaging is demonstrated. The microscope enables spectral-domain optical coherence microscopy based on optical backscatter, and multi-photon microscopy for the detection of two-photon fluorescence and second harmonic generation signals. The unique configuration of this integrated microscope allows for the simultaneous acquisition of both anatomical (structural) and functional imaging information with particular emphasis for applications in the fields of tissue engineering and cell biology. In addition, the contemporary analysis of the spectroscopic features can enhance contrast by differentiating among different tissue components.

  3. Imaging via complete cantilever dynamic detection: general dynamic mode imaging and spectroscopy in scanning probe microscopy

    Science.gov (United States)

    Somnath, Suhas; Collins, Liam; Matheson, Michael A.; Sukumar, Sreenivas R.; Kalinin, Sergei V.; Jesse, Stephen

    2016-10-01

    We develop and implement a multifrequency spectroscopy and spectroscopic imaging mode, referred to as general dynamic mode (GDM), that captures the complete spatially- and stimulus dependent information on nonlinear cantilever dynamics in scanning probe microscopy (SPM). GDM acquires the cantilever response including harmonics and mode mixing products across the entire broadband cantilever spectrum as a function of excitation frequency. GDM spectra substitute the classical measurements in SPM, e.g. amplitude and phase in lock-in detection. Here, GDM is used to investigate the response of a purely capacitively driven cantilever. We use information theory techniques to mine the data and verify the findings with governing equations and classical lock-in based approaches. We explore the dependence of the cantilever dynamics on the tip–sample distance, AC and DC driving bias. This approach can be applied to investigate the dynamic behavior of other systems within and beyond dynamic SPM. GDM is expected to be useful for separating the contribution of different physical phenomena in the cantilever response and understanding the role of cantilever dynamics in dynamic AFM techniques.

  4. Molecular recognition imaging using tuning fork-based transverse dynamic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Hofer, Manuel; Adamsmaier, Stefan [University of Linz, Institute for Biophysics, Altenbergerstr. 69, 4040 Linz (Austria); Zanten, Thomas S. van [IBEC-Institute for Bioengineering of Catalonia and CIBER-Bbn, Baldiri i Reixac 15-21, Barcelona 08028 (Spain); Chtcheglova, Lilia A. [University of Linz, Institute for Biophysics, Altenbergerstr. 69, 4040 Linz (Austria); Manzo, Carlo [IBEC-Institute for Bioengineering of Catalonia and CIBER-Bbn, Baldiri i Reixac 15-21, Barcelona 08028 (Spain); Duman, Memed [University of Linz, Institute for Biophysics, Altenbergerstr. 69, 4040 Linz (Austria); Mayer, Barbara [Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Institute for Biophysics, University of Linz, Altenbergerstr. 69, 4040 Linz (Austria); Ebner, Andreas [University of Linz, Institute for Biophysics, Altenbergerstr. 69, 4040 Linz (Austria); Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Institute for Biophysics, University of Linz, Altenbergerstr. 69, 4040 Linz (Austria); Moertelmaier, Manuel; Kada, Gerald [Agilent Technologies Austria GmbH, Aubrunnerweg 11, 4040 Linz (Austria); Garcia-Parajo, Maria F. [IBEC-Institute for Bioengineering of Catalonia and CIBER-Bbn, Baldiri i Reixac 15-21, Barcelona 08028 (Spain); ICREA-Institucio Catalana de Recerca i Estudis Avancats, 08010 Barcelona (Spain); Hinterdorfer, Peter, E-mail: peter.hinterdorfer@jku.at [University of Linz, Institute for Biophysics, Altenbergerstr. 69, 4040 Linz (Austria); Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Institute for Biophysics, University of Linz, Altenbergerstr. 69, 4040 Linz (Austria); Kienberger, Ferry [Agilent Technologies Austria GmbH, Aubrunnerweg 11, 4040 Linz (Austria)

    2010-05-15

    We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging using tuning forks as piezoelectric sensors. Tapered aluminum-coated glass fibers were chemically functionalized with biotin and anti-lysozyme molecules and attached to one of the prongs of a 32 kHz tuning fork. The lateral oscillation amplitude of the tuning fork was used as feedback signal for topographical imaging of avidin aggregates and lysozyme molecules on mica substrate. The phase difference between the excitation and detection signals of the tuning fork provided molecular recognition between avidin/biotin or lysozyme/anti-lysozyme. Aggregates of avidin and lysozyme molecules appeared as features with heights of 1-4 nm in the topographic images, consistent with single molecule atomic force microscopy imaging. Recognition events between avidin/biotin or lysozyme/anti-lysozyme were detected in the phase image at high signal-to-noise ratio with phase shifts of 1-2{sup o}. Because tapered glass fibers and shear-force microscopy based on tuning forks are commonly used for near-field scanning optical microscopy (NSOM), these results open the door to the exciting possibility of combining optical, topographic and biochemical recognition at the nanometer scale in a single measurement and in liquid conditions.

  5. 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.

  6. Single-exposure quantitative phase imaging in color-coded LED microscopy (Conference Presentation)

    Science.gov (United States)

    Lee, Wonchan; Jung, Daeseong; Joo, Chulmin

    2017-02-01

    Quantitative phase-gradient or phase imaging in LED microscopy has been recently demonstrated. The methods enable measurement of phase distribution of transparent specimens in a simple and cost-effective manner, but require multiple image acquisitions with different source or pupil configurations to improve phase accuracy. Here, we demonstrate a strategy for single-shot quantitative phase imaging in color-coded LED microscopy. We employ a circular LED illumination pattern that is trisected into subregions with equal area, assigned to red, green and blue colors, respectively. Additional color filter is also employed to mitigate the color leakage of light into different color channels of the image sensor. Image acquisition with a color image sensor and subsequent computation based on the weak object transfer function allow for quantitative amplitude and phase measurements of a specimen. We describe computational model and single-shot quantitative phase imaging capability of our method by presenting phase images of calibrated phase sample and dynamics of cells. Phase measurement accuracy is validated with pre-characterized phase plate, and single-shot phase imaging capability is demonstrated with time-lapse imaging of cells acquired at 30 Hz.

  7. A software framework for the analysis of complex microscopy image data.

    Science.gov (United States)

    Chao, Jerry; Ward, E Sally; Ober, Raimund J

    2010-07-01

    Technological advances in both hardware and software have made possible the realization of sophisticated biological imaging experiments using the optical microscope. As a result, modern microscopy experiments are capable of producing complex image datasets. For a given data analysis task, the images in a set are arranged, based on the requirements of the task, by attributes such as the time and focus levels at which they were acquired. Importantly, different tasks performed over the course of an analysis are often facilitated by the use of different arrangements of the images. We present a software framework that supports the use of different logical image arrangements to analyze a physical set of images. This framework, called the Microscopy Image Analysis Tool (MIATool), realizes the logical arrangements using arrays of pointers to the images, thereby removing the need to replicate and manipulate the actual images in their storage medium. In order that they may be tailored to the specific requirements of disparate analysis tasks, these logical arrangements may differ in size and dimensionality, with no restrictions placed on the number of dimensions and the meaning of each dimension. MIATool additionally supports processing flexibility, extensible image processing capabilities, and data storage management.

  8. Performance of a malaria microscopy image analysis slide reading device

    Directory of Open Access Journals (Sweden)

    Prescott William R

    2012-05-01

    Full Text Available Abstract Background Viewing Plasmodium in Romanovsky-stained blood has long been considered the gold standard for diagnosis and a cornerstone in management of the disease. This method however, requires a subjective evaluation by trained, experienced diagnosticians and establishing proficiency of diagnosis is fraught with many challenges. Reported here is an evaluation of a diagnostic system (a “device” consisting of a microscope, a scanner, and a computer algorithm that evaluates scanned images of standard Giemsa-stained slides and reports species and parasitaemia. Methods The device was challenged with two independent tests: a 55 slide, expert slide reading test the composition of which has been published by the World Health Organization (“WHO55” test, and a second test in which slides were made from a sample of consenting subjects participating in a malaria incidence survey conducted in Equatorial Guinea (EGMIS test. These subjects’ blood was tested by malaria RDT as well as having the blood smear diagnosis unequivocally determined by a worldwide panel of a minimum of six reference microscopists. Only slides with unequivocal microscopic diagnoses were used for the device challenge, n = 119. Results On the WHO55 test, the device scored a “Level 4” using the WHO published grading scheme. Broken down by more traditional analysis parameters this result was translated to 89% and 70% sensitivity and specificity, respectively. Species were correctly identified in 61% of the slides and the quantification of parasites fell within acceptable range of the validated parasitaemia in 10% of the cases. On the EGMIS test it scored 100% and 94% sensitivity/specificity, with 64% of the species correct and 45% of the parasitaemia within an acceptable range. A pooled analysis of the 174 slides used for both tests resulted in an overall 92% sensitivity and 90% specificity with 61% species and 19% quantifications correct. Conclusions In its

  9. Pixel timing correction in time-lapsed calcium imaging using point scanning microscopy.

    Science.gov (United States)

    Boiroux, Dimitri; Oke, Yoshihiko; Miwakeichi, Fumikazu; Oku, Yoshitaka

    2014-11-30

    In point scanning imaging, data are acquired by sequentially scanning each pixel of a predetermined area. This way of scanning leads to time delays between pixels, especially for lower scanning speed or large scanned areas. Therefore, experiments are often performed at lower framerates in order to ensure a sufficient signal-to-noise ratio, even though framerates above 30 frames per second are technically feasible. For these framerates, we suggest that it becomes crucial to correct the time delay between image pixels prior to analyses. In this paper, we apply temporal interpolation (or pixel timing correction) for calcium imaging in two-photon microscopy as an example of fluorescence imaging. We present and compare three interpolation methods (linear, Lanczos and cubic B-spline). We test these methods on a simulated network of coupled bursting neurons at different framerates. In this network, we introduce a time delay to simulate a scanning by point scanning microscopy. We also assess these methods on actual microscopic calcium imaging movies recorded at usual framerates. Our numerical results suggest that point scanning microscopy imaging introduces statistically significant time delays between image pixels at low frequency. However, we demonstrate that pixel timing correction compensates for these time delays, regardless of the used interpolation method.

  10. Two-photon autofluorescence/FLIM/SHG endoscopy to study the oral cavity and wound healing in humans (Conference Presentation)

    Science.gov (United States)

    König, Karsten

    2016-03-01

    Monitoring the oral cavity noninvasively with superior 3D resolution is realized by clinical multiphoton tomography and high NA two-photon endoscopy without the need of additional contrast agents. The technology behind this investigation is based on nonlinear optical contrast of the multiphoton tomograph MPTflex®. Furthermore, the miniaturized GRIN endoscope was used to realize more accessibility for more demanding wound conditions in skin. The MPTflex® distinguishes autofluorescence (AF) signals from second harmonic generation (SHG) signals simultaneously. Fluorescence lifetime imaging (FLIM) based on time correlated single photon counting (TCSPC) technology offers additional information on the functional level of the intratissue fluorophores, their binding status, and the contribution of SHG signals in chronic wounds.

  11. Simultaneous whole-animal 3D-imaging of neuronal activity using light field microscopy

    CERN Document Server

    Prevedel, R; Hoffmann, M; Pak, N; Wetzstein, G; Kato, S; Schrödel, T; Raskar, R; Zimmer, M; Boyden, E S; Vaziri, A

    2014-01-01

    3D functional imaging of neuronal activity in entire organisms at single cell level and physiologically relevant time scales faces major obstacles due to trade-offs between the size of the imaged volumes, and spatial and temporal resolution. Here, using light-field microscopy in combination with 3D deconvolution, we demonstrate intrinsically simultaneous volumetric functional imaging of neuronal population activity at single neuron resolution for an entire organism, the nematode Caenorhabditis elegans. The simplicity of our technique and possibility of the integration into epi-fluoresence microscopes makes it an attractive tool for high-speed volumetric calcium imaging.

  12. Laser Doppler holographic microscopy in transmission: application to fish embryo imaging

    CERN Document Server

    Verrier, Nicolas; Gross, Michel

    2014-01-01

    We have extended Laser Doppler holographic microscopy to transmission geometry. The technique is validated with living fish embryos imaged by a modified upright bio-microcope. By varying the frequency of the holographic reference beam, and the combination of frames used to calculate the hologram, multimodal imaging has been performed. Doppler images of the blood vessels for different Doppler shifts, images where the flow direction is coded in RGB colors or movies showing blood cells individual motion have been obtained as well. The ability to select the Fourier space zone that is used to calculate the signal, makes the method quantitative.

  13. X-ray imaging microscopy at 25 keV with Fresnel zone plate optics

    CERN Document Server

    Awaji, M; Takeuchi, A; Takano, H; Kamijo, N; Tamura, S; Yasumoto, M

    2001-01-01

    X-ray imaging microscopy with a sputtered-sliced Fresnel zone plate (SS-FZP) has been developed at an X-ray energy of 25 keV. Objects were imaged in transmission with the SS-FZP as an objective with a magnification of 10.2 times, and detected with a X-ray image sensor. The performance of the imaging microscope has been tested with a gold mesh and a resolution test pattern at an undulator beamline 47XU of SPring-8. The resolution test patterns up to 0.5 mu m line-and-space structures have been resolved.

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

    Directory of Open Access Journals (Sweden)

    Christian Schou Oxvig

    2014-10-01

    Full Text Available 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 provides researchers in compressed sensing with a selection of algorithms for reconstructing undersampled general images, and offers a consistent and rigorous way to efficiently evaluate the researchers own developed reconstruction algorithms in terms of phase transitions. The package also serves as a convenient platform for researchers in compressed sensing aiming at obtaining a high degree of reproducibility of their research.

  15. IMAGING WOOD PULP FIBRE SURFACE LIGNIN BY FLUORESCENCE CONFOCAL LASER SCANNING MICROSCOPY

    Institute of Scientific and Technical Information of China (English)

    Kecheng Li; Douglas W. Reeve

    2004-01-01

    A novel methodology for imaging wood pulp fibre surface lignin by fluorescence confocal laser scanning microscopy was developed. Various imaging modes and imaging conditions were explored for quantitative analysis. Acridine Orange was used for labelling lignin and the orthochromatic labelling condition was developed. Withthe thusly established methodology, the distribution of lignin across the fibre wall was clearly imaged. It was found that surface lignin concentration is about 2-4 times higher than bulk lignin concentration, and that high concentration of lignin was also found on the fibre lumen surfaces and pit borders.

  16. IMAGING WOOD PULP FIBRE SURFACE LIGNIN BY FLUORESCENCE CONFOCAL LASER SCANNING MICROSCOPY

    Institute of Scientific and Technical Information of China (English)

    KechengLi; DouglasW.Reeve

    2004-01-01

    A novel methodology for imaging wood pulp fibre surface lignin by fluorescence confocal laser scanning microscopy was developed. Various imaging modes and imaging conditions were explored for quantitative analysis. Acridine Orange was used for labelling lignin and the orthochromatic labelling condition was developed. With the thusly established methodology, the distribution of lignin across the fibre wall was clearly imaged. It was found that surface lignin concentration is about 2-4 times higher than bulk lignin concentration and that high concentration of lignin was also found on the fibre lumen surfaces and pit borders.

  17. The Open Microscopy Environment: open image informatics for the biological sciences

    Science.gov (United States)

    Blackburn, Colin; Allan, Chris; Besson, Sébastien; Burel, Jean-Marie; Carroll, Mark; Ferguson, Richard K.; Flynn, Helen; Gault, David; Gillen, Kenneth; Leigh, Roger; Leo, Simone; Li, Simon; Lindner, Dominik; Linkert, Melissa; Moore, Josh; Moore, William J.; Ramalingam, Balaji; Rozbicki, Emil; Rustici, Gabriella; Tarkowska, Aleksandra; Walczysko, Petr; Williams, Eleanor; Swedlow, Jason R.

    2016-07-01

    Despite significant advances in biological imaging and analysis, major informatics challenges remain unsolved: file formats are proprietary, storage and analysis facilities are lacking, as are standards for sharing image data and results. While the open FITS file format is ubiquitous in astronomy, astronomical imaging shares many challenges with biological imaging, including the need to share large image sets using secure, cross-platform APIs, and the need for scalable applications for processing and visualization. The Open Microscopy Environment (OME) is an open-source software framework developed to address these challenges. OME tools include: an open data model for multidimensional imaging (OME Data Model); an open file format (OME-TIFF) and library (Bio-Formats) enabling free access to images (5D+) written in more than 145 formats from many imaging domains, including FITS; and a data management server (OMERO). The Java-based OMERO client-server platform comprises an image metadata store, an image repository, visualization and analysis by remote access, allowing sharing and publishing of image data. OMERO provides a means to manage the data through a multi-platform API. OMERO's model-based architecture has enabled its extension into a range of imaging domains, including light and electron microscopy, high content screening, digital pathology and recently into applications using non-image data from clinical and genomic studies. This is made possible using the Bio-Formats library. The current release includes a single mechanism for accessing image data of all types, regardless of original file format, via Java, C/C++ and Python and a variety of applications and environments (e.g. ImageJ, Matlab and R).

  18. Fluorescence lifetime imaging in biosciences: technologies and applications

    Institute of Scientific and Technical Information of China (English)

    Raluca NIESNER; Karl-Heinz GERICKE

    2008-01-01

    The biosciences require the development of methods that allow a non-invasive and rapid investigation of biological systems. In this aspect, high-end imaging tech-niques allow intravital microscopy in real-time, providing information on a molecular basis. Far-field fluorescence imaging techniques are some of the most adequate methods for such investigations. However, there are great differences between the common fluorescence imaging techniques, i.e., wide-field, confocal one-photon and two-photon microscopy, as far as their applicability in diverse bioscientific research areas is concerned. In the first part of this work, we briefly compare these techniques. Standard methods used in the biosciences, i.e., steady-state techniques based on the analy-sis of the total fluorescence signal originating from the sam-ple, can successfully be employed in the study of cell, tissue and organ morphology as well as in monitoring the macro-scopic tissue function. However, they are mostly inadequate for the quantitative investigation of the cellular function at the molecular level. The intrinsic disadvantages of steady-state techniques are countered by using time-resolved tech-niques. Among these fluorescence lifetime imaging (FLIM) is currently the most common. Different FLIM principles as well as applications of particular relevance for the biosci-ences, especially for fast intravital studies are discussed in this work.

  19. Imaging stability in force-feedback high-speed atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Byung I., E-mail: ByungKim@boisestate.edu [Department of Physics, Boise State University, 1910 University Drive Boise, ID 83725-1570, United States of America (United States); Boehm, Ryan D. [Department of Physics, Boise State University, 1910 University Drive Boise, ID 83725-1570, United States of America (United States)

    2013-02-15

    We studied the stability of force-feedback high-speed atomic force microscopy (HSAFM) by imaging soft, hard, and biological sample surfaces at various applied forces. The HSAFM images showed sudden topographic variations of streaky fringes with a negative applied force when collected on a soft hydrocarbon film grown on a grating sample, whereas they showed stable topographic features with positive applied forces. The instability of HSAFM images with the negative applied force was explained by the transition between contact and noncontact regimes in the force–distance curve. When the grating surface was cleaned, and thus hydrophilic by removing the hydrocarbon film, enhanced imaging stability was observed at both positive and negative applied forces. The higher adhesive interaction between the tip and the surface explains the improved imaging stability. The effects of imaging rate on the imaging stability were tested on an even softer adhesive Escherichia coli biofilm deposited onto the grating structure. The biofilm and planktonic cell structures in HSAFM images were reproducible within the force deviation less than ∼0.5 nN at the imaging rate up to 0.2 s per frame, suggesting that the force-feedback HSAFM was stable for various imaging speeds in imaging softer adhesive biological samples. - Highlights: ► We investigated the imaging stability of force-feedback HSAFM. ► Stable–unstable imaging transitions rely on applied force and sample hydrophilicity. ► The stable–unstable transitions are found to be independent of imaging rate.

  20. 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.

  1. 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.

  2. Strip mosaicing confocal microscopy for rapid imaging over large areas of excised tissue

    Science.gov (United States)

    Abeytunge, Sanjee; Li, Yongbiao; Larson, Bjorg; Peterson, Gary; Toledo-Crow, Ricardo; Rajadhyaksha, Milind

    2012-03-01

    Confocal mosaicing microscopy is a developing technology platform for imaging tumor margins directly in fresh tissue, without the processing that is required for conventional pathology. Previously, basal cell carcinoma margins were detected by mosaicing of confocal images of 12 x 12 mm2 of excised tissue from Mohs surgery. This mosaicing took 9 minutes. Recently we reported the initial feasibility of a faster approach called "strip mosaicing" on 10 x 10 mm2 of tissue that was demonstrated in 3 minutes. In this paper we report further advances in instrumentation and software. Rapid mosaicing of confocal images on large areas of fresh tissue potentially offers a means to perform pathology at the bedside. Thus, strip mosaicing confocal microscopy may serve as an adjunct to pathology for imaging tumor margins to guide surgery.

  3. Lossless compression of JPEG2000 whole slide images is not required for diagnostic virtual microscopy.

    Science.gov (United States)

    Kalinski, Thomas; Zwönitzer, Ralf; Grabellus, Florian; Sheu, Sien-Yi; Sel, Saadettin; Hofmann, Harald; Roessner, Albert

    2011-12-01

    The use of lossy compression in medical imaging is controversial, although it is inevitable to reduce large data amounts. In contrast with lossy compression, lossless compression does not impair image quality. In addition to our previous studies, we evaluated virtual 3-dimensional microscopy using JPEG2000 whole slide images of gastric biopsy specimens with or without Helicobacter pylori gastritis using lossless compression (1:1) or lossy compression with different compression levels: 5:1, 10:1, and 20:1. The virtual slides were diagnosed in a blinded manner by 3 pathologists using the updated Sydney classification. The results showed no significant differences in the diagnosis of H pylori between the different levels of compression in virtual microscopy. We assume that lossless compression is not required for diagnostic virtual microscopy. The limits of lossy compression in virtual microscopy without a loss of diagnostic quality still need to be determined. Analogous to the processes in radiology, recommendations for the use of lossy compression in diagnostic virtual microscopy have to be worked out by pathology societies.

  4. Fluorescence lifetime imaging for the characterization of the biochemical composition of atherosclerotic plaques

    Science.gov (United States)

    Phipps, Jennifer; Sun, Yinghua; Saroufeem, Ramez; Hatami, Nisa; Fishbein, Michael C.; Marcu, Laura

    2011-09-01

    This study investigates the ability of a flexible fiberoptic-based fluorescence lifetime imaging microscopy (FLIM) technique to resolve biochemical features in plaque fibrotic cap associated with plaque instability and based solely on fluorescence decay characteristics. Autofluorescence of atherosclerotic human aorta (11 autopsy samples) was measured at 48 locations through two filters, F377: 377/50 and F460: 460/60 nm (center wavelength/bandwidth). The fluorescence decay dynamic was described by average lifetime (τ) and four Laguerre coefficients (LECs) retrieved through a Laguerre deconvolution technique. FLIM-derived parameters discriminated between four groups [elastin-rich (ER), elastin and macrophage-rich (E+M), collagen-rich (CR), and lipid-rich (LR)]. For example, τF377 discriminated ER from CR (R = 0.84); τF460 discriminated E+M from CR and ER (R = 0.60 and 0.54, respectively); LEC-1F377 discriminated CR from LR and E+M (R = 0.69 and 0.77, respectively); P 87% (all cases) and sensitivity as high as 86%. Current results demonstrate for the first time that clinically relevant features (e.g., ratios of lipid versus collagen versus elastin) can be evaluated with a flexible-fiber based FLIM technique without the need for fluorescence intensity information or contrast agents.

  5. Three-dimensional microscopy and sectional image reconstruction using optical scanning holography.

    Science.gov (United States)

    Lam, Edmund Y; Zhang, Xin; Vo, Huy; Poon, Ting-Chung; Indebetouw, Guy

    2009-12-01

    Fast acquisition and high axial resolution are two primary requirements for three-dimensional microscopy. However, they are sometimes conflicting: imaging modalities such as confocal imaging can deliver superior resolution at the expense of sequential acquisition at different axial planes, which is a time-consuming process. Optical scanning holography (OSH) promises to deliver a good trade-off between these two goals. With just a single scan, we can capture the entire three-dimensional volume in a digital hologram; the data can then be processed to obtain the individual sections. An accurate modeling of the imaging system is key to devising an appropriate image reconstruction algorithm, especially for real data where random noise and other imaging imperfections must be taken into account. In this paper we demonstrate sectional image reconstruction by applying an inverse imaging sectioning technique to experimental OSH data of biological specimens and visualizing the sections using the OSA Interactive Science Publishing software.

  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. Coherent anti-stokes Raman scattering microscopy: chemical imaging for biology and medicine.

    Science.gov (United States)

    Evans, Conor L; Xie, X Sunney

    2008-01-01

    Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free imaging technique that is capable of real-time, nonperturbative examination of living cells and organisms based on molecular vibrational spectroscopy. Recent advances in detection schemes, understanding of contrast mechanisms, and developments of laser sources have enabled superb sensitivity and high time resolution. Emerging applications, such as metabolite and drug imaging and tumor identification, raise many exciting new possibilities for biology and medicine.

  8. Fluorescence microscopy studies of a peripheral-benzodiazepine-receptor-targeted molecular probe for brain tumor imaging

    Science.gov (United States)

    Marcu, Laura; Vernier, P. Thomas; Manning, H. Charles; Salemi, Sarah; Li, Aimin; Craft, Cheryl M.; Gundersen, Martin A.; Bornhop, Darryl J.

    2003-10-01

    This study investigates the potential of a new multi-modal lanthanide chelate complex for specifically targeting brain tumor cells. We report here results from ongoing studies of up-take, sub-cellular localization and binding specificity of this new molecular imaging probe. Fluorescence microscopy investigations in living rat C6 glioma tumor cells demonstrate that the new imaging agent has affinity for glioma cells and binds to mitochondria.

  9. Joint denoising and distortion correction of atomic scale scanning transmission electron microscopy images

    OpenAIRE

    Berkels, Benjamin; Wirth, Benedikt

    2016-01-01

    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 th...

  10. 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 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.

  11. 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.

  12. 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 a...

  13. EVOLUTION OF MICROSTRUCTURE IN LASER CLAD COATINGS STUDIED BY ORIENTATION IMAGING MICROSCOPY

    NARCIS (Netherlands)

    Ocelik, Vaclav; Furar, Ivan; De Hosson, Jeff Th. M.

    2010-01-01

    Laser powder deposition of thick metallic coatings is a surface engineering technique that provides coatings resistant against high loading impact, severe wear and corrosion at high temperatures. In this work Orientation Imaging Microscopy (OIM) based on Electron Back Scatter Diffraction in a Scanni

  14. Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy.

    Science.gov (United States)

    Lauterbach, Marcel A; Ronzitti, Emiliano; Sternberg, Jenna R; Wyart, Claire; Emiliani, Valentina

    2015-01-01

    Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.

  15. 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

  16. Atomic scale imaging of hydroxyapatite and brushite in air by force microscopy

    Science.gov (United States)

    Siperko, Lorraine M.; Landis, William J.

    1992-11-01

    A method for obtaining atomic scale images of powder samples by force microscopy has been used to determine surface structures of hydroxyapatite and brushite. From isolated hydroxyapatite crystal clusters, two crystal planes have been identified. The and spacings obtained agree well with published crystallographic values. Groups of brushite platelets yielded atomic spacings which are presumed to be those of the crystal plane.

  17. A Study of the Probe Effect on the Apparent Image of Biological Atomic Force Microscopy

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The probe effect on the apparent image of biological atomic force microscopy was explored in this study, and the potential of AFM in conformational study of gene related biological processes was illustrated by the specific nanostructural information of a new antitumor drug binding to DNA.

  18. 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 fermente...

  19. A MONTE CARLO SIMULATION OF SECONDARY ELECTRON AND BACKSCATTERED ELECTRON IMAGES IN SCANNING ELECTRON MICROSCOPY

    Institute of Scientific and Technical Information of China (English)

    H.M. Li; Z.J. Ding

    2005-01-01

    A new parallel Monte Carlo simulation method of secondary electron (SE) and backscattered electron images (BSE) of scanning electron microscopy (SEM) for a complex geometric structure has been developed. This paper describes briefly the simulation method and the modification to the conventional sampling method for the step length. Example simulation results have been obtained for several artificial structures.

  20. EVOLUTION OF MICROSTRUCTURE IN LASER CLAD COATINGS STUDIED BY ORIENTATION IMAGING MICROSCOPY

    NARCIS (Netherlands)

    Ocelik, Vaclav; Furar, Ivan; De Hosson, Jeff Th. M.

    2010-01-01

    Laser powder deposition of thick metallic coatings is a surface engineering technique that provides coatings resistant against high loading impact, severe wear and corrosion at high temperatures. In this work Orientation Imaging Microscopy (OIM) based on Electron Back Scatter Diffraction in a

  1. 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

    2016-03-01

    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.

  2. Live imaging of nervous system development and function using light-sheet microscopy.

    Science.gov (United States)

    Lemon, William C; Keller, Philipp J

    2015-01-01

    In vivo imaging applications typically require carefully balancing conflicting parameters. Often it is necessary to achieve high imaging speed, low photo-bleaching, and photo-toxicity, good three-dimensional resolution, high signal-to-noise ratio, and excellent physical coverage at the same time. Light-sheet microscopy provides good performance in all of these categories, and is thus emerging as a particularly powerful live imaging method for the life sciences. We see an outstanding potential for applying light-sheet microscopy to the study of development and function of the early nervous system in vertebrates and higher invertebrates. Here, we review state-of-the-art approaches to live imaging of early development, and show how the unique capabilities of light-sheet microscopy can further advance our understanding of the development and function of the nervous system. We discuss key considerations in the design of light-sheet microscopy experiments, including sample preparation and fluorescent marker strategies, and provide an outlook for future directions in the field.

  3. EVOLUTION OF MICROSTRUCTURE IN LASER CLAD COATINGS STUDIED BY ORIENTATION IMAGING MICROSCOPY

    NARCIS (Netherlands)

    Ocelik, Vaclav; Furar, Ivan; De Hosson, Jeff Th. M.

    2010-01-01

    Laser powder deposition of thick metallic coatings is a surface engineering technique that provides coatings resistant against high loading impact, severe wear and corrosion at high temperatures. In this work Orientation Imaging Microscopy (OIM) based on Electron Back Scatter Diffraction in a Scanni

  4. Applying two-photon excitation fluorescence lifetime imaging microscopy to study photosynthesis in plant leaves

    NARCIS (Netherlands)

    Broess, K.; Borst, J.W.; Amerongen, van H.

    2009-01-01

    This study investigates to which extent two-photon excitation (TPE) fluorescence lifetime imaging microscopy can be applied to study picosecond fluorescence kinetics of individual chloroplasts in leaves. Using femtosecond 860 nm excitation pulses, fluorescence lifetimes can be measured in leaves of

  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

    2015-11-01

    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. 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

  7. 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

  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. Parallel deconvolution of large 3D images obtained by confocal laser scanning microscopy.

    Science.gov (United States)

    Pawliczek, Piotr; Romanowska-Pawliczek, Anna; Soltys, Zbigniew

    2010-03-01

    Various deconvolution algorithms are often used for restoration of digital images. Image deconvolution is especially needed for the correction of three-dimensional images obtained by confocal laser scanning microscopy. Such images suffer from distortions, particularly in the Z dimension. As a result, reliable automatic segmentation of these images may be difficult or even impossible. Effective deconvolution algorithms are memory-intensive and time-consuming. In this work, we propose a parallel version of the well-known Richardson-Lucy deconvolution algorithm developed for a system with distributed memory and implemented with the use of Message Passing Interface (MPI). It enables significantly more rapid deconvolution of two-dimensional and three-dimensional images by efficiently splitting the computation across multiple computers. The implementation of this algorithm can be used on professional clusters provided by computing centers as well as on simple networks of ordinary PC machines.

  10. Deep-brain imaging via epi-fluorescence Computational Cannula Microscopy

    Science.gov (United States)

    Kim, Ganghun; Nagarajan, Naveen; Pastuzyn, Elissa; Jenks, Kyle; Capecchi, Mario; Shepherd, Jason; Menon, Rajesh

    2017-03-01

    Here we demonstrate widefield (field diameter = 200 μm) fluorescence microscopy and video imaging inside the rodent brain at a depth of 2 mm using a simple surgical glass needle (cannula) of diameter 0.22 mm as the primary optical element. The cannula guides excitation light into the brain and the fluorescence signal out of the brain. Concomitant image-processing algorithms are utilized to convert the spatially scrambled images into fluorescent images and video. The small size of the cannula enables minimally invasive imaging, while the long length (>2 mm) allow for deep-brain imaging with no additional complexity in the optical system. Since no scanning is involved, widefield fluorescence video at the native frame rate of the camera can be achieved.

  11. Enhanced image reconstruction of three-dimensional fluorescent assays by subtractive structured-light illumination microscopy.

    Science.gov (United States)

    Choi, Jong-ryul; Kim, Donghyun

    2012-10-01

    We investigate improved image reconstruction of structured light illumination for high-resolution imaging of three-dimensional (3D) cell-based assays. For proof of concept, an in situ fluorescence optical detection system was built with a digital micromirror device as a spatial light modulator, for which phase and tilting angle in a grid pattern were varied to implement specific image reconstruction schemes. Subtractive reconstruction algorithms based on structured light illumination were used to acquire images of fluorescent microbeads deposited as a two-dimensional monolayer or in 3D alginate matrix. We have confirmed that an optical subtraction algorithm improves axial and lateral resolution by effectively removing out-of-focus fluorescence. The results suggest that subtractive image reconstruction can be useful for structured illumination microscopy of broad types of cell-based assays with high image resolution.

  12. Deep-brain imaging via epi-fluorescence Computational Cannula Microscopy

    Science.gov (United States)

    Kim, Ganghun; Nagarajan, Naveen; Pastuzyn, Elissa; Jenks, Kyle; Capecchi, Mario; Shepherd, Jason; Menon, Rajesh

    2017-01-01

    Here we demonstrate widefield (field diameter = 200 μm) fluorescence microscopy and video imaging inside the rodent brain at a depth of 2 mm using a simple surgical glass needle (cannula) of diameter 0.22 mm as the primary optical element. The cannula guides excitation light into the brain and the fluorescence signal out of the brain. Concomitant image-processing algorithms are utilized to convert the spatially scrambled images into fluorescent images and video. The small size of the cannula enables minimally invasive imaging, while the long length (>2 mm) allow for deep-brain imaging with no additional complexity in the optical system. Since no scanning is involved, widefield fluorescence video at the native frame rate of the camera can be achieved. PMID:28317915

  13. 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

    2016-03-01

    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.

  14. Label-free imaging of gold nanoparticles in single live cells by photoacoustic microscopy

    Science.gov (United States)

    Tian, Chao; Qian, Wei; Shao, Xia; Xie, Zhixing; Cheng, Xu; Liu, Shengchun; Cheng, Qian; Liu, Bing; Wang, Xueding

    2016-03-01

    Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.

  15. Non-invasive imaging of skin cancer with fluorescence lifetime imaging using two photon tomography

    Science.gov (United States)

    Patalay, Rakesh; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Breunig, Hans Georg; König, Karsten; Warren, Sean; Neil, Mark A. A.; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Christopher

    2011-07-01

    Multispectral fluorescence lifetime imaging (FLIM) using two photon microscopy as a non-invasive technique for the diagnosis of skin lesions is described. Skin contains fluorophores including elastin, keratin, collagen, FAD and NADH. This endogenous contrast allows tissue to be imaged without the addition of exogenous agents and allows the in vivo state of cells and tissues to be studied. A modified DermaInspect® multiphoton tomography system was used to excite autofluorescence at 760 nm in vivo and on freshly excised ex vivo tissue. This instrument simultaneously acquires fluorescence lifetime images in four spectral channels between 360-655 nm using time-correlated single photon counting and can also provide hyperspectral images. The multispectral fluorescence lifetime images were spatially segmented and binned to determine lifetimes for each cell by fitting to a double exponential lifetime model. A comparative analysis between the cellular lifetimes from different diagnoses demonstrates significant diagnostic potential.

  16. From morphology to clinical pathophysiology: multiphoton fluorescence lifetime imaging at patients' bedside

    Science.gov (United States)

    Mess, Christian; Zens, Katharina; Gorzelanny, Christian; Metze, Dieter; Luger, Thomas A.; König, Karsten; Schneider, Stefan W.; Huck, Volker

    2017-02-01

    Application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of skin diseases. By means of multiphoton excitation, endogenous biomolecules like NADH, collagen or elastin show autofluorescence or second harmonic generation. Thus, these molecules provide information about the subcellular morphology, epidermal architecture and physiological condition of the skin. To gain a deeper understanding of the linkage between cellular structure and physiological processes, non-invasive multiphotonbased intravital tomography (MPT) and fluorescence lifetime imaging (FLIM) were combined within the scopes of inflammatory skin, chronic wounds and drug delivery in clinical application. The optical biopsies generated via MPT were morphologically analyzed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Independent morphometric algorithms reliably showed a perinuclear accumulation in lesional skin in contrast to an even distribution in healthy skin. Confirmatively, MPT-FLIM showed an obvious metabolic shift in lesions. Moreover, detection of the onset and progression of inflammatory processes could be achieved. The feasibility of primary in vivo tracking of applied therapeutic agents further broadened our scope: We examined the permeation and subsequent distribution of agents directly visualized in patientś skin in short-term repetitive measurements. Furthermore, we performed MPT-FLIM follow-up investigations in the long-term course of therapy. Therefore, clinical MPT-FLIM application offers new insights into the pathophysiology and the individual therapeutic course of skin diseases, facilitating a better understanding of the processes of inflammation and wound healing.

  17. Scanning superlens microscopy for non-invasive large field-of-view visible light nanoscale imaging

    Science.gov (United States)

    Wang, Feifei; Liu, Lianqing; Yu, Haibo; Wen, Yangdong; Yu, Peng; Liu, Zhu; Wang, Yuechao; Li, Wen Jung

    2016-12-01

    Nanoscale correlation of structural information acquisition with specific-molecule identification provides new insight for studying rare subcellular events. To achieve this correlation, scanning electron microscopy has been combined with super-resolution fluorescent microscopy, despite its destructivity when acquiring biological structure information. Here we propose time-efficient non-invasive microsphere-based scanning superlens microscopy that enables the large-area observation of live-cell morphology or sub-membrane structures with sub-diffraction-limited resolution and is demonstrated by observing biological and non-biological objects. This microscopy operates in both non-invasive and contact modes with ~200 times the acquisition efficiency of atomic force microscopy, which is achieved by replacing the point of an atomic force microscope tip with an imaging area of microspheres and stitching the areas recorded during scanning, enabling sub-diffraction-limited resolution. Our method marks a possible path to non-invasive cell imaging and simultaneous tracking of specific molecules with nanoscale resolution, facilitating the study of subcellular events over a total cell period.

  18. Perfect optical vortex enhanced surface plasmon excitation for plasmonic structured illumination microscopy imaging

    Science.gov (United States)

    Zhang, Chonglei; Min, Changjun; Du, Luping; Yuan, X.-C.

    2016-05-01

    We propose an all-optical technique for plasmonic structured illumination microscopy (PSIM) with perfect optical vortex (POV). POV can improve the efficiency of the excitation of surface plasma and reduce the background noise of the excited fluorescence. The plasmonic standing wave patterns are excited by POV with fractional topological charges for accurate phase shift of {-2π/3, 0, and 2π/3}. The imaging resolution of less than 200 nm was produced. This PSIM technique is expected to be used as a wide field, super resolution imaging technique in dynamic biological imaging.

  19. Origin and compensation of imaging artefacts in localization-based super-resolution microscopy.

    Science.gov (United States)

    Erdélyi, M; Sinkó, J; Kákonyi, R; Kelemen, A; Rees, E; Varga, D; Szabó, G

    2015-10-15

    Interpretation of high resolution images provided by localization-based microscopy techniques is a challenge due to imaging artefacts that can be categorized by their origin. They can be introduced by the optical system, by the studied sample or by the applied algorithms. Some artefacts can be eliminated via precise calibration procedures, others can be reduced only below a certain value. Images studied both theoretically and experimentally are qualified either by pattern specific metrics or by a more general metric based on fluorescence correlation spectroscopy.

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

    Energy Technology Data Exchange (ETDEWEB)

    Findlay, S.D., E-mail: scott@sigma.t.u-tokyo.ac.jp [Institute of Engineering Innovation, The University of Tokyo, Tokyo 116-0013 (Japan); Shibata, N. [Institute of Engineering Innovation, The University of Tokyo, Tokyo 116-0013 (Japan); PRESTO, Japan Science and Technology Agency, Saitama 332-0012 (Japan); Sawada, H.; Okunishi, E.; Kondo, Y. [JEOL Ltd., Tokyo 196-8558 (Japan); Ikuhara, Y. [Institute of Engineering Innovation, The University of Tokyo, Tokyo 116-0013 (Japan); Nanostructures Research Laboratory, Japan Fine Ceramics Center, Nagoya 456-8587 (Japan); WPI Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577 (Japan)

    2010-06-15

    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.

  1. 22 nm node wafer inspection using diffraction phase microscopy and image post-processing

    Science.gov (United States)

    Zhou, Renjie; Popescu, Gabriel; Goddard, Lynford L.

    2013-04-01

    We applied epi-illumination diffraction phase microscopy to measure the amplitude and phase of the scattered field from a SEMATECH 22 nm node intentional defect array (IDA) wafer. We used several imaging processing techniques to remove the wafer's underlying structure and reduce both the spatial and temporal noise and eliminate the system calibration error to produce stretched panoramic amplitude and phase images. From the stretched images, we detected defects down to 20 nm × 160 nm for a parallel bridge, 20 nm × 100 nm for perpendicular bridge, and 35 nm × 70 nm for an isolated dot.

  2. Submolecular Resolution Imaging of Molecules by Atomic Force Microscopy: The Influence of the Electrostatic Force

    Science.gov (United States)

    van der Lit, Joost; Di Cicco, Francesca; Hapala, Prokop; Jelinek, Pavel; Swart, Ingmar

    2016-03-01

    The forces governing the contrast in submolecular resolution imaging of molecules with atomic force microscopy (AFM) have recently become a topic of intense debate. Here, we show that the electrostatic force is essential to understand the contrast in atomically resolved AFM images of polar molecules. Specifically, we image strongly polarized molecules with negatively and positively charged tips. A contrast inversion is observed above the polar groups. By taking into account the electrostatic forces between tip and molecule, the observed contrast differences can be reproduced using a molecular mechanics model. In addition, we analyze the height dependence of the various force components contributing to the high-resolution AFM contrast.

  3. 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.

  4. Advances in electron microscopy: A qualitative view of instrumentation development for macromolecular imaging and tomography.

    Science.gov (United States)

    Schröder, Rasmus R

    2015-09-01

    Macromolecular imaging and tomography of ice embedded samples has developed into a mature imaging technology, in structural biology today widely referred to simply as cryo electron microscopy.(1) While the pioneers of the technique struggled with ill-suited instruments, state-of-the-art cryo microscopes are now readily available and an increasing number of groups are producing excellent high-resolution structural data of macromolecular complexes, of cellular organelles, or the morphology of whole cells. Instrumentation developers, however, are offering yet more novel electron optical devices, such as energy filters and monochromators, aberration correctors or physical phase plates. Here we discuss how current instrumentation has already changed cryo EM, and how newly available instrumentation - often developed in other fields of electron microscopy - may further develop the use and applicability of cryo EM to the imaging of single isolated macromolecules of smaller size or molecules embedded in a crowded cellular environment.

  5. 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.

  6. 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.

  7. Tip radius preservation for high resolution imaging in amplitude modulation atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Ramos, Jorge R., E-mail: jorge.rr@cea.cu [Instituto de Ciencia de Materiales de Madrid, Sor Juana Inés de la Cruz 3, Canto Blanco, 28049 Madrid, España (Spain)

    2014-07-28

    The acquisition of high resolution images in atomic force microscopy (AFM) is correlated to the cantilever's tip shape, size, and imaging conditions. In this work, relative tip wear is quantified based on the evolution of a direct experimental observable in amplitude modulation atomic force microscopy, i.e., the critical amplitude. We further show that the scanning parameters required to guarantee a maximum compressive stress that is lower than the yield/fracture stress of the tip can be estimated via experimental observables. In both counts, the optimized parameters to acquire AFM images while preserving the tip are discussed. The results are validated experimentally by employing IgG antibodies as a model system.

  8. Selective-plane illumination microscopy for high-content volumetric biological imaging

    Science.gov (United States)

    McGorty, Ryan; Huang, Bo

    2016-03-01

    Light-sheet microscopy, also named selective-plane illumination microscopy, enables optical sectioning with minimal light delivered to the sample. Therefore, it allows one to gather volumetric datasets of developing embryos and other light-sensitive samples over extended times. We have configured a light-sheet microscope that, unlike most previous designs, can image samples in formats compatible with high-content imaging. Our microscope can be used with multi-well plates or with microfluidic devices. In designing our optical system to accommodate these types of sample holders we encounter large optical aberrations. We counter these aberrations with both static optical components in the imaging path and with adaptive optics. Potential applications of this microscope include studying the development of a large number of embryos in parallel and over long times with subcellular resolution and doing high-throughput screens on organisms or cells where volumetric data is necessary.

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

    Science.gov (United States)

    Xie, Xiaoliang Sunney; Freudiger, Christian; Min, Wei

    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.

  10. Total 3D imaging of phase objects using defocusing microscopy: application to red blood cells

    CERN Document Server

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

    2014-01-01

    We present Defocusing Microscopy (DM), a bright-field optical microscopy technique able to perform total 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 new methodology using DM and apply it to red blood cells subject to different osmolality conditions: hypotonic, isotonic and hypertonic solutions. For each situation the shape of the upper and lower cell surface-membranes (lipid bilayer/cytoskeleton) are completely recovered, displaying the deformation of RBCs 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 volume, superficial area, sphericity index and RBCs refractive index for each osmotic condition.

  11. Label-free imaging of cellular malformation using high resolution photoacoustic microscopy

    Science.gov (United States)

    Chen, Zhongjiang; Li, Bingbing; Yang, Sihua

    2014-09-01

    A label-free high resolution photoacoustic microscopy (PAM) system for imaging cellular malformation is presented. The carbon fibers were used to testify the lateral resolution of the PAM. Currently, the lateral resolution is better than 2.7 μm. The human normal red blood cells (RBCs) were used to prove the imaging capability of the system, and a single red blood cell was mapped with high contrast. Moreover, the iron deficiency anemia RBCs were clearly distinguished from the cell morphology by using the PAM. The experimental results demonstrate that the photoacoustic microscopy system can accomplish label-free photoacoustic imaging and that it has clinical potential for use in the detection of erythrocytes and blood vessels malformation.

  12. 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.

  13. 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.

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

    Science.gov (United States)

    Skytte, Jacob L; Ghita, Ovidiu; Whelan, Paul F; Andersen, Ulf; Møller, Flemming; Dahl, Anders B; Larsen, Rasmus

    2015-06-01

    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 dairy products. When studying such networks, hundreds of images can be obtained, and here image analysis methods are essential for using the images in statistical analysis. Previously, methods including gray level co-occurrence matrix analysis and fractal analysis have been used with success. However, a range of other image texture characterization methods exists. These methods describe an image by a frequency distribution of predefined image features (denoted textons). Our contribution is an investigation of the choice of image analysis methods by performing a comparative study of 7 major approaches 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, and cluster analysis. Our investigation suggests that the texton-based descriptors provide a fuller description of the images compared to gray-level co-occurrence matrix descriptors and fractal analysis, while still being as applicable and in some cases as easy to tune.

  15. Label-free imaging of human breast tissues using coherent anti-Stokes Raman scattering microscopy

    Science.gov (United States)

    Yang, Yaliang; Gao, Liang; Wang, Zhiyong; Thrall, Michael J.; Luo, Pengfei; Wong, Kelvin K.; Wong, Stephen T.

    2011-03-01

    Breast cancer is a common disease in women. Current imaging and diagnostic methods for breast cancer confront several limitations, like time-consuming, invasive and with a high cost. Alternative strategies are in high demand to alleviate patients' trauma and lower medical expenses. Coherent anti-Stokes Raman scattering (CARS) imaging technique offers many advantages, including label-free, sub-wavelength spatial resolution and video-rate imaging speed. Therefore, it has been demonstrated as a powerful tool for various biomedical applications. In this study, we present a label-free fast imaging method to identify breast cancer and its subtypes using CARS microscopy. Human breast tissues, including normal, benign and invasive carcinomas, were imaged ex vivo using a custom-built CARS microscope. Compared with results from corresponding hematoxylin and eosin (H&E) stains, the CARS technique has demonstrated its capability in identifying morphological features in a similar way as in H&E stain. These features can be used to distinguish breast cancer from normal and benign tissues, and further separate cancer subtypes from each other. Our pilot study suggests that CARS microscopy could be used as a routine examination tool to characterize breast cancer ex vivo. Moreover, its label-free and fast imaging properties render this technique as a promising approach for in vivo and real-time imaging and diagnosis of breast cancer.

  16. Accumulative difference image protocol for particle tracking in fluorescence microscopy tested in mouse lymphonodes.

    Science.gov (United States)

    Villa, Carlo E; Caccia, Michele; Sironi, Laura; D'Alfonso, Laura; Collini, Maddalena; Rivolta, Ilaria; Miserocchi, Giuseppe; Gorletta, Tatiana; Zanoni, Ivan; Granucci, Francesca; Chirico, Giuseppe

    2010-08-17

    The basic research in cell biology and in medical sciences makes large use of imaging tools mainly based on confocal fluorescence and, more recently, on non-linear excitation microscopy. Substantially the aim is the recognition of selected targets in the image and their tracking in time. We have developed a particle tracking algorithm optimized for low signal/noise images with a minimum set of requirements on the target size and with no a priori knowledge of the type of motion. The image segmentation, based on a combination of size sensitive filters, does not rely on edge detection and is tailored for targets acquired at low resolution as in most of the in-vivo studies. The particle tracking is performed by building, from a stack of Accumulative Difference Images, a single 2D image in which the motion of the whole set of the particles is coded in time by a color level. This algorithm, tested here on solid-lipid nanoparticles diffusing within cells and on lymphocytes diffusing in lymphonodes, appears to be particularly useful for the cellular and the in-vivo microscopy image processing in which few a priori assumption on the type, the extent and the variability of particle motions, can be done.

  17. Accumulative difference image protocol for particle tracking in fluorescence microscopy tested in mouse lymphonodes.

    Directory of Open Access Journals (Sweden)

    Carlo E Villa

    Full Text Available The basic research in cell biology and in medical sciences makes large use of imaging tools mainly based on confocal fluorescence and, more recently, on non-linear excitation microscopy. Substantially the aim is the recognition of selected targets in the image and their tracking in time. We have developed a particle tracking algorithm optimized for low signal/noise images with a minimum set of requirements on the target size and with no a priori knowledge of the type of motion. The image segmentation, based on a combination of size sensitive filters, does not rely on edge detection and is tailored for targets acquired at low resolution as in most of the in-vivo studies. The particle tracking is performed by building, from a stack of Accumulative Difference Images, a single 2D image in which the motion of the whole set of the particles is coded in time by a color level. This algorithm, tested here on solid-lipid nanoparticles diffusing within cells and on lymphocytes diffusing in lymphonodes, appears to be particularly useful for the cellular and the in-vivo microscopy image processing in which few a priori assumption on the type, the extent and the variability of particle motions, can be done.

  18. Potential of ultraviolet widefield imaging and multiphoton microscopy for analysis of dehydroergosterol in cellular membranes

    DEFF Research Database (Denmark)

    Wüstner, Daniel; Brewer, Jonathan R.; Bagatolli, Luis

    2011-01-01

    Dehydroergosterol (DHE) is an intrinsically fluorescent sterol with absorption/emission in the ultraviolet (UV) region and biophysical properties similar to those of cholesterol. We compared the potential of UV-sensitive low-light-level wide-field (UV-WF) imaging with that of multiphoton (MP...... found that the lateral resolution of MP microscopy is ∼1.5-fold higher than that of UV-WF deconvolution microscopy, allowing for improved spatiotemporal analysis of plasma membrane sterol distribution. Surface intensity patterns of DHE with a diameter of 0.2 μm persisting over several minutes could...

  19. Interferometric backward third harmonic generation microscopy for axial imaging with accuracy beyond the diffraction limit.

    Directory of Open Access Journals (Sweden)

    Daaf Sandkuijl

    Full Text Available A new nonlinear microscopy technique based on interference of backward-reflected third harmonic generation (I-THG from multiple interfaces is presented. The technique is used to measure height variations or changes of a layer thickness with an accuracy of up to 5 nm. Height variations of a patterned glass surface and thickness variations of fibroblasts are visualized with the interferometric epi-THG microscope with an accuracy at least two orders of magnitude better than diffraction limit. The microscopy technique can be broadly applied for measuring distance variations between membranes or multilayer structures inside biological tissue and for surface height variation imaging.

  20. 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.

  1. Enhanced CellClassifier: a multi-class classification tool for microscopy images

    Directory of Open Access Journals (Sweden)

    Horvath Peter

    2010-01-01

    Full Text Available Abstract Background Light microscopy is of central importance in cell biology. The recent introduction of automated high content screening has expanded this technology towards automation of experiments and performing large scale perturbation assays. Nevertheless, evaluation of microscopy data continues to be a bottleneck in many projects. Currently, among open source software, CellProfiler and its extension Analyst are widely used in automated image processing. Even though revolutionizing image analysis in current biology, some routine and many advanced tasks are either not supported or require programming skills of the researcher. This represents a significant obstacle in many biology laboratories. Results We have developed a tool, Enhanced CellClassifier, which circumvents this obstacle. Enhanced CellClassifier starts from images analyzed by CellProfiler, and allows multi-class classification using a Support Vector Machine algorithm. Training of objects can be done by clicking directly "on the microscopy image" in several intuitive training modes. Many routine tasks like out-of focus exclusion and well summary are also supported. Classification results can be integrated with other object measurements including inter-object relationships. This makes a detailed interpretation of the image possible, allowing the differentiation of many complex phenotypes. For the generation of the output, image, well and plate data are dynamically extracted and summarized. The output can be generated as graphs, Excel-files, images with projections of the final analysis and exported as variables. Conclusion Here we describe Enhanced CellClassifier which allows multiple class classification, elucidating complex phenotypes. Our tool is designed for the biologist who wants both, simple and flexible analysis of images without requiring programming skills. This should facilitate the implementation of automated high-content screening.

  2. X-ray microscopy as an approach to increasing accuracy and efficiency of serial block-face imaging for correlated light and electron microscopy of biological specimens.

    Science.gov (United States)

    Bushong, Eric A; Johnson, Donald D; Kim, Keun-Young; Terada, Masako; Hatori, Megumi; Peltier, Steven T; Panda, Satchidananda; Merkle, Arno; Ellisman, Mark H

    2015-02-01

    The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging.

  3. Comparison of calcium imaging in dorsal root ganglion neurons by using laser scanning confocal and two-photon microscopy

    Science.gov (United States)

    Huang, Yimei; Yang, Hongqin; Chen, Jiangxu; Shen, Xiuqiu; Zheng, Liqin; Wang, Yuhua; Xie, Shusen

    2012-03-01

    As one of the most important second messengers, calcium in nerve cells plays a critical role in neuronal processes, including excitability, neurotransmitter release, synaptic plasticity. Modulation of the calcium concentration is an important means of regulating diverse neuronal functions. To evaluate the role of calcium, quantitative measurement of cytosolic free calcium concentrations is necessary. There are several optical techniques that are available for measurement of calcium in live cells. Laser scanning confocal microscopy and two-photon microscopy are two prevalent techniques for their advantage in spatial resolution. In this paper, calcium in dorsal root ganglion neurons was imaged by laser scanning confocal microscopy and two-photon microscopy with Fluo-3, a calcium specific fluorescence probe. Both of spatial resolution and photobleaching, two common limitations of optical image modality, were compared between laser scanning confocal microscopy and two-photon microscopy, respectively. Three dimension images showed that laser scanning confocal microscopy and two-photon microscopy had not only similar lateral resolution but also parallel vertical resolution. However, Laser scanning confocal microscopy had an advantage over the two-photon microcopy in photobleaching. These results indicated that laser scanning confocal microscopy was more suitable than two-photon microscopy to be applied in imaging calcium in dorsal root ganglion neurons with Fluo-3.

  4. Soft X-ray Tomography and Cryogenic Light Microscopy: The Cool Combination in Cellular Imaging

    Science.gov (United States)

    McDermott, Gerry; Le Gros, Mark A.; Knoechel, Christian G.; Uchida, Maho; Larabell, Carolyn A.

    2012-01-01

    Soft x-ray tomography (SXT) is ideally suited to imaging sub-cellular architecture and organization, particularly in eukaryotic cells. SXT is similar in concept to the well-established medical diagnostic technique computed axial tomography (CAT), except SXT is capable of imaging with a spatial resolution of 50 nm, or better. In soft x-ray tomography (SXT) cells are imaged using photons from a region of the spectrum known as the ‘water window’. This results in quantitative, high-contrast images of intact, fully hydrated cells without the need to use contrast-enhancing agents. Cells are therefore visualized very close to their native, fully functional state. The utility of SXT has recently been enhanced by the development of high numerical aperture cryogenic light microscopy for correlated imaging. Taking this multi-modal approach now allows labeled molecules to be localized in the context of a high-resolution 3-dimensional tomographic reconstruction of the cell. PMID:19818625

  5. Motion artefact detection in structured illumination microscopy for live cell imaging.

    Science.gov (United States)

    Förster, Ronny; Wicker, Kai; Müller, Walter; Jost, Aurélie; Heintzmann, Rainer

    2016-09-19

    The reconstruction process of structured illumination microscopy (SIM) creates substantial artefacts if the specimen has moved during the acquisition. This reduces the applicability of SIM for live cell imaging, because these artefacts cannot always be recognized as such in the final image. A movement is not necessarily visible in the raw data, due to the varying excitation patterns and the photon noise. We present a method to detect motion by extracting and comparing two independent 3D wide-field images out of the standard SIM raw data without needing additional images. Their difference reveals moving objects overlaid with noise, which are distinguished by a probability theory-based analysis. Our algorithm tags motion-artefacts in the final high-resolution image for the first time, preventing the end-user from misinterpreting the data. We show and explain different types of artefacts and demonstrate our algorithm on a living cell.

  6. Label-free imaging of developing vasculature in zebrafish with phase variance optical coherence microscopy

    Science.gov (United States)

    Chen, Yu; Fingler, Jeff; Trinh, Le A.; Fraser, Scott E.

    2016-03-01

    A phase variance optical coherence microscope (pvOCM) has been created to visualize blood flow in the vasculature of zebrafish embryos, without using exogenous labels. The pvOCM imaging system has axial and lateral resolutions of 2 μm in tissue, and imaging depth of more than 100 μm. Imaging of 2-5 days post-fertilization zebrafish embryos identified the detailed structures of somites, spinal cord, gut and notochord based on intensity contrast. Visualization of the blood flow in the aorta, veins and intersegmental vessels was achieved with phase variance contrast. The pvOCM vasculature images were confirmed with corresponding fluorescence microscopy of a zebrafish transgene that labels the vasculature with green fluorescent protein. The pvOCM images also revealed functional information of the blood flow activities that is crucial for the study of vascular development.

  7. 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 sections. Characteristic features that suggest malignant lesions, such as complex papillary architecture, microfollicules, psammomatous calcifications, or replacement of normal follicular architecture with sheets/nests of tumor cells, can be identified from OCT and OCM images and are clearly differentiable from normal or benign 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.

  8. Dual tree complex wavelet transform based denoising of optical microscopy images.

    Science.gov (United States)

    Bal, Ufuk

    2012-12-01

    Photon shot noise is the main noise source of optical microscopy images and can be modeled by a Poisson process. Several discrete wavelet transform based methods have been proposed in the literature for denoising images corrupted by Poisson noise. However, the discrete wavelet transform (DWT) has disadvantages such as shift variance, aliasing, and lack of directional selectivity. To overcome these problems, a dual tree complex wavelet transform is used in our proposed denoising algorithm. Our denoising algorithm is based on the assumption that for the Poisson noise case threshold values for wavelet coefficients can be estimated from the approximation coefficients. Our proposed method was compared with one of the state of the art denoising algorithms. Better results were obtained by using the proposed algorithm in terms of image quality metrics. Furthermore, the contrast enhancement effect of the proposed method on collagen fıber images is examined. Our method allows fast and efficient enhancement of images obtained under low light intensity conditions.

  9. 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.

  10. Methodology for Quantitative Characterization of Fluorophore Photoswitching to Predict Superresolution Microscopy Image Quality

    Science.gov (United States)

    Bittel, Amy M.; Nickerson, Andrew; Saldivar, Isaac S.; Dolman, Nick J.; Nan, Xiaolin; Gibbs, Summer L.

    2016-07-01

    Single-molecule localization microscopy (SMLM) image quality and resolution strongly depend on the photoswitching properties of fluorophores used for sample labeling. Development of fluorophores with optimized photoswitching will considerably improve SMLM spatial and spectral resolution. Currently, evaluating fluorophore photoswitching requires protein-conjugation before assessment mandating specific fluorophore functionality, which is a major hurdle for systematic characterization. Herein, we validated polyvinyl alcohol (PVA) as a single-molecule environment to efficiently quantify the photoswitching properties of fluorophores and identified photoswitching properties predictive of quality SMLM images. We demonstrated that the same fluorophore photoswitching properties measured in PVA films and using antibody adsorption, a protein-conjugation environment analogous to labeled cells, were significantly correlated to microtubule width and continuity, surrogate measures of SMLM image quality. Defining PVA as a fluorophore photoswitching screening platform will facilitate SMLM fluorophore development and optimal image buffer assessment through facile and accurate photoswitching property characterization, which translates to SMLM fluorophore imaging performance.

  11. 4D scanning transmission ultrafast electron microscopy: Single-particle imaging and spectroscopy.

    Science.gov (United States)

    Ortalan, Volkan; Zewail, Ahmed H

    2011-07-20

    We report the development of 4D scanning transmission ultrafast electron microscopy (ST-UEM). The method was demonstrated in the imaging of silver nanowires and gold nanoparticles. For the wire, the mechanical motion and shape morphological dynamics were imaged, and from the images we obtained the resonance frequency and the dephasing time of the motion. Moreover, we demonstrate here the simultaneous acquisition of dark-field images and electron energy loss spectra from a single gold nanoparticle, which is not possible with conventional methods. The local probing capabilities of ST-UEM open new avenues for probing dynamic processes, from single isolated to embedded nanostructures, without being affected by the heterogeneous processes of ensemble-averaged dynamics. Such methodology promises to have wide-ranging applications in materials science and in single-particle biological imaging.

  12. A Comparison of Image Quality Evaluation Techniques for Transmission X-Ray Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Bolgert, Peter J; /Marquette U. /SLAC

    2012-08-31

    Beamline 6-2c at Stanford Synchrotron Radiation Lightsource (SSRL) is capable of Transmission X-ray Microscopy (TXM) at 30 nm resolution. Raw images from the microscope must undergo extensive image processing before publication. Since typical data sets normally contain thousands of images, it is necessary to automate the image processing workflow as much as possible, particularly for the aligning and averaging of similar images. Currently we align images using the 'phase correlation' algorithm, which calculates the relative offset of two images by multiplying them in the frequency domain. For images containing high frequency noise, this algorithm will align noise with noise, resulting in a blurry average. To remedy this we multiply the images by a Gaussian function in the frequency domain, so that the algorithm ignores the high frequency noise while properly aligning the features of interest (FOI). The shape of the Gaussian is manually tuned by the user until the resulting average image is sharpest. To automatically optimize this process, it is necessary for the computer to evaluate the quality of the average image by quantifying its sharpness. In our research we explored two image sharpness metrics, the variance method and the frequency threshold method. The variance method uses the variance of the image as an indicator of sharpness while the frequency threshold method sums up the power in a specific frequency band. These metrics were tested on a variety of test images, containing both real and artificial noise. To apply these sharpness metrics, we designed and built a MATLAB graphical user interface (GUI) called 'Blur Master.' We found that it is possible for blurry images to have a large variance if they contain high amounts of noise. On the other hand, we found the frequency method to be quite reliable, although it is necessary to manually choose suitable limits for the frequency band. Further research must be performed to design an

  13. 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.

  14. 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.

  15. 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.

  16. Development of carbon electrodes for electrochemistry, solid-state electronics and multimodal atomic force microscopy imaging

    Science.gov (United States)

    Morton, Kirstin Claire

    Carbon is one of the most remarkable elements due to its wide abundance on Earth and its many allotropes, which include diamond and graphite. Many carbon allotropes are conductive and in recent decades scientists have discovered and synthesized many new forms of carbon, including graphene and carbon nanotubes. The work in this thesis specifically focuses on the fabrication and characterization of pyrolyzed parylene C (PPC), a conductive pyrocarbon, as an electrode material for diodes, as a conductive coating for atomic force microscopy (AFM) probes and as an ultramicroelectrode (UME) for the electrochemical interrogation of cellular systems in vitro. Herein, planar and three-dimensional (3D) PPC electrodes were microscopically, spectroscopically and electrochemically characterized. First, planar PPC films and PPC-coated nanopipettes were utilized to detect a model redox species, Ru(NH3) 6Cl3. Then, free-standing PPC thin films were chemically doped, with hydrazine and concentrated nitric acid, to yield p- and n-type carbon films. Doped PPC thin films were positioned in conjunction with doped silicon to create Schottky and p-n junction diodes for use in an alternating current half-wave rectifier circuit. Pyrolyzed parylene C has found particular merit as a 3D electrode coating of AFM probes. Current sensing-atomic force microscopy imaging in air of nanoscale metallic features was undertaken to demonstrate the electronic imaging applicability of PPC AFM probes. Upon further insulation with parylene C and modification with a focused ion beam, a PPC UME was microfabricated near the AFM probe apex and utilized for electrochemical imaging. Subsequently, scanning electrochemical microscopy-atomic force microscopy imaging was undertaken to electrochemically quantify and image the spatial location of dopamine exocytotic release, elicited mechanically via the AFM probe itself, from differentiated pheochromocytoma 12 cells in vitro.

  17. 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.

  18. Photon flux requirements for EUV reticle imaging microscopy in the 22 and 16 nm nodes

    Energy Technology Data Exchange (ETDEWEB)

    Wintz, D.; Goldberg, K. A.; Mochi, I.; Huh, S.

    2010-03-12

    EUV-wavelength actinic microscopy yields detailed information about EUV mask patterns, architectures, defects, and the performance of defect repair strategies, without the complications of photoresist imaging. The measured aerial image intensity profiles provide valuable feedback to improve mask and lithography system modeling methods. In order to understand the photon-flux-dependent pattern measurement limits of EUV mask-imaging microscopy, we have investigated the effects of shot noise on aerial image linewidth measurements for lines in the 22 and 16-nm generations. Using a simple model of image formation near the resolution limit, we probe the influence of photon shot noise on the measured, apparent line roughness. With this methodology, we arrive at general flux density requirements independent of the specific EUV microscope configurations. Analytical and statistical analysis of aerial image simulations in the 22 and 16-nm generations reveal the trade-offs between photon energy density (controllable with exposure time), effective pixel dimension on the CCO (controlled by the microscope's magnification ratio), and image log slope (ILS). We find that shot-noise-induced linewidth roughness (LWR) varies imersely with the square root of the photon energy density, and is proportional to the imaging magnification ratio. While high magnification is necessary for adequate spatial resolution, for a given flux density, higher magnification ratios have diminishing benefits. With practical imaging parameters, we find that in order to achieve an LWR (3{sigma}) value of 5% of linewidth for dense, 88-nm mask features with 80% aerial image contrast and 13.5-nm effective pixel width (1000x magnification ratio), a peak photon flux of approximately 1400 photons per pixel per exposure is required.

  19. Acoustic Imaging Frequency Dynamics of Ferroelectric Domains by Atomic Force Microscopy

    Institute of Scientific and Technical Information of China (English)

    ZHAO Kun-Yu; Shunji Takekawa; Kenji Kitamura; ZENG Hua-Rong; SONG Hong-Zhang; HUI Sen-Xing; LI Guo-Rong; YIN Qing-Rui; Kiyoshi Shimamura; Chinna Venkadasamy Kannan; Encarnacion Antonia Garcia Villora

    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.h kHz by AFM-based acoustic microscopy. The frequency-dependent acoustic signal revealed a strong acoustic response in the frequency range from 7 kHz to lO kHz, and reached maximum at 8.1 kHz. 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.

  20. 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

  1. A novel multimodal optical imaging system for early detection of oral cancer

    Science.gov (United States)

    Malik, Bilal H.; Jabbour, Joey M.; Cheng, Shuna; Cuenca, Rodrigo; Cheng, Yi-Shing Lisa; Wright, John M.; Jo, Javier A.; Maitland, Kristen C.

    2015-01-01

    Objectives Several imaging techniques have been advocated as clinical adjuncts to improve identification of suspicious oral lesions. However, these have not yet shown superior sensitivity or specificity over conventional oral examination techniques. We developed a multimodal, multi-scale optical imaging system that combines macroscopic biochemical imaging of fluorescence lifetime imaging (FLIM) with subcellular morphologic imaging of reflectance confocal microscopy (RCM) for early detection of oral cancer. We tested our system on excised human oral tissues. Study Design A total of four tissue specimen were imaged. These specimens were diagnosed as one each: clinically normal, oral lichen planus, gingival hyperplasia, and superficially-invasive squamous cell carcinoma (SCC). The optical and fluorescence lifetime properties of each specimen were recorded. Results Both quantitative and qualitative differences between normal, benign and SCC lesions can be resolved with FLIM-RCM imaging. The results demonstrate that an integrated approach based on these two methods can potentially enable rapid screening and evaluation of large areas of oral epithelial tissue. Conclusions Early results from ongoing studies of imaging human oral cavity illustrate the synergistic combination of the two modalities. An adjunct device based on such optical characterization of oral mucosa can potentially be used to detect oral carcinogenesis in early stages. PMID:26725720

  2. 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

    2012-07-17

    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.

  3. Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy.

    Science.gov (United States)

    Bushby, Andrew J; P'ng, Kenneth M Y; Young, Robert D; Pinali, Christian; Knupp, Carlo; Quantock, Andrew J

    2011-06-01

    In this protocol, we describe a 3D imaging technique known as 'volume electron microscopy' or 'focused ion beam scanning electron microscopy (FIB/SEM)' applied to biological tissues. A scanning electron microscope equipped with a focused gallium ion beam, used to sequentially mill away the sample surface, and a backscattered electron (BSE) detector, used to image the milled surfaces, generates a large series of images that can be combined into a 3D rendered image of stained and embedded biological tissue. Structural information over volumes of tens of thousands of cubic micrometers is possible, revealing complex microanatomy with subcellular resolution. Methods are presented for tissue processing, for the enhancement of contrast with osmium tetroxide/potassium ferricyanide, for BSE imaging, for the preparation and platinum deposition over a selected site in the embedded tissue block, and for sequential data collection with ion beam milling; all this takes approximately 90 h. The imaging conditions, procedures for alternate milling and data acquisition and techniques for processing and partitioning the 3D data set are also described; these processes take approxiamtely 30 h. The protocol is illustrated by application to developing chick cornea, in which cells organize collagen fibril bundles into complex, multilamellar structures essential for transparency in the mature connective tissue matrix. The techniques described could have wide application in a range of fields, including pathology, developmental biology, microstructural anatomy and regenerative medicine.

  4. Neurosurgical confocal endomicroscopy: A review of contrast agents, confocal systems, and future imaging modalities

    Directory of Open Access Journals (Sweden)

    Aqib H Zehri

    2014-01-01

    Full Text Available Background: The clinical application of fluorescent contrast agents (fluorescein, indocyanine green, and aminolevulinic acid with intraoperative microscopy has led to advances in intraoperative brain tumor imaging. Their properties, mechanism of action, history of use, and safety are analyzed in this report along with a review of current laser scanning confocal endomicroscopy systems. Additional imaging modalities with potential neurosurgical utility are also analyzed. Methods: A comprehensive literature search was performed utilizing PubMed and key words: In vivo confocal microscopy, confocal endomicroscopy, fluorescence imaging, in vivo diagnostics/neoplasm, in vivo molecular imaging, and optical imaging. Articles were reviewed that discussed clinically available fluorophores in neurosurgery, confocal endomicroscopy instrumentation, confocal microscopy systems, and intraoperative cancer diagnostics. Results: Current clinically available fluorescent contrast agents have specific properties that provide microscopic delineation of tumors when imaged with laser scanning confocal endomicroscopes. Other imaging modalities such as coherent anti-Stokes Raman scattering (CARS microscopy, confocal reflectance microscopy, fluorescent lifetime imaging (FLIM, two-photon microscopy, and second harmonic generation may also have potential in neurosurgical applications. Conclusion: In addition to guiding tumor resection, intraoperative fluorescence and microscopy have the potential to facilitate tumor identification and complement frozen section analysis during surgery by providing real-time histological assessment. Further research, including clinical trials, is necessary to test the efficacy of fluorescent contrast agents and optical imaging instrumentation in order to establish their role in neurosurgery.

  5. Cell motility dynamics: a novel segmentation algorithm to quantify multi-cellular bright field microscopy images.

    Directory of Open Access Journals (Sweden)

    Assaf Zaritsky

    Full Text Available Confocal microscopy analysis of fluorescence and morphology is becoming the standard tool in cell biology and molecular imaging. Accurate quantification algorithms are required to enhance the understanding of different biological phenomena. We present a novel approach based on image-segmentation of multi-cellular regions in bright field images demonstrating enhanced quantitative analyses and better understanding of cell motility. We present MultiCellSeg, a segmentation algorithm to separate between multi-cellular and background regions for bright field images, which is based on classification of local patches within an image: a cascade of Support Vector Machines (SVMs is applied using basic image features. Post processing includes additional classification and graph-cut segmentation to reclassify erroneous regions and refine the segmentation. This approach leads to a parameter-free and robust algorithm. Comparison to an alternative algorithm on wound healing assay images demonstrates its superiority. The proposed approach was used to evaluate common cell migration models such as wound healing and scatter assay. It was applied to quantify the acceleration effect of Hepatocyte growth factor/scatter factor (HGF/SF on healing rate in a time lapse confocal microscopy wound healing assay and demonstrated that the healing rate is linear in both treated and untreated cells, and that HGF/SF accelerates the healing rate by approximately two-fold. A novel fully automated, accurate, zero-parameters method to classify and score scatter-assay images was developed and demonstrated that multi-cellular texture is an excellent descriptor to measure HGF/SF-induced cell scattering. We show that exploitation of textural information from differential interference contrast (DIC images on the multi-cellular level can prove beneficial for the analyses of wound healing and scatter assays. The proposed approach is generic and can be used alone or alongside traditional

  6. Imaging latex-carbon nanotube composites by subsurface electrostatic force microscopy

    Science.gov (United States)

    Patel, Sajan; Petty, Clayton W.; Krafcik, Karen; Loyola, Bryan; O'Bryan, Greg; Friddle, Raymond W.

    2016-10-01

    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. 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.

  7. Image edge-enhancement in optical microscopy with a phase mismatched spiral phase plate

    Institute of Scientific and Technical Information of China (English)

    Shibiao Wei; Jing Bu; Siwei Zhu; Xiaocong Yuan

    2011-01-01

    @@ We present a spiral phase filtering system with a large tolerance for edge enhancement of both phase and amplitude objects in optical microscopy.The method is based on a Fourier 4-f spatial filtering system.A phase mismatched spiral phase plate (SPP) fabricated by electron beam lithography is employed as the radial Hilbert transform for image edge enhancement.Compared with holography, SPP is simple,economical, reliable, and easy to integrate.%We present a spiral phase filtering system with a large tolerance for edge enhancement of both phase and amplitude objects in optical microscopy. The method is based on a Fourier 4-f spatial filtering system.A phase mismatched spiral phase plate (SPP) fabricated by electron beam lithography is employed as the radial Hilbert transform for image edge enhancement. Compared with holography, SPP is simple,economical, reliable, and easy to integrate.

  8. 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.

  9. High-Resolution Imaging of Dendrimers Used in Drug Delivery via Scanning Probe Microscopy

    Directory of Open Access Journals (Sweden)

    Lifang Shi

    2011-01-01

    Full Text Available Dendrimers and telodendrimer micelles represent two new classes of vehicles for drug delivery that have attracted much attention recently. Their structural characterization at the molecular and submolecular level remains a challenge due to the difficulties in reaching high resolution when imaging small particles in their native media. This investigation offers a new approach towards this challenge, using scanning tunneling microscopy (STM and atomic force microscopy (AFM. By using new sample preparation protocols, this work demonstrates that (a intramolecular features such as drug molecules and dendrimer termini can be resolved; and (b telodendrimer micelles can be immobilized on the surface without compromising structural integrity, and as such, high resolution AFM imaging may be performed to attain 3D information. This high-resolution structural information should enhance our knowledge of the nanocarrier structure and nanocarrier-drug interaction and, therefore, facilitate design and optimization of the efficiency in drug delivery.

  10. 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.

  11. Sub-diffraction imaging on standard microscopes through photobleaching microscopy with non-linear processing.

    Science.gov (United States)

    Munck, Sebastian; Miskiewicz, Katarzyna; Sannerud, Ragna; Menchon, Silvia A; Jose, Liya; Heintzmann, Rainer; Verstreken, Patrik; Annaert, Wim

    2012-05-01

    Visualization of organelles and molecules at nanometer resolution is revolutionizing the biological sciences. However, such technology is still limited for many cell biologists. We present here a novel approach using photobleaching microscopy with non-linear processing (PiMP) for sub-diffraction imaging. Bleaching of fluorophores both within the single-molecule regime and beyond allows visualization of stochastic representations of sub-populations of fluorophores by imaging the same region over time. Our method is based on enhancing the probable positions of the fluorophores underlying the images. The random nature of the bleached fluorophores is assessed by calculating the deviation of the local actual bleached fluorescence intensity to the average bleach expectation as given by the overall decay of intensity. Subtracting measured from estimated decay images yields differential images. Non-linear enhancement of maxima in these diffraction-limited differential images approximates the positions of the underlying structure. Summing many such processed differential images yields a super-resolution PiMP image. PiMP allows multi-color, three-dimensional sub-diffraction imaging of cells and tissues using common fluorophores and can be implemented on standard wide-field or confocal systems.

  12. Single-wavelength reflected confocal and multiphoton microscopy for tissue imaging

    Science.gov (United States)

    Chen, Wei-Liang; Chou, Chen-Kuan; Lin, Ming-Gu; Chen, Yang-Fang; Jee, Shiou-Hwa; Tan, Hsin-Yuan; Tsai, Tsung-Hua; Kim, Ki-Hean; Kim, Daekeun; So, Peter T. C.; Lin, Sung-Jan; Dong, Chen-Yuan

    2009-09-01

    Both reflected confocal and multiphoton microscopy can have clinical diagnostic applications. The successful combination of both modalities in tissue imaging enables unique image contrast to be achieved, especially if a single laser excitation wavelength is used. We apply this approach for skin and corneal imaging using the 780-nm output of a femtosecond, titanium-sapphire laser. We find that the near-IR, reflected confocal (RC) signal is useful in characterizing refractive index varying boundaries in bovine cornea and porcine skin, while the multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) intensities can be used to image cytoplasm and connective tissues (collagen), respectively. In addition, quantitative analysis shows that we are able to detect MAF from greater imaging depths than with the near-IR RC signal. Furthermore, by performing RC imaging at 488, 543, and 633 nm, we find that a longer wavelength leads to better image contrast for deeper imaging of the bovine cornea and porcine skin tissue. Finally, by varying power of the 780-nm source, we find that comparable RC image quality was achieved in the 2.7 to 10.7-mW range.

  13. 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.

  14. 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.

  15. NI-78LABEL-FREE MULTIPHOTON MICROSCOPY: A NOVEL TOOL FOR THE IMAGING OF BRAIN TUMORS

    Science.gov (United States)

    Uckermann, Ortrud; Galli, Roberta; Geiger, Kathrin; Koch, Edmund; Schackert, Gabriele; Steiner, Gerald; Kirsch, Matthias

    2014-01-01

    Changes in tissue composition caused by brain tumor growth involve a series of complex biochemical alterations which can be imaged on unstained native tissue using multiphoton microscopy: We used coherent anti-Stokes Raman scattering (CARS) imaging that resonantly excites the symmetric stretching vibration of CH2 groups at 2850 cm−1 and visualizes lipid content in combination with imaging of endogenous two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) to discern different types of tumors from normal tissue in unstained, native brain samples. Experimental brain tumors were induced in nude mice NMRI nu/nu (n = 25) by stereotactic implantation of glioblastoma (U87), melanoma (A375) and breast cancer (MCF-7) cell lines. Label-free multiphoton microscopy of brain cryosections provided exhaustive information of the tumor morphochemistry. The tumor border was defined with cellular resolution by a strong reduction of CARS signal intensity to 61% (glioblastoma), 71% (melanoma) and 68% (breast cancer). This reduction of lipid content within the tumor was confirmed by Raman spectroscopy. Micrometastases infiltrating normal tissue (size 50 - 200 µm) were identified in glioblastoma and melanoma. Additionally, multiphoton microscopy proved a reduction of CARS signal intensity in all human glioblastoma samples analyzed (to 72%, n = 6). Additionally, relevant SHG and TPEF signals were detected in human primary and secondary brain tumor samples and enabled to image variations in tumor associated vasculature, fibrosis, necrosis and nuclear size and density. All primary or secondary brain tumors investigated were characterized by a lower intensity of the CARS signal, therefore offering a simple tool for objective tumor detection and delineation. The combination of techniques allows retrieving a quantity of information on native unstained tissue which is comparable to H&E staining. Therefore, label-free multiphoton microscopy has the potential to become a

  16. Ferroelectric Domain Imaging Mechanism in High-Vacuum Scanning Force Microscopy

    Institute of Scientific and Technical Information of China (English)

    ZENG Hua-Rong; YU Han-Feng; CHU Rui-Qing; LI Guo-Rong; YIN Qing-Rui

    2005-01-01

    @@ High-vacuum scanning force microscopy of the domain structures in PMN-PT single crystals is investigated. It has been shown that under high vacuum conditions, the polarization charges are not effectively compensated for by intrinsic screening charges from the ferroelectrics. This result suggests that the electrostatic tip-sample interaction plays a great contribution to the domain imaging mechanism in PMN-PT ferroelectric single crystals under high vacuum conditions.

  17. Pulse splitter-based nonlinear microscopy for live-cardiomyocyte imaging

    OpenAIRE

    Wang, Zhonghai; Qin, Wan; Shao, Yonghong; Ma, Siyu; Borg, Thomas K.; GAO, BRUCE Z.

    2014-01-01

    Second harmonic generation (SHG) microscopy is a new imaging technique used in sarcomeric-addition studies. However, during the early stage of cell culture in which sarcomeric additions occur, the neonatal cardiomyocytes that we have been working with are very sensitive to photodamage, the resulting high rate of cell death prevents systematic study of sarcomeric addition using a conventional SHG system. To address this challenge, we introduced use of the pulse-splitter system developed by Na ...

  18. Magnetic soft x-ray microscopy-imaging fast spin dynamics inmagnetic nanostructures

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, Peter; Kim, Dong-Hyun; Mesler, Brooke L.; Chao, Weilun; Sakdinawat, Anne E.; Anderson, Erik H.

    2007-06-01

    Magnetic soft X-ray microscopy combines 15nm spatial resolution with 70ps time resolution and elemental sensitivity. Fresnel zone plates are used as X-ray optics and X-ray magnetic circular dichroism serves as magnetic contrast mechanism. Thus scientifically interesting and technologically relevant low dimensional nanomagnetic systems can be imaged at fundamental length and ultrafast time scales in a unique way. Studies include magnetization reversal in magnetic multilayers, nanopatterned systems, vortex dynamics in nanoelements and spin current induced phenomena.

  19. Super-resolution imaging of plasmodesmata using three-dimensional structured illumination microscopy

    OpenAIRE

    Fitzgibbon, Jessica; Bell,Karen; King, Emma; Oparka, Karl

    2010-01-01

    We used three-dimensional structured illumination microscopy (3D-SIM) to obtain subdiffraction ("super-resolution") images of plasmodesmata (PD) expressing a green fluorescent protein-tagged viral movement protein (MP) in tobacco (Nicotiana tabacum). In leaf parenchyma cells, we were able to resolve individual components of PD (neck and central cavities) at twice the resolution of a confocal microscope. Within the phloem, MP-green fluorescent protein filaments extended outward from the specia...

  20. 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

  1. Carbon Nanostructure Examined by Lattice Fringe Analysis of High Resolution Transmission Electron Microscopy Images

    Science.gov (United States)

    VanderWal, Randy L.; Tomasek, Aaron J.; Street, Kenneth; Thompson, William K.; Hull, David R.

    2003-01-01

    The dimensions of graphitic layer planes directly affect the reactivity of soot towards oxidation and growth. Quantification of graphitic structure could be used to develop and test correlations between the soot nanostructure and its reactivity. Based upon transmission electron microscopy images, this paper provides a demonstration of the robustness of a fringe image analysis code for determining the level of graphitic structure within nanoscale carbon, i.e., soot. Results, in the form of histograms of graphitic layer plane lengths, are compared to their determination through Raman analysis.

  2. In situ scanning FTIR microscopy and IR imaging of Pt electrode surface towards CO adsorption

    Institute of Scientific and Technical Information of China (English)

    孙世刚; 洪双进; 陈声培; 卢国强; 戴鸿平; 肖晓银

    1999-01-01

    In situ scanning FTIR microscopy was built up for the first time in the present work, which consists of an FTIR apparatus, an IR microscope, an X-Y mapping stage, and the specially designed electrochemical IR cell and computer software. It has been demonstrated that this new space-resolvd in situ IR technique can be used to study vibration properties of micro-area, and to perform IR imaging of electrode surface. The chemical image obtained using this technique fur CO adsorption on Pt electrode illustrated, at a space-resolution of 10-2 cm, the inhomogeneity and the distribution of reactivity of micro-area of electrode surface.

  3. Monte Carlo simulation of secondary electron images for real sample structures in scanning electron microscopy.

    Science.gov (United States)

    Zhang, P; Wang, H Y; Li, Y G; Mao, S F; Ding, Z J

    2012-01-01

    Monte Carlo simulation methods for the study of electron beam interaction with solids have been mostly concerned with specimens of simple geometry. In this article, we propose a simulation algorithm for treating arbitrary complex structures in a real sample. The method is based on a finite element triangular mesh modeling of sample geometry and a space subdivision for accelerating simulation. Simulation of secondary electron image in scanning electron microscopy has been performed for gold particles on a carbon substrate. Comparison of the simulation result with an experiment image confirms that this method is effective to model complex morphology of a real sample.

  4. Holographic microscopy reconstruction in both object and image half spaces with undistorted 3D grid

    CERN Document Server

    Verrier, Nicolas; Tessier, Gilles; Gross, Michel

    2015-01-01

    We propose a holographic microscopy reconstruction method, which propagates the hologram, in the object half space, in the vicinity of the object. The calibration yields reconstructions with an undistorted reconstruction grid i.e. with orthogonal x, y and z axis and constant pixels pitch. The method is validated with an USAF target imaged by a x60 microscope objective, whose holograms are recorded and reconstructed for different USAF locations along the longitudinal axis:-75 to +75 {\\mu}m. Since the reconstruction numerical phase mask, the reference phase curvature and MO form an afocal device, the reconstruction can be interpreted as occurring equivalently in the object or in image half space.

  5. Development of a fast electromagnetic shutter for compressive sensing imaging in scanning transmission electron microscopy

    CERN Document Server

    Béché, Armand; Freitag, Bert; Verbeeck, Jo

    2015-01-01

    The concept of compressive sensing was recently proposed to significantly reduce the electron dose in scanning transmission electron microscopy (STEM) while still maintaining the main features in the image. Here, an experimental setup based on an electromagnetic shutter placed in the condenser plane of a STEM is proposed. The shutter blanks the beam following a random pattern while the scanning coils are moving the beam in the usual scan pattern. Experimental images at both medium scale and high resolution are acquired and then reconstructed based on a discrete cosine algorithm. The obtained results confirm the predicted usefulness of compressive sensing in experimental STEM even though some remaining artifacts need to be resolved.

  6. Imaging of surface spin textures on bulk crystals by scanning electron microscopy

    OpenAIRE

    Hiroshi Akamine; So Okumura; Sahar Farjami; Yasukazu Murakami; Minoru Nishida

    2016-01-01

    Direct observation of magnetic microstructures is vital for advancing spintronics and other technologies. Here we report a method for imaging surface domain structures on bulk samples by scanning electron microscopy (SEM). Complex magnetic domains, referred to as the maze state in CoPt/FePt alloys, were observed at a spatial resolution of less than 100 nm by using an in-lens annular detector. The method allows for imaging almost all the domain walls in the mazy structure, whereas the visualis...

  7. Imaging the bipolarity of myosin filaments with Interferometric Second Harmonic Generation microscopy.

    Science.gov (United States)

    Rivard, Maxime; Couture, Charles-André; Miri, Amir K; Laliberté, Mathieu; Bertrand-Grenier, Antony; Mongeau, Luc; Légaré, François

    2013-01-01

    We report that combining interferometry with Second Harmonic Generation (SHG) microscopy provides valuable information about the relative orientation of noncentrosymmetric structures composing tissues. This is confirmed through the imaging of rat medial gastrocnemius muscle. The inteferometric Second Harmonic Generation (ISHG) images reveal that each side of the myosin filaments composing the A band of the sarcomere generates π phase shifted SHG signal which implies that the myosin proteins at each end of the filaments are oriented in opposite directions. This highlights the bipolar structural organization of the myosin filaments and shows that muscles can be considered as a periodically poled biological structure.

  8. Real-time in vivo imaging collagen in lymphedematous skin using multiphoton microscopy.

    Science.gov (United States)

    Wu, Xiufeng; Zhuo, Shuangmu; Chen, Jianxin; Liu, Ningfei

    2011-01-01

    Changes of dermal collagen are characteristic for chronic lymphedema. To evaluate these changes, a real-time imaging based on two-photon excited fluorescence and second-harmonic generation was developed for investigating collagen of lymphedematous mouse and rat tail skin in vivo. Our findings showed that the technique could image the morphological changes and distribution of collagen in lymphedematous mouse and rat tail skin in vivo. More importantly, it may allow visualization of dynamic collagen alteration during the progression of lymphedema. Our findings demonstrated that multiphoton microscopy may have potential in a clinical setting as an in vivo diagnostic and monitoring system for therapy in lymphology.

  9. Non-rigid image registration to reduce beam-induced blurring of cryo-electron microscopy images

    Energy Technology Data Exchange (ETDEWEB)

    Karimi Nejadasl, Fatemeh; Karuppasamy, Manikandan [Leiden University Medical Center, PO Box 9600, 2300RC Leiden (Netherlands); Newman, Emily R.; McGeehan, John E. [University of Portsmouth, Portsmouth PO1 2DY (United Kingdom); Ravelli, Raimond B. G., E-mail: raimond.nl@gmail.com [Leiden University Medical Center, PO Box 9600, 2300RC Leiden (Netherlands)

    2013-01-01

    Cryo-electron microscopy images of vitrified large macromolecular complexes can become blurred due to beam-induced specimen alterations. Exposure series are examined, and rigid and non-rigid image registration schemes are applied to reduce such blurring. The typical dose used to record cryo-electron microscopy images from vitrified biological specimens is so high that radiation-induced structural alterations are bound to occur during data acquisition. Integration of all scattered electrons into one image can lead to significant blurring, particularly if the data are collected from an unsupported thin layer of ice suspended over the holes of a support film. Here, the dose has been fractioned and exposure series have been acquired in order to study beam-induced specimen movements under low dose conditions, prior to bubbling. Gold particles were added to the protein sample as fiducial markers. These were automatically localized and tracked throughout the exposure series and showed correlated motions within small patches, with larger amplitudes of motion vectors at the start of a series compared with the end of each series. A non-rigid scheme was used to register all images within each exposure series, using natural neighbor interpolation with the gold particles as anchor points. The procedure increases the contrast and resolution of the examined macromolecules.

  10. Digital holographic microscopy for imaging growth and treatment response in 3D tumor models

    Science.gov (United States)

    Li, Yuyu; Petrovic, Ljubica; Celli, Jonathan P.; Yelleswarapu, Chandra S.

    2014-03-01

    While three-dimensional tumor models have emerged as valuable tools in cancer research, the ability to longitudinally visualize the 3D tumor architecture restored by these systems is limited with microscopy techniques that provide only qualitative insight into sample depth, or which require terminal fixation for depth-resolved 3D imaging. Here we report the use of digital holographic microscopy (DHM) as a viable microscopy approach for quantitative, non-destructive longitudinal imaging of in vitro 3D tumor models. Following established methods we prepared 3D cultures of pancreatic cancer cells in overlay geometry on extracellular matrix beds and obtained digital holograms at multiple timepoints throughout the duration of growth. The holograms were digitally processed and the unwrapped phase images were obtained to quantify nodule thickness over time under normal growth, and in cultures subject to chemotherapy treatment. In this manner total nodule volumes are rapidly estimated and demonstrated here to show contrasting time dependent changes during growth and in response to treatment. This work suggests the utility of DHM to quantify changes in 3D structure over time and suggests the further development of this approach for time-lapse monitoring of 3D morphological changes during growth and in response to treatment that would otherwise be impractical to visualize.

  11. 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.

  12. 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

  13. 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.

  14. Photo-imprint Photoacoustic Microscopy for Three-dimensional Label-free Sub-diffraction Imaging

    Science.gov (United States)

    Yao, Junjie; Wang, Lidai; Li, Chiye; Zhang, Chi; Wang, Lihong V.

    2014-01-01

    Sub-diffraction optical microscopy allows the imaging of cellular and subcellular structures with resolution finer than the diffraction limit. Here, combining the absorption-based photoacoustic effect and intensity-dependent photobleaching effect, we demonstrate a simple method for sub-diffraction photoacoustic imaging of both fluorescent and non-fluorescent samples. Our method is based on a double-excitation process, where the first excitation pulse partially and inhomogeneously bleaches the molecules in the diffraction-limited excitation volume, thus biasing the signal contributions from a second excitation pulse striking the same region. The differential signal between the two excitations preserves the signal contribution mostly from the center of the excitation volume, and dramatically sharpens the lateral resolution. Moreover, due to the nonlinear nature of the signal, our method offers inherent optical sectioning capability, which is lacking in conventional photoacoustic microscopy. By scanning the excitation beam, we performed three-dimensional sub-diffraction imaging of varied fluorescent and non-fluorescent species. As any molecules have absorption, this technique has the potential to enable label-free sub-diffraction imaging, and can be transferred to other optical imaging modalities or combined with other sub-diffraction methods. PMID:24483902

  15. 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.

  16. Imaging arterial cells, atherosclerosis, and restenosis by multimodal nonlinear optical microscopy

    Science.gov (United States)

    Wang, Han-Wei; Simianu, Vlad; Locker, Matthew J.; Sturek, Michael; Cheng, Ji-Xin

    2008-02-01

    By integrating sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on a coherent anti-Stokes Raman scattering (CARS) microscope platform, multimodal nonlinear optical (NLO) imaging of arteries and atherosclerotic lesions was demonstrated. CARS signals arising from CH II-rich membranes allowed visualization of endothelial cells and smooth muscle cells in a carotid artery. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are rich in CH II bonds in their cross-linking residues. The extracellular matrix organization was further confirmed by TPEF signals arising from elastin's autofluorescence and SFG signals arising from collagen fibrils' non-centrosymmetric structure. The system is capable of identifying different atherosclerotic lesion stages with sub-cellular resolution. The stages of atherosclerosis, such as macrophage infiltration, lipid-laden foam cell accumulation, extracellular lipid distribution, fibrous tissue deposition, plaque establishment, and formation of other complicated lesions could be viewed by our multimodal CARS microscope. Collagen percentages in the region adjacent to coronary artery stents were resolved. High correlation between NLO and histology imaging evidenced the validity of the NLO imaging. The capability of imaging significant components of an arterial wall and distinctive stages of atherosclerosis in a label-free manner suggests the potential application of multimodal nonlinear optical microscopy to monitor the onset and progression of arterial diseases.

  17. A workflow for the automatic segmentation of organelles in electron microscopy image stacks.

    Science.gov (United States)

    Perez, Alex J; Seyedhosseini, Mojtaba; Deerinck, Thomas J; Bushong, Eric A; Panda, Satchidananda; Tasdizen, Tolga; Ellisman, Mark H

    2014-01-01

    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.

  18. 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.

  19. A novel Kalman filter based video image processing scheme for two-photon fluorescence microscopy

    Science.gov (United States)

    Sun, Wenqing; Huang, Xia; Li, Chunqiang; Xiao, Chuan; Qian, Wei

    2016-03-01

    Two-photon fluorescence microscopy (TPFM) is a perfect optical imaging equipment to monitor the interaction between fast moving viruses and hosts. However, due to strong unavoidable background noises from the culture, videos obtained by this technique are too noisy to elaborate this fast infection process without video image processing. In this study, we developed a novel scheme to eliminate background noises, recover background bacteria images and improve video qualities. In our scheme, we modified and implemented the following methods for both host and virus videos: correlation method, round identification method, tree-structured nonlinear filters, Kalman filters, and cell tracking method. After these procedures, most of noises were eliminated and host images were recovered with their moving directions and speed highlighted in the videos. From the analysis of the processed videos, 93% bacteria and 98% viruses were correctly detected in each frame on average.

  20. Analysis of cancer cell morphology in fluorescence microscopy image exploiting shape descriptor

    Science.gov (United States)

    Kang, Mi-Sun; Kim, Hye-Ryun; Kim, Sudong; Ryu, Gyu Ha; Kim, Myoung-Hee

    2016-04-01

    Cancer cell morphology is closely related to their phenotype and activity. These characteristics are important in drug-response prediction for personalized cancer therapeutics. We used multi-channel fluorescence microscopy images to analyze the morphology of highly cohesive cancer cells. First, we detected individual nuclei regions in single-channel images using advanced simple linear iterative clustering. The center points of the nuclei regions were used as seeds for the Voronoi diagram method to extract spatial arrangement features from cell images. Human cancer cell populations form irregularly shaped aggregates, making their detection more difficult. We overcame this problem by identifying individual cells using an image-based shape descriptor. Finally, we analyzed the correlation between cell agglutination and cell shape.

  1. Calcium imaging of neural circuits with extended depth-of-field light-sheet microscopy.

    Science.gov (United States)

    Quirin, Sean; Vladimirov, Nikita; Yang, Chao-Tsung; Peterka, Darcy S; Yuste, Rafael; Ahrens, Misha B

    2016-03-01

    Increasing the volumetric imaging speed of light-sheet microscopy will improve its ability to detect fast changes in neural activity. Here, a system is introduced for brain-wide imaging of neural activity in the larval zebrafish by coupling structured illumination with cubic phase extended depth-of-field (EDoF) pupil encoding. This microscope enables faster light-sheet imaging and facilitates arbitrary plane scanning-removing constraints on acquisition speed, alignment tolerances, and physical motion near the sample. The usefulness of this method is demonstrated by performing multi-plane calcium imaging in the fish brain with a 416×832×160  μm field of view at 33 Hz. The optomotor response behavior of the zebrafish is monitored at high speeds, and time-locked correlations of neuronal activity are resolved across its brain.

  2. Memory-effect based deconvolution microscopy for super-resolution imaging through scattering media

    Science.gov (United States)

    Edrei, Eitan; Scarcelli, Giuliano

    2016-09-01

    High-resolution imaging through turbid media is a fundamental challenge of optical sciences that has attracted a lot of attention in recent years for its wide range of potential applications. Here, we demonstrate that the resolution of imaging systems looking behind a highly scattering medium can be improved below the diffraction-limit. To achieve this, we demonstrate a novel microscopy technique enabled by the optical memory effect that uses a deconvolution image processing and thus it does not require iterative focusing, scanning or phase retrieval procedures. We show that this newly established ability of direct imaging through turbid media provides fundamental and practical advantages such as three-dimensional refocusing and unambiguous object reconstruction.

  3. Imaging of apoptotic HeLa cells by using scanning near-field optical microscopy

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    By using scanning near-field optical microscopy (SNOM), HeLa cells in apoptosis process are imaged with a higher optical resolution beyond the diffraction limit. Since SNOM provides both topographic and transmitted light intensity information of a cell, it can correlate the structural characteristics and optical properties with the spatial position of the apoptotic cells. Wavelength imaging by using near-field spectroscopy shows that there is a great difference in light propagation and absorption in the cell. This unique technique can be applied to the super high resolution imaging of different components in the cell. The observations by near-field optical imaging and near-field spectroscopy indicate an inhomogeneous aggregation of the inner structure in the apoptotic HeLa cells and the change of transmission intensity of light with the apoptosis status.

  4. Video-rate imaging of microcirculation with single-exposure oblique back-illumination microscopy

    Science.gov (United States)

    Ford, Tim N.; Mertz, Jerome

    2013-06-01

    Oblique back-illumination microscopy (OBM) is a new technique for simultaneous, independent measurements of phase gradients and absorption in thick scattering tissues based on widefield imaging. To date, OBM has been used with sequential camera exposures, which reduces temporal resolution, and can produce motion artifacts in dynamic samples. Here, a variation of OBM that allows single-exposure operation with wavelength multiplexing and image splitting with a Wollaston prism is introduced. Asymmetric anamorphic distortion induced by the prism is characterized and corrected in real time using a graphics-processing unit. To demonstrate the capacity of single-exposure OBM to perform artifact-free imaging of blood flow, video-rate movies of microcirculation in ovo in the chorioallantoic membrane of the developing chick are presented. Imaging is performed with a high-resolution rigid Hopkins lens suitable for endoscopy.

  5. 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.

  6. Techniques for imaging human metaphase chromosomes in liquid conditions by atomic force microscopy

    Science.gov (United States)

    Ushiki, Tatsuo; Shigeno, Masatsugu; Hoshi, Osamu

    2008-09-01

    The purpose of this study was to obtain three-dimensional images of wet chromosomes by atomic force microscopy (AFM) in liquid conditions. Human metaphase chromosomes—obtained either by chromosome spreads or by an isolation technique—were observed in a dynamic mode by AFM in a buffer solution. Under suitable operating conditions with a soft triangular cantilever (with the spring constant of 0.08-0.4 N m-1), clear images of fixed chromosomes in the chromosome spread were obtained by AFM. For imaging isolated chromosomes with the height of more than 400 nm, a cantilever with a high aspect ratio probing tip was required. The combination of a Q-control system and the sampling intelligent scan (SIS) system in dynamic force mode AFM was useful for obtaining high-quality images of the isolated chromosomes, in which globular or cord-like structures about 50 nm thick were clearly observed on the surface of each chromatid.

  7. Prospects for lithium imaging using annular bright field scanning transmission electron microscopy: A theoretical study

    Energy Technology Data Exchange (ETDEWEB)

    Findlay, S.D., E-mail: scott@sigma.t.u-tokyo.ac.jp [Institute of Engineering Innovation, The University of Tokyo, Tokyo 116-0013 (Japan); Lugg, N.R. [School of Physics, University of Melbourne, Parkville, Victoria 3010 (Australia); Shibata, N. [Institute of Engineering Innovation, The University of Tokyo, Tokyo 116-0013 (Japan); PRESTO, Japan Science and Technology Agency, Saitama 332-0012 (Japan); Allen, L.J. [School of Physics, University of Melbourne, Parkville, Victoria 3010 (Australia); Ikuhara, Y. [Institute of Engineering Innovation, The University of Tokyo, Tokyo 116-0013 (Japan); Nanostructures Research Laboratory, Japan Fine Ceramic Center, Nagoya 456-8587 (Japan); WPI Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577 (Japan)

    2011-07-15

    There is strong interest in lithium imaging, particularly because of its significance in battery materials. However, light atoms only scatter electrons weakly and atomic resolution direct imaging of lithium has proven difficult. This paper explores theoretically the conditions under which lithium columns can be expected to be directly visible using annular bright field scanning transmission electron microscopy. A detailed discussion is given of the controllable parameters and the conditions most favourable for lithium imaging. -- Highlights: {yields} Optimum conditions to image Li columns in Li-bearing materials with ABF are explored. {yields} Higher accelerating voltages give better contrast at a given resolution. {yields} Aperture size must compromise between resolution and good coupling to the column. {yields} Samples with small along-column interatomic spacing between Li atoms are best. {yields} The trends observed are consistent with prediction based on the s-state model.

  8. 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.

  9. Non-destructive electron microscopy imaging and analysis of biological samples with graphene coating

    Science.gov (United States)

    Park, Jong Bo; Kim, Yong-Jin; Kim, Seong-Min; Yoo, Je Min; Kim, Youngsoo; Gorbachev, Roman; Barbolina, I. I.; Kim, Sang Jin; Kang, Sangmin; Yoon, Myung-Han; Cho, Sung-Pyo; Novoselov, Konstantin S.; Hong, Byung Hee

    2016-12-01

    In electron microscopy (EM), charging of non-conductive biological samples by focused electron beams hinders their high-resolution imaging. Gold or platinum coatings have been commonly used to prevent such sample charging, but it disables further quantitative and qualitative chemical analyses such as energy dispersive spectroscopy (EDS). Here we report that graphene-coating on biological samples enables non-destructive high-resolution imaging by EM as well as chemical analysis by EDS, utilizing graphene’s transparency to electron beams, high conductivity, outstanding mechanical strength and flexibility. We believe that the graphene-coated imaging and analysis would provide us a new opportunity to explore various biological phenomena unseen before due to the limitation in sample preparation and image resolution, which will broaden our understanding on the life mechanism of various living organisms.

  10. 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.

  11. 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.

  12. 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.

  13. Conditions to minimize soft single biomolecule deformation when imaging with atomic force microscopy.

    Science.gov (United States)

    Godon, Christian; Teulon, Jean-Marie; Odorico, Michael; Basset, Christian; Meillan, Matthieu; Vellutini, Luc; Chen, Shu-Wen W; Pellequer, Jean-Luc

    2016-12-23

    A recurrent interrogation when imaging soft biomolecules using atomic force microscopy (AFM) is the putative deformation of molecules leading to a bias in recording true topographical surfaces. Deformation of biomolecules comes from three sources: sample instability, adsorption to the imaging substrate, and crushing under tip pressure. To disentangle these causes, we measured the maximum height of a well-known biomolecule, the tobacco mosaic virus (TMV), under eight different experimental conditions positing that the maximum height value is a specific indicator of sample deformations. Six basic AFM experimental factors were tested: imaging in air (AIR) versus in liquid (LIQ), imaging with flat minerals (MICA) versus flat organic surfaces (self-assembled monolayers, SAM), and imaging forces with oscillating tapping mode (TAP) versus PeakForce tapping (PFT). The results show that the most critical parameter in accurately measuring the height of TMV in air is the substrate. In a liquid environment, regardless of the substrate, the most critical parameter is the imaging mode. Most importantly, the expected TMV height values were obtained with both imaging with the PeakForce tapping mode either in liquid or in air at the condition of using self-assembled monolayers as substrate. This study unambiguously explains previous poor results of imaging biomolecules on mica in air and suggests alternative methodologies for depositing soft biomolecules on well organized self-assembled monolayers.

  14. 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.

  15. Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues.

    Science.gov (United States)

    Durr, Nicholas J; Weisspfennig, Christian T; Holfeld, Benjamin A; Ben-Yakar, Adela

    2011-02-01

    Endogenous fluorescence provides morphological, spectral, and lifetime contrast that can indicate disease states in tissues. Previous studies have demonstrated that two-photon autofluorescence microscopy (2PAM) can be used for noninvasive, three-dimensional imaging of epithelial tissues down to approximately 150 μm beneath the skin surface. We report ex-vivo 2PAM images of epithelial tissue from a human tongue biopsy down to 370 μm below the surface. At greater than 320 μm deep, the fluorescence generated outside the focal volume degrades the image contrast to below one. We demonstrate that these imaging depths can be reached with 160 mW of laser power (2-nJ per pulse) from a conventional 80-MHz repetition rate ultrafast laser oscillator. To better understand the maximum imaging depths that we can achieve in epithelial tissues, we studied image contrast as a function of depth in tissue phantoms with a range of relevant optical properties. The phantom data agree well with the estimated contrast decays from time-resolved Monte Carlo simulations and show maximum imaging depths similar to that found in human biopsy results. This work demonstrates that the low staining inhomogeneity (∼ 20) and large scattering coefficient (∼ 10 mm(-1)) associated with conventional 2PAM limit the maximum imaging depth to 3 to 5 mean free scattering lengths deep in epithelial tissue.

  16. The optical microscopy with virtual image breaks a record: 50-nm resolution imaging is demonstrated

    CERN Document Server

    Wang, Zengbo; Li, Lin; Liu, Zhu; Luk'yanchuk, Boris; Chen, Zaichun; Hong, Minghui

    2010-01-01

    We demonstrate a new 'microsphere nanoscope' that uses ordinary SiO2 microspheres as superlenses to create a virtual image of the object in near field. The magnified virtual image greatly overcomes the diffraction limit. We are able to resolve clearly 50-nm objects under a standard white light source in both transmission and reflection modes. The resolution achieved for white light opens a new opportunity to image viruses, DNA and molecules in real time.

  17. Secretory glands and microvascular systems imaged in aqueous solution by atmospheric scanning electron microscopy (ASEM).

    Science.gov (United States)

    Yamazawa, Toshiko; Nakamura, Naotoshi; Sato, Mari; Sato, Chikara

    2016-12-01

    Exocrine glands, e.g., salivary and pancreatic glands, play an important role in digestive enzyme secretion, while endocrine glands, e.g., pancreatic islets, secrete hormones that regulate blood glucose levels. The dysfunction of these secretory organs immediately leads to various diseases, such as diabetes or Sjögren's syndrome, by poorly understood mechanisms. Gland-related diseases have been studied by optical microscopy (OM), and at higher resolution by transmission electron microscopy (TEM) of Epon embedded samples, which necessitates hydrophobic sample pretreatment. Here, we report the direct observation of tissue in aqueous solution by atmospheric scanning electron microscopy (ASEM). Salivary glands, lacrimal glands, and pancreas were fixed, sectioned into slabs, stained with phosphotungstic acid (PTA), and inspected in radical scavenger d-glucose solution from below by an inverted scanning electron microscopy (SEM), guided by optical microscopy from above to target the tissue substructures. A 2- to 3-µm specimen thickness was visualized by the SEM. In secretory cells, cytoplasmic vesicles and other organelles were clearly imaged at high resolution, and the former could be classified according to the degree of PTA staining. In islets of Langerhans, the microvascular system used as an outlet by the secretory cells was also clearly observed. Microvascular system is also critically involved in the onset of diabetic complications and was clearly visible in subcutaneous tissue imaged by ASEM. The results suggest the use of in-solution ASEM for histology and to study vesicle secretion systems. Further, the high-throughput of ASEM makes it a potential tool for the diagnosis of exocrine and endocrine-related diseases.

  18. 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.

  19. High-resolution MR imaging of triangular fibrocartilage complex (TFCC): comparison of microscopy coils and a conventional small surface coil

    Energy Technology Data Exchange (ETDEWEB)

    Yoshioka, Hiroshi [Department of Radiology, University of Tsukuba, Tsukuba (Japan); Department of Radiology, Brigham and Women' s Hospital, 75 Francis Street, 02115, Boston, MA (United States); Ueno, Teruko; Itai, Yuji [Department of Radiology, University of Tsukuba, Tsukuba (Japan); Tanaka, Toshikazu [Department of Orthopedic Surgery, Tsukuba Kinen Hospital, Tsukuba (Japan); Shindo, Masashi [Tsukuba University Hospital, Tsukuba (Japan)

    2003-10-01

    To compare MR images of the triangular fibrocartilage complex (TFCC) using microscopy coils with those using a conventional surface coil qualitatively and quantitatively. Proton density-weighted images and T2*-weighted images of the TFCC from ten normal volunteers were obtained with a conventional surface coil (C4 coil; 80 mm in diameter), a 47-mm microscopy surface coil and a 23-mm microscopy surface coil at 1.5 T. Qualitative image analysis of MR images with three coils was performed by two radiologists who assigned one of five numerical scores (0, nonvisualization; 1, poor; 2, average; 3, good; 4, excellent) for five TFCC components, which were disc proper, triangular ligament, meniscus homologue, ulnotriquetral and ulnolunate ligament. Quantitative analysis included the signal-to-noise ratio (S/N) of the disc proper of TFCC, the lunate cartilage, the lunate bone and the contrast-noise-ratio (C/N) between articular cartilage and disc proper or bone marrow were measured. All structures show higher scores qualitatively on MR with microscopy coils than those with a C4 coil, and the difference was significant with the exception of the ulnolunate ligament. MR with microscopy coils showed significantly higher S/N values than those with a conventional surface coil (P<0.05 to P<0.001). T2*-weighted images using microscopy coils showed significantly higher cartilage-disc proper C/N and cartilage-bone marrow C/N (P<0.01 to P<0.001). On proton density-weighted images, the C/N between cartilage and disc proper with two microscopy coils was significantly higher (P<0.01) than that with a conventional coil. High-resolution MR images of the normal wrist using microscopy coils were superior to those using a conventional surface coil qualitatively and quantitatively. High-resolution MR imaging with a microscopy coil would be a promising method to diagnose TFCC lesions. (orig.)

  20. Bright field microscopy as an alternative to whole cell fluorescence in automated analysis of macrophage images.

    Directory of Open Access Journals (Sweden)

    Jyrki Selinummi

    Full Text Available BACKGROUND: 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. METHODOLOGY: 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. SIGNIFICANCE: 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.