Mapping microscopic order in plant and mammalian cells and tissues: novel differential polarization attachment for new generation confocal microscopes (DP-LSM)

Science.gov (United States)

Steinbach, G.; Pawlak, K.; Pomozi, I.; Tóth, E. A.; Molnár, A.; Matkó, J.; Garab, G.

2014-03-01

Elucidation of the molecular architecture of complex, highly organized molecular macro-assemblies is an important, basic task for biology. Differential polarization (DP) measurements, such as linear (LD) and circular dichroism (CD) or the anisotropy of the fluorescence emission (r), which can be carried out in a dichrograph or spectrofluorimeter, respectively, carry unique, spatially averaged information about the molecular organization of the sample. For inhomogeneous samples—e.g. cells and tissues—measurements on macroscopic scale are not satisfactory, and in some cases not feasible, thus microscopic techniques must be applied. The microscopic DP-imaging technique, when based on confocal laser scanning microscope (LSM), allows the pixel by pixel mapping of anisotropy of a sample in 2D and 3D. The first DP-LSM configuration, which, in fluorescence mode, allowed confocal imaging of different DP quantities in real-time, without interfering with the ‘conventional’ imaging, was built on a Zeiss LSM410. It was demonstrated to be capable of determining non-confocally the linear birefringence (LB) or LD of a sample and, confocally, its FDLD (fluorescence detected LD), the degree of polarization (P) and the anisotropy of the fluorescence emission (r), following polarized and non-polarized excitation, respectively (Steinbach et al 2009 Acta Histochem.111 316-25). This DP-LSM configuration, however, cannot simply be adopted to new generation microscopes with considerably more compact structures. As shown here, for an Olympus FV500, we designed an easy-to-install DP attachment to determine LB, LD, FDLD and r, in new-generation confocal microscopes, which, in principle, can be complemented with a P-imaging unit, but specifically to the brand and type of LSM.

Mapping microscopic order in plant and mammalian cells and tissues: novel differential polarization attachment for new generation confocal microscopes (DP-LSM)

International Nuclear Information System (INIS)

Steinbach, G; Pawlak, K; Garab, G; Pomozi, I; Tóth, E A; Molnár, A; Matkó, J

2014-01-01

Elucidation of the molecular architecture of complex, highly organized molecular macro-assemblies is an important, basic task for biology. Differential polarization (DP) measurements, such as linear (LD) and circular dichroism (CD) or the anisotropy of the fluorescence emission (r), which can be carried out in a dichrograph or spectrofluorimeter, respectively, carry unique, spatially averaged information about the molecular organization of the sample. For inhomogeneous samples—e.g. cells and tissues—measurements on macroscopic scale are not satisfactory, and in some cases not feasible, thus microscopic techniques must be applied. The microscopic DP-imaging technique, when based on confocal laser scanning microscope (LSM), allows the pixel by pixel mapping of anisotropy of a sample in 2D and 3D. The first DP-LSM configuration, which, in fluorescence mode, allowed confocal imaging of different DP quantities in real-time, without interfering with the ‘conventional’ imaging, was built on a Zeiss LSM410. It was demonstrated to be capable of determining non-confocally the linear birefringence (LB) or LD of a sample and, confocally, its FDLD (fluorescence detected LD), the degree of polarization (P) and the anisotropy of the fluorescence emission (r), following polarized and non-polarized excitation, respectively (Steinbach et al 2009 Acta Histochem.111 316–25). This DP-LSM configuration, however, cannot simply be adopted to new generation microscopes with considerably more compact structures. As shown here, for an Olympus FV500, we designed an easy-to-install DP attachment to determine LB, LD, FDLD and r, in new-generation confocal microscopes, which, in principle, can be complemented with a P-imaging unit, but specifically to the brand and type of LSM. (paper)

Color Laser Microscope

Science.gov (United States)

Awamura, D.; Ode, T.; Yonezawa, M.

1987-04-01

A color laser microscope utilizing a new color laser imaging system has been developed for the visual inspection of semiconductors. The light source, produced by three lasers (Red; He-Ne, Green; Ar, Blue; He-Cd), is deflected horizontally by an AOD (Acoustic Optical Deflector) and vertically by a vibration mirror. The laser beam is focused in a small spot which is scanned over the sample at high speed. The light reflected back from the sample is reformed to contain linear information by returning to the original vibration mirror. The linear light is guided to the CCD image sensor where it is converted into a video signal. Individual CCD image sensors are used for each of the three R, G, or B color image signals. The confocal optical system with its laser light source yields a color TV monitor image with no flaring and a much sharper resolution than that of the conventional optical microscope. The AOD makes possible a high speed laser scan and a NTSC or PAL TV video signal is produced in real time without any video memory. Since the light source is composed of R, G, and B laser beams, color separation superior to that of white light illumination is achieved. Because of the photometric linearity of the image detector, the R, G, and B outputs of the system are most suitably used for hue analysis. The CCD linear image sensors in the optical system produce no geometrical distortion, and good color registration is available principally. The output signal can be used for high accuracy line width measuring. The many features of the color laser microscope make it ideally suited for the visual inspection of semiconductor processing. A number of these systems have already been installed in such a capacity. The Color Laser Microscope can also be a very useful tool for the fields of material engineering and biotechnology.

Digital Position Encoding Of Galvanometer Scanner In A Laser Microscope

Science.gov (United States)

Liljeborg, Anders

1988-09-01

An account is given of a realization of a feedback method to digitize the analog position signal from a moving iron galvanometer. It is employed in a confocal scanning laser microscope for generating digital images. The photometric sampling has to be closely coupled to the position of a mirror that scans a focused laser beam across a microscope specimen. Pictures with low geometric distortion are obtained up to the size 1024 x 1024 pixels.

Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture.

Science.gov (United States)

Diaspro, A; Corosu, M; Ramoino, P; Robello, M

1999-11-01

Within the framework of a national National Institute of Physics of Matter (INFM) project, we have realised a two-photon excitation (TPE) fluorescence microscope based on a new generation commercial confocal scanning head. The core of the architecture is a mode-locked Ti:Sapphire laser (Tsunami 3960, Spectra Physics Inc., Mountain View, CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.) and an ultracompact confocal scanning head, Nikon PCM2000 (Nikon Instruments, Florence, Italy) using a single-pinhole design. Three-dimensional point-spread function has been measured to define spatial resolution performances. The TPE microscope has been used with a wide range of excitable fluorescent molecules (DAPI, Fura-2, Indo-1, DiOC(6)(3), fluoresceine, Texas red) covering a single photon spectral range from UV to green. An example is reported on 3D imaging of the helical structure of the sperm head of the Octopus Eledone cirrhosa labelled with an UV excitable dye, i.e., DAPI. The system can be easily switched for operating both in conventional and two-photon mode. Copyright 1999 Wiley-Liss, Inc.

Fluorescence (Multiwave) Confocal Microscopy.

Science.gov (United States)

Welzel, J; Kästle, Raphaela; Sattler, Elke C

2016-10-01

In addition to reflectance confocal microscopy, multiwave confocal microscopes with different laser wavelengths in combination with exogenous fluorophores allow fluorescence mode confocal microscopy in vivo and ex vivo. Fluorescence mode confocal microscopy improves the contrast between the epithelium and the surrounding soft tissue and allows the depiction of certain structures, like epithelial tumors, nerves, and glands. Copyright © 2016 Elsevier Inc. All rights reserved.

Confocal Laser Endomicroscopy in Inflammatory Bowel Disease

DEFF Research Database (Denmark)

Rasmussen, Ditlev Nytoft; Karstensen, John Gásdal; Riis, Lene Buhl

2015-01-01

included. Next, eligible studies were analysed with respect to several parameters, such as technique and clinical aim and definitions of outcomes. RESULTS: Confocal laser endomicroscopy has been used for a wide range of purposes in inflammatory bowel disease, covering assessment of inflammatory severity...... of confocal laser endomicroscopy for inflammatory bowel disease. METHODS: Available literature was searched systematically for studies applying confocal laser endomicroscopy in Crohn's disease or ulcerative colitis. Relevant literature was reviewed and only studies reporting original clinical data were...... of histological features such as colonic crypts, epithelial gaps and epithelial leakiness to fluorescein. CONCLUSIONS: Confocal laser endomicroscopy remains an experimental but emerging tool for assessment of inflammatory bowel disease. It is the only method that enables in vivo functional assessment...

Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics.

Science.gov (United States)

Hayashi, Shinichi; Okada, Yasushi

2015-05-01

Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of micro-tubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30-100 frames/s. Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging. © 2015 Hayashi and Okada. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Evaluation and purchase of confocal microscopes: Numerous factors to consider

Science.gov (United States)

The purchase of a confocal microscope can be a complex and difficult decision for an individual scientist, group or evaluation committee. This is true even for scientists that have used confocal technology for many years. The task of reaching the optimal decision becomes almost i...

Live Imaging of Shoot Meristems on an Inverted Confocal Microscope Using an Objective Lens Inverter Attachment

Science.gov (United States)

Nimchuk, Zachary L.; Perdue, Tony D.

2017-01-01

Live imaging of above ground meristems can lead to new insights in plant development not possible from static imaging of fixed tissue. The use of an upright confocal microscope offers several technical and biological advantages for live imaging floral or shoot meristems. However, many departments and core facilities possess only inverted confocal microscopes and lack the funding for an additional upright confocal microscope. Here we show that imaging of living apical meristems can be performed on existing inverted confocal microscopes with the use of an affordable and detachable InverterScope accessory. PMID:28579995

Live Imaging of Shoot Meristems on an Inverted Confocal Microscope Using an Objective Lens Inverter Attachment.

Science.gov (United States)

Nimchuk, Zachary L; Perdue, Tony D

2017-01-01

Live imaging of above ground meristems can lead to new insights in plant development not possible from static imaging of fixed tissue. The use of an upright confocal microscope offers several technical and biological advantages for live imaging floral or shoot meristems. However, many departments and core facilities possess only inverted confocal microscopes and lack the funding for an additional upright confocal microscope. Here we show that imaging of living apical meristems can be performed on existing inverted confocal microscopes with the use of an affordable and detachable InverterScope accessory.

High resolution 3D confocal microscope imaging of volcanic ash particles.

Science.gov (United States)

Wertheim, David; Gillmore, Gavin; Gill, Ian; Petford, Nick

2017-07-15

We present initial results from a novel high resolution confocal microscopy study of the 3D surface structure of volcanic ash particles from two recent explosive basaltic eruptions, Eyjafjallajökull (2010) and Grimsvötn (2011), in Iceland. The majority of particles imaged are less than 100μm in size and include PM 10 s, known to be harmful to humans if inhaled. Previous studies have mainly used 2D microscopy to examine volcanic particles. The aim of this study was to test the potential of 3D laser scanning confocal microscopy as a reliable analysis tool for these materials and if so to what degree high resolution surface and volume data could be obtained that would further aid in their classification. First results obtained using an Olympus LEXT scanning confocal microscope with a ×50 and ×100 objective lens are highly encouraging. They reveal a range of discrete particle types characterised by sharp or concave edges consistent with explosive formation and sudden rupture of magma. Initial surface area/volume ratios are given that may prove useful in subsequent modelling of damage to aircraft engines and human tissue where inhalation has occurred. Copyright © 2017 Elsevier B.V. All rights reserved.

Confocal laser endomicroscopy

DEFF Research Database (Denmark)

Karstensen, John Gásdal; Săftoiu, Adrian; Brynskov, Jørn

2016-01-01

BACKGROUND AND STUDY AIMS: Confocal laser endomicroscopy (CLE) has been shown to predict relapse in ulcerative colitis in remission, but little is currently known about its role in Crohn's disease. The aim of this study was to identify reproducible CLE features in patients with Crohn's disease...

Methods for studying biofilm formation: flow cells and confocal laser scanning microscopy

DEFF Research Database (Denmark)

Tolker-Nielsen, Tim; Sternberg, Claus

2014-01-01

In this chapter methods for growing and analyzing biofilms under hydrodynamic conditions in flow cells are described. Use of flow cells allows for direct microscopic investigation of biofilm formation. The flow in these chambers is essentially laminar, which means that the biofilms can be grown u......, inoculation of the flow cells, running of the system, confocal laser scanning microscopy and image analysis, and disassembly and cleaning of the system....

A portable confocal hyperspectral microscope without any scan or tube lens and its application in fluorescence and Raman spectral imaging

Science.gov (United States)

Li, Jingwei; Cai, Fuhong; Dong, Yongjiang; Zhu, Zhenfeng; Sun, Xianhe; Zhang, Hequn; He, Sailing

2017-06-01

In this study, a portable confocal hyperspectral microscope is developed. In traditional confocal laser scanning microscopes, scan lens and tube lens are utilized to achieve a conjugate relationship between the galvanometer and the back focal plane of the objective, in order to achieve a better resolution. However, these lenses make it difficult to scale down the volume of the system. In our portable confocal hyperspectral microscope (PCHM), the objective is placed directly next to the galvomirror. Thus, scan lens and tube lens are not included in our system and the size of this system is greatly reduced. Furthermore, the resolution is also acceptable in many biomedical and food-safety applications. Through reducing the optical length of the system, the signal detection efficiency is enhanced. This is conducive to realizing both the fluorescence and Raman hyperspectral imaging. With a multimode fiber as a pinhole, an improved image contrast is also achieved. Fluorescent spectral images for HeLa cells/fingers and Raman spectral images of kumquat pericarp are present. The spectral resolution and spatial resolutions are about 0.4 nm and 2.19 μm, respectively. These results demonstrate that this portable hyperspectral microscope can be used in in-vivo fluorescence imaging and in situ Raman spectral imaging.

Confocal endomicroscopy for in vivo microscopic analysis of upper gastrointestinal tract premalignant and malignant lesions.

Science.gov (United States)

Gheorghe, Cristian; Iacob, Razvan; Becheanu, Gabriel; Dumbrav Abreve, Mona

2008-03-01

Confocal LASER endomicroscopy (CLE) is a new endoscopic technique which allows subsurface in vivo microscopic analysis during ongoing endoscopy, using systemically or topically administered fluorescent agents. It allows targeted biopsies to be taken, potentially improving the diagnostic rate in certain gastrointestinal diseases. Worldwide experience with CLE for upper gastrointestinal malignant and premalignant lesions is still reduced. Potential clinical applications are presented, including diagnosis of NERD, Barrett's esophagus, atrophic gatritis, gastric intestinal metaplasia and dysplasia, gastric adenomatous or hyperplastic polyps, gastric cancer.

Configurations of the Re-scan Confocal Microscope (RCM) for biomedical applications

NARCIS (Netherlands)

de Luca, G. M. R.; Desclos, E.; Breedijk, R. M. P.; Dolz-Edo, L.; Smits, G. J.; Bielefeld, P.; Picavet, L.; Fitzsimons, C. P.; Hoebe, R.; Manders, E. M. M.

2017-01-01

The new high-sensitive and high-resolution technique, Re-scan Confocal Microscopy (RCM), is based on a standard confocal microscope extended with a re-scan detection unit. The re-scan unit includes a pair of re-scanning mirrors that project the emission light onto a camera in a scanning manner. The

Configurations of the Re-scan Confocal Microscope (RCM) for biomedical applications

NARCIS (Netherlands)

De Luca, G.M.R.; Desclos, E.; Breedijk, R.M.P.; Dolz-Edo, L.; Smits, G.J.; Nahidiazar, L.; Bielefeld, P.; Picavet, L.; Fitzsimons, C.P.; Hoebe, R.; Manders, E.M.M.

The new high-sensitive and high-resolution technique, Re-scan Confocal Microscopy (RCM), is based on a standard confocal microscope extended with a re-scan detection unit. The re-scan unit includes a pair of re-scanning mirrors that project the emission light onto a camera in a scanning manner. The

Confocal laser endomicroscopy in ulcerative colitis

DEFF Research Database (Denmark)

Karstensen, John Gásdal; Săftoiu, Adrian; Brynskov, Jørn

2016-01-01

BACKGROUND AND AIMS: Confocal laser endomicroscopy enables real-time in vivo microscopy during endoscopy and can predict relapse in patients with inflammatory bowel disease in remission. However, little is known about how endomicroscopic features change with time. The aim of this longitudinal study...... was to correlate colonic confocal laser endomicroscopy (CLE) in ulcerative colitis with histopathology and macroscopic appearance before and after intensification of medical treatment. METHODS: Twenty-two patients with ulcerative colitis in clinical relapse and 7 control subjects referred for colonoscopy were...

Confocal Microscope Alignment of Nanocrystals for Coherent Diffraction Imaging

International Nuclear Information System (INIS)

Beitra, Loren; Watari, Moyu; Matsuura, Takashi; Shimamoto, Naonobu; Harder, Ross; Robinson, Ian

2010-01-01

We have installed and tested an Olympus LEXT confocal microscope at the 34-ID-C beamline of the Advanced Photon Source (APS). The beamline is for Coherent X-ray Diffraction (CXD) experiments in which a nanometre-sized crystal is aligned inside a focussed X-ray beam. The microscope was required for three-dimensional (3D) sample alignment to get around sphere-of-confusion issues when locating Bragg peaks in reciprocal space. In this way, and by use of strategic sample preparations, we have succeeded in measuring six Bragg peaks from a single 200 nm gold crystal and obtained six projections of its internal displacement field. This enables the clear identification of stacking-fault bands within the crystal. The confocal alignment method will allow a full determination of the strain tensor provided three or more Bragg reflections from the same crystal are found.

In-situ Crystallization of Highly Volatile Commercial Mold Flux Using an Isolated Observation System in the Confocal Laser Scanning Microscope

Science.gov (United States)

Park, Jun-Yong; Ryu, Jae Wook; Sohn, Il

2014-08-01

The in situ crystallization behavior of highly volatile commercial mold fluxes for medium carbon steels was investigated using the confocal laser scanning microscope (CLSM) equipped with an optimized isolated observation system. The highly volatile compounds of the mold flux were suppressed during heating allowing direct observation in the CLSM. Cooling rates of 25, 50, 100, 400, and 800 K/min were incorporated and continuous cooling transformation (CCT) diagrams of 4 different commercial mold fluxes for medium carbon steels were developed. Identification of the crystalline phase was conducted with XRD and SEM-EDS analysis. A cuspidine crystalline was observed in all samples at various cooling rates. With higher basicity, CaF2, and NaF, the crystallization of the fluxes was enhanced according to the CCT diagram. As the slag structure becomes depolymerized, the diffusion rate of the cathodic ions seems to increase.

Confocal laser microscopic imaging of conspicuous facial pores in vivo: relation between the appearance and the internal structure of skin.

Science.gov (United States)

Sugata, Keiichi; Nishijima, Takafumi; Kitahara, Takashi; Takema, Yoshinori

2008-05-01

Conspicuous facial pores are one of the more serious esthetic defects of most concern to women. Previous microscopic observations of the skin surface around conspicuous pores have discovered large hollows and uneven skin tone. In this study, the observation area was extended from the skin surface to deeper skin to find the characteristic features of conspicuous pores in a wider spectrum. First, a magnified surface image of the cheek skin was obtained using a video microscope. Second, replicas were collected from the same area. Third, the horizontal cross-sectioned images of the epidermis and papillary dermis in different depths were non-invasively obtained using in vivo confocal laser scanning microscopy. These images were compared with each other to find a correlation between features of the skin surface and those of deeper layers. In cross-sectioned images of conspicuous pores, a strongly undulated epidermal-dermal junction was commonly observed around a pore's opening. Areas with this feature correlated well to the areas with larger hollows and an uneven skin tone. Our results indicate that there is a positive correlation between the incidence of the characteristic feature at the epidermal-dermal junction and the visual appearance of a pore.

A Clinical and Confocal Microscopic Comparison of Transepithelial PRK and LASEK for Myopia

Directory of Open Access Journals (Sweden)

Safak Korkmaz

2014-01-01

Full Text Available Purpose. To compare the clinical and confocal microscopic results of transepithelial PRK versus LASEK for correction of myopia. Materials and Methods. Twelve patients with myopia received transepithelial PRK in one eye and LASEK in the other. In transepithelial PRK-treated eyes, the corneal epithelium was removed with 40 microns of excimer laser ablation and in LASEK-treated eyes with 25-second application of 18% ethanol. Time to epithelial healing, ocular discomfort, uncorrected and best corrected visual acuities, manifest refraction, haze, greyscale value, and keratocyte apoptosis in confocal microscopy were recorded. Results. The mean time to epithelial healing was significantly longer after LASEK (4.00 ± 0.43 versus 3.17 ± 0.6 days. On day 1, ocular discomfort was significantly higher after transepithelial PRK. The grade of haze, keratocyte apoptosis, and greyscale value in confocal microscopy were significantly higher in transepithelial PRK-treated eyes at 1 month. All transepithelial PRK- and LASEK-treated eyes achieved 20/25 or better UCVA and were within ±1.00 D of emmetropia at final visits. Conclusions. Both transepithelial PRK and LASEK offer effective correction of myopia at 1 year. However, LASEK appeared to induce less discomfort and less intense wound healing in the early postoperative period.

A Clinical and Confocal Microscopic Comparison of Transepithelial PRK and LASEK for Myopia.

Science.gov (United States)

Korkmaz, Safak; Bilgihan, Kamil; Sul, Sabahattin; Hondur, Ahmet

2014-01-01

Purpose. To compare the clinical and confocal microscopic results of transepithelial PRK versus LASEK for correction of myopia. Materials and Methods. Twelve patients with myopia received transepithelial PRK in one eye and LASEK in the other. In transepithelial PRK-treated eyes, the corneal epithelium was removed with 40 microns of excimer laser ablation and in LASEK-treated eyes with 25-second application of 18% ethanol. Time to epithelial healing, ocular discomfort, uncorrected and best corrected visual acuities, manifest refraction, haze, greyscale value, and keratocyte apoptosis in confocal microscopy were recorded. Results. The mean time to epithelial healing was significantly longer after LASEK (4.00 ± 0.43 versus 3.17 ± 0.6 days). On day 1, ocular discomfort was significantly higher after transepithelial PRK. The grade of haze, keratocyte apoptosis, and greyscale value in confocal microscopy were significantly higher in transepithelial PRK-treated eyes at 1 month. All transepithelial PRK- and LASEK-treated eyes achieved 20/25 or better UCVA and were within ±1.00 D of emmetropia at final visits. Conclusions. Both transepithelial PRK and LASEK offer effective correction of myopia at 1 year. However, LASEK appeared to induce less discomfort and less intense wound healing in the early postoperative period.

3-D reconstruction of neurons from multichannel confocal laser scanning image series.

Science.gov (United States)

Wouterlood, Floris G

2014-04-10

A confocal laser scanning microscope (CLSM) collects information from a thin, focal plane and ignores out-of-focus information. Scanning of a specimen, with stepwise axial (Z-) movement of the stage in between each scan, produces Z-series of confocal images of a tissue volume, which then can be used to 3-D reconstruct structures of interest. The operator first configures separate channels (e.g., laser, filters, and detector settings) for each applied fluorochrome and then acquires Z-series of confocal images: one series per channel. Channel signal separation is extremely important. Measures to avoid bleaching are vital. Post-acquisition deconvolution of the image series is often performed to increase resolution before 3-D reconstruction takes place. In the 3-D reconstruction programs described in this unit, reconstructions can be inspected in real time from any viewing angle. By altering viewing angles and by switching channels off and on, the spatial relationships of 3-D-reconstructed structures with respect to structures visualized in other channels can be studied. Since each brand of CLSM, computer program, and 3-D reconstruction package has its own proprietary set of procedures, a general approach is provided in this protocol wherever possible. Copyright © 2014 John Wiley & Sons, Inc.

Living Matter Observations with a Novel Hyperspectral Supercontinuum Confocal Microscope for VIS to Near-IR Reflectance Spectroscopy

Directory of Open Access Journals (Sweden)

Francesca R. Bertani

2013-10-01

Full Text Available A broad range hyper-spectroscopic microscope fed by a supercontinuum laser source and equipped with an almost achromatic optical layout is illustrated with detailed explanations of the design, implementation and data. The real novelty of this instrument, a confocal spectroscopic microscope capable of recording high resolution reflectance data in the VIS-IR spectral range from about 500 nm to 2.5 μm wavelengths, is the possibility of acquiring spectral data at every physical point as defined by lateral coordinates, X and Y, as well as at a depth coordinate, Z, as obtained by the confocal optical sectioning advantage. With this apparatus we collect each single scanning point as a whole spectrum by combining two linear spectral detector arrays, one CCD for the visible range, and one InGaAs infrared array, simultaneously available at the sensor output channel of the home made instrument. This microscope has been developed for biomedical analysis of human skin and other similar applications. Results are shown illustrating the technical performances of the instrument and the capability in extracting information about the composition and the structure of different parts or compartments in biological samples as well as in solid statematter. A complete spectroscopic fingerprinting of samples at microscopic level is shown possible by using statistical analysis on raw data or analytical reflectance models based on Abelés matrix transfer methods.

A multiphoton laser scanning microscope setup for transcranial in vivo brain imaging on mice

Science.gov (United States)

Nase, Gabriele; Helm, P. Johannes; Reppen, Trond; Ottersen, Ole Petter

2005-12-01

We describe a multiphoton laser scanning microscope setup for transcranial in vivo brain imaging in mice. The modular system is based on a modified industrial standard Confocal Scanning Laser Microscope (CSLM) and is assembled mainly from commercially available components. A special multifunctional stage, which is optimized for both laser scanning microscopic observation and preparative animal surgery, has been developed and built. The detection unit includes a highly efficient photomultiplier tube installed in a Peltier-cooled thermal box shielding the detector from changes in room temperature and from distortions caused by external electromagnetic fields. The images are recorded using a 12-bit analog-to-digital converter. Depending on the characteristics of the staining, individual nerve cells can be imaged down to at least 100μm below the intact cranium and down to at least 200μm below the opened cranium.

Design and analysis of a cross-type structured-illumination confocal microscope for high speed and high resolution

International Nuclear Information System (INIS)

Kim, Young-Duk; Ahn, MyoungKi; Kim, Taejoong; Gweon, DaeGab; Yoo, Hongki

2012-01-01

There have been many studies about a super resolution microscope for many years. A super resolution microscope can detect the physical phenomena or morphology of a biological sample more precisely than conventional microscopes. The structured-illumination microscope (SIM) is one of the technologies that demonstrate super resolution. However, the conventional SIM requires more time to obtain one resolution-enhanced image than other super resolution microscopes. More specifically, the conventional SIM uses three images with a 120° phase difference for each direction and three different directions are image-processed to make one resolution enhancement by increasing the optical transfer function in three directions. In this paper, we present a novel cross structured-illumination confocal microscope (CSICM) that takes the advantage of the technology of both SIM and the confocal microscope. The CSICM uses only two directions with three phase difference images, for a total of six images. By reducing the number of images that must be obtained, the total image acquisition time and image reconstruction time in obtaining the final output images can be decreased, and the confocal microscope provides axial information of the sample automatically. We demonstrate our method of cross illumination and evaluate the performance of the CSICM and compare it to the conventional SIM and the confocal microscope. (paper)

CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: SPECTROSCOPY

Science.gov (United States)

The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of emitted signals. The accuracy of these measurements demands that...

Spectral confocal reflection microscopy using a white light source

Science.gov (United States)

Booth, M.; Juškaitis, R.; Wilson, T.

2008-08-01

We present a reflection confocal microscope incorporating a white light supercontinuum source and spectral detection. The microscope provides images resolved spatially in three-dimensions, in addition to spectral resolution covering the wavelength range 450-650nm. Images and reflection spectra of artificial and natural specimens are presented, showing features that are not normally revealed in conventional microscopes or confocal microscopes using discrete line lasers. The specimens include thin film structures on semiconductor chips, iridescent structures in Papilio blumei butterfly scales, nacre from abalone shells and opal gemstones. Quantitative size and refractive index measurements of transparent beads are derived from spectral interference bands.

Laser-induced cartilage damage: an ex-vivo model using confocal microscopy

Science.gov (United States)

Frenz, Martin; Zueger, Benno J.; Monin, D.; Weiler, C.; Mainil-Varlet, P. M.; Weber, Heinz P.; Schaffner, Thomas

1999-06-01

Although there is an increasing popularity of lasers in orthopedic surgery, there is a growing concern about negative side effects of this therapy e.g. prolonged restitution time, radiation damage to adjacent cartilage or depth effects like bone necrosis. Despite case reports and experimental investigations over the last few years little is known about the extent of acute cartilage damage induced by different lasers types and energies. Histological examination offers only limited insights in cell viability and metabolism. Ho:YAG and Er:YAG lasers emitting at 2.1 micrometer and 2.94 micrometer, respectively, are ideally suited for tissue treatment because these wavelengths are strongly absorbed in water. The Purpose of the present study is to evaluate the effect of laser type and energy on chondrocyte viability in an ex vivo model. Free running Er:YAG (E equals 100 and 150 mJ) and Ho:YAG (E equals 500 and 800 mJ) lasers were used at different energy levels using a fixed pulse length of 400 microseconds. The energy was delivered at 8 Hz through optical fibers. Fresh bovine hyaline cartilage samples were mounted in a water bath at room temperature and the fiber was positioned at 30 degree and 180 degree angles relative to the tissue surface. After laser irradiation the samples were assessed by a life-dead cell viability test using a confocal microscope and by standard histology. Thermal damage was much deeper with Ho:YAG (up to 1800 micrometer) than with the Er:YAG laser (up to 70 micrometer). The cell viability test revealed a damage zone about twice the one determined by standard histology. Confocal microscopy is a powerful tool for assessing changes in tissue structure after laser treatment. In addition this technique allows to quantify these alterations without necessitating time consuming and expensive animal experiments.

Evaluation and purchase of confocal microscopes: numerous factors to consider.

Science.gov (United States)

Zucker, Robert M; Chua, Michael

2010-10-01

The purchase of a confocal microscope is a difficult decision. Many factors need to be considered, which include hardware, software, company, support, service, and price. These issues are discussed to help guide the purchasing process. © 2010 by John Wiley & Sons, Inc.

Dark-field scanning confocal microscope for vertical particle tracks in nuclear emulsion

International Nuclear Information System (INIS)

Astakhov, A.Ya.; Batusov, Yu.A.; Soroko, L.M.; Tereshchenko, S.V.; Tereshchenko, V.V.

1999-01-01

The principle of the DArk-FIeld Scanning CONfocal (DAFISCON) microscope for selective observation of the vertical particle tracks in nuclear emulsion is described. The construction of the DAFISCON microscope, built on the basis of the 2D measurement microscope, is described. The results of the experimental testing of the DAFISCON microscope, accomplished at high density of the vertical particle tracks, are presented. The 2D plot and the 1D plot of the CCD dark-field image are given. The spatial resolution of our microscope can be increased by using the objective with higher aperture

Three-dimensional imaging of porous media using confocal laser scanning microscopy.

Science.gov (United States)

Shah, S M; Crawshaw, J P; Boek, E S

2017-02-01

In the last decade, imaging techniques capable of reconstructing three-dimensional (3-D) pore-scale model have played a pivotal role in the study of fluid flow through complex porous media. In this study, we present advances in the application of confocal laser scanning microscopy (CLSM) to image, reconstruct and characterize complex porous geological materials with hydrocarbon reservoir and CO 2 storage potential. CLSM has a unique capability of producing 3-D thin optical sections of a material, with a wide field of view and submicron resolution in the lateral and axial planes. However, CLSM is limited in the depth (z-dimension) that can be imaged in porous materials. In this study, we introduce a 'grind and slice' technique to overcome this limitation. We discuss the practical and technical aspects of the confocal imaging technique with application to complex rock samples including Mt. Gambier and Ketton carbonates. We then describe the complete workflow of image processing to filtering and segmenting the raw 3-D confocal volumetric data into pores and grains. Finally, we use the resulting 3-D pore-scale binarized confocal data obtained to quantitatively determine petrophysical pore-scale properties such as total porosity, macro- and microporosity and single-phase permeability using lattice Boltzmann (LB) simulations, validated by experiments. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: SPECTROSCOPY AND FOUNDATIONS FOR QUANTITATION

Science.gov (United States)

The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The reliability of the CLSM to obtain specific measurements and quantify fluorescence data is dependent on using a correctly aligned machine that contains a stable laser power. For man...

Assessment of statistical agreement of three techniques for the study of cut marks: 3D digital microscope, laser scanning confocal microscopy and micro-photogrammetry.

Science.gov (United States)

Maté-González, Miguel Ángel; Aramendi, Julia; Yravedra, José; Blasco, Ruth; Rosell, Jordi; González-Aguilera, Diego; Domínguez-Rodrigo, Manuel

2017-09-01

In the last few years, the study of cut marks on bone surfaces has become fundamental for the interpretation of prehistoric butchery practices. Due to the difficulties in the correct identification of cut marks, many criteria for their description and classification have been suggested. Different techniques, such as three-dimensional digital microscope (3D DM), laser scanning confocal microscopy (LSCM) and micro-photogrammetry (M-PG) have been recently applied to the study of cut marks. Although the 3D DM and LSCM microscopic techniques are the most commonly used for the 3D identification of cut marks, M-PG has also proved to be very efficient and a low-cost method. M-PG is a noninvasive technique that allows the study of the cortical surface without any previous preparation of the samples, and that generates high-resolution models. Despite the current application of microscopic and micro-photogrammetric techniques to taphonomy, their reliability has never been tested. In this paper, we compare 3D DM, LSCM and M-PG in order to assess their resolution and results. In this study, we analyse 26 experimental cut marks generated with a metal knife. The quantitative and qualitative information registered is analysed by means of standard multivariate statistics and geometric morphometrics to assess the similarities and differences obtained with the different methodologies. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Orbital single particle tracking on a commercial confocal microscope using piezoelectric stage feedback

International Nuclear Information System (INIS)

Lanzanò, L; Gratton, E

2014-01-01

Single Particle Tracking (SPT) is a technique used to locate fluorescent particles with nanometer precision. In the orbital tracking method the position of a particle is obtained analyzing the distribution of intensity along a circular orbit scanned around the particle. In combination with an active feedback this method allows tracking of particles in 2D and 3D with millisecond temporal resolution. Here we describe a SPT setup based on a feedback approach implemented with minimal modification of a commercially available confocal laser scanning microscope, the Zeiss LSM 510, in combination with an external piezoelectric stage scanner. The commercial microscope offers the advantage of a user-friendly software interface and pre-calibrated hardware components. The use of an external piezo-scanner allows the addition of feedback into the system but also represents a limitation in terms of its mechanical response. We describe in detail this implementation of the orbital tracking method and discuss advantages and limitations. As an example of application to live cell experiments we perform the 3D tracking of acidic vesicles in live polarized epithelial cells. (paper)

Evaluation of Enterococcus faecalis adhesion, penetration, and method to prevent the penetration of Enterococcus faecalis into root cementum: Confocal laser scanning microscope and scanning electron microscope analysis.

Science.gov (United States)

Halkai, Rahul S; Hegde, Mithra N; Halkai, Kiran R

2016-01-01

To ascertain the role of Enterococcus faecalis in persistent infection and a possible method to prevent the penetration of E. faecalis into root cementum. One hundred and twenty human single-rooted extracted teeth divided into five groups. Group I (control): intact teeth, Group II: no apical treatment done, Group III divided into two subgroups. In Groups IIIa and IIIb, root apex treated with lactic acid of acidic and neutral pH, respectively. Group IV: apical root cementum exposed to lactic acid and roughened to mimic the apical resorption. Group V: apical treatment done same as Group IV and root-end filling done using mineral trioxide aggregate (MTA). Apical one-third of all samples immersed in E. faecalis broth for 8 weeks followed by bone morphogenetic protein and obturation and again immersed into broth for 8 weeks. Teeth split into two halves and observed under confocal laser scanning microscope and scanning electron microscope, organism identified by culture and polymerase chain reaction techniques. Adhesion and penetration was observed in Group IIIa and Group IV. Only adhesion in Group II and IIIB and no adhesion and penetration in Group I and V. Adhesion and penetration of E. faecalis into root cementum providing a long-term nidus for subsequent infection are the possible reason for persistent infection and root-end filling with MTA prevents the adhesion and penetration.

Confocal Laser Endomicroscopy in Neurosurgery: A New Technique with Much Potential

Directory of Open Access Journals (Sweden)

David Breuskin

2013-01-01

Full Text Available Technical innovations in brain tumour diagnostic and therapy have led to significant improvements of patient outcome and recurrence free interval. The use of technical devices such as surgical microscopes as well as neuronavigational systems have helped localising tumours as much as fluorescent agents, such as 5-aminolaevulinic acid, have helped visualizing pathologically altered tissue. Nonetheless, intraoperative instantaneous frozen sections and histological diagnosis remain the only method of gaining certainty of the nature of the resected tissue. This technique is time consuming and does not provide close-to-real-time information. In gastroenterology, confocal endoscopy closed the gap between tissue resection and histological examination, providing an almost real-time histological diagnosis. The potential of this technique using a confocal laser endoscope EndoMAG1 by Karl Storz Company was evaluated by our group on pig brains, tumour tissue cell cultures, and fresh human tumour specimen. Here, the authors report for the first time on the results of applying this new technique and provide first confocal endoscopic images of various brain and tumour structures. In all, the technique harbours a very promising potential to provide almost real-time intraoperative diagnosis, but further studies are needed to provide evidence for the technique’s potential.

Automated Microscopy: Macro Language Controlling a Confocal Microscope and its External Illumination: Adaptation for Photosynthetic Organisms.

Science.gov (United States)

Steinbach, Gábor; Kaňa, Radek

2016-04-01

Photosynthesis research employs several biophysical methods, including the detection of fluorescence. Even though fluorescence is a key method to detect photosynthetic efficiency, it has not been applied/adapted to single-cell confocal microscopy measurements to examine photosynthetic microorganisms. Experiments with photosynthetic cells may require automation to perform a large number of measurements with different parameters, especially concerning light conditions. However, commercial microscopes support custom protocols (through Time Controller offered by Olympus or Experiment Designer offered by Zeiss) that are often unable to provide special set-ups and connection to external devices (e.g., for irradiation). Our new system combining an Arduino microcontroller with the Cell⊕Finder software was developed for controlling Olympus FV1000 and FV1200 confocal microscopes and the attached hardware modules. Our software/hardware solution offers (1) a text file-based macro language to control the imaging functions of the microscope; (2) programmable control of several external hardware devices (light sources, thermal controllers, actuators) during imaging via the Arduino microcontroller; (3) the Cell⊕Finder software with ergonomic user environment, a fast selection method for the biologically important cells and precise positioning feature that reduces unwanted bleaching of the cells by the scanning laser. Cell⊕Finder can be downloaded from http://www.alga.cz/cellfinder. The system was applied to study changes in fluorescence intensity in Synechocystis sp. PCC6803 cells under long-term illumination. Thus, we were able to describe the kinetics of phycobilisome decoupling. Microscopy data showed that phycobilisome decoupling appears slowly after long-term (>1 h) exposure to high light.

Spatially resolved analyses of uranium species using a coupled system made up of confocal laser-scanning microscopy (CLSM) and laser induced fluorescence spectroscopy (LIFS)

International Nuclear Information System (INIS)

Brockmann, S.; Grossmann, K.; Arnold, T.

2014-01-01

The fluorescent properties of uranium when excited by UV light are used increasingly for spectroscope analyses of uranium species within watery samples. Here, alongside the fluorescent properties of the hexavalent oxidation phases, the tetra and pentavalent oxidation phases also play an increasingly important role. The detection of fluorescent emission spectrums on solid and biological samples using (time-resolved) laser induced fluorescence spectroscopy (TRLFS or LIFS respectively) has, however, the disadvantage that no statements regarding the spatial localisation of the uranium can be made. However, particularly in complex, biological samples, such statements on the localisation of the uranium enrichment in the sample are desired, in order to e.g. be able to distinguish between intra and extra-cellular uranium bonds. The fluorescent properties of uranium (VI) compounds and minerals can also be used to detect their localisation within complex samples. So the application of fluorescent microscopic methods represents one possibility to localise and visualise uranium precipitates and enrichments in biological samples, such as biofilms or cells. The confocal laser-scanning microscopy (CLSM) is especially well suited to this purpose. Coupling confocal laser-scanning microscopy (CLSM) with laser induced fluorescence spectroscopy (LIFS) makes it possible to localise and visualise fluorescent signals spatially and three-dimensionally, while at the same time being able to detect spatially resolved, fluorescent-spectroscopic data. This technology is characterised by relatively low detection limits from up to 1.10 -6 M for uranium (VI) compounds within the confocal volume. (orig.)

Confocal laser scanning microscopy in study of bone calcification

Science.gov (United States)

Nishikawa, Tetsunari; Kokubu, Mayu; Kato, Hirohito; Imai, Koichi; Tanaka, Akio

2012-12-01

Bone regeneration in mandible and maxillae after extraction of teeth or tumor resection and the use of rough surface implants in bone induction must be investigated to elucidate the mechanism of calcification. The calcified tissues are subjected to chemical decalcification or physical grinding to observe their microscopic features with light microscopy and transmission electron microscopy where the microscopic tissue morphology is significantly altered. We investigated the usefulness of confocal laser scanning microscopy (CLSM) for this purpose. After staggering the time of administration of calcein and alizarin red to experimental rats and dogs, rat alveolar bone and dog femur grafted with coral as scaffold or dental implants were observed with CLSM. In rat alveolar bone, the calcification of newly-formed bone and net-like canaliculi was observed at the mesial bone from the roots progressed at the rate of 15 μm/day. In dog femur grafted with coral, newly-formed bones along the space of coral were observed in an orderly manner. In dog femur with dental implants, after 8 weeks, newly-formed bone proceeded along the rough surface of the implants. CLSM produced high-magnification images of newly-formed bone and thin sections were not needed.

Molecular confocal laser endomicroscopy

DEFF Research Database (Denmark)

Karstensen, John Gásdal; Klausen, Pia Helene; Saftoiu, Adrian

2014-01-01

While flexible endoscopy is essential for macroscopic evaluation, confocal laser endomicroscopy (CLE) has recently emerged as an endoscopic method enabling visualization at a cellular level. Two systems are currently available, one based on miniprobes that can be inserted via a conventional...... during on-going endoscopy), a novel world of molecular evaluation opens up. The method of molecular CLE could potentially be used for estimating the expression of important receptors in carcinomas, subsequently resulting in immediate individualization of treatment regimens, but also for improving...

In-vivo diagnosis and non-inasive monitoring of Imiquimod 5% cream for non-melanoma skin cancer using confocal laser scanning microscopy

International Nuclear Information System (INIS)

Dietterle, S; Lademann, J; Röwert-Huber, H-J; Stockfleth, E; Astner, S; Antoniou, C; Sterry, W

2008-01-01

Basal cell carcinoma (BCC) is the most common cutaneous malignancy with increasing incidence rates worldwide. A number of established treatments are available, including surgical excision. The emergence of new non-invasive treatment modalities has prompted the development of non-invasive optical devices for therapeutic monitoring and evaluating treatment efficacy. This study was aimed to evaluate the clinical applicability of a fluorescence confocal laser scanning microscope (CFLSM) for non-invasive therapeutic monitoring of basal cell carcinoma treated with Imiquimod (Aldara®) as topical immune-response modifier. Eight participants with a diagnosis of basal cell carcinoma (BCC) were enrolled in this investigation. Sequential evaluation during treatment with Imiquimod showed progressive normalization of the confocal histomorphologic parameters in correlation with normal skin. Confocal laser scanning microscopy was able to identify characteristic features of BCC and allowed the visualization of therapeutic effects over time. Thus our results indicate the clinical applicability of CFLSM imaging to evaluate treatment efficacy in vivo and non-invasively

Confocal Laser Endomicroscopy in the Study of Colonic Mucosa in IBD Patients: A Review

Directory of Open Access Journals (Sweden)

Francesca Salvatori

2012-01-01

Full Text Available Confocal laser endomicroscopy (CLE is one of several novel methods that provide real-time, high-resolution imaging at a micronscale via endoscopes. CLE and related technologies are often termed “virtual biopsy” as they simulate the images seen in traditional histology. Recently, the use of CLE was reported in the study of colonic mucosa in patients with inflammatory bowel diseases and in particular in patients affected by ulcerative colitis. CLE has the potential to have an important role in management of IBD patients as it can be used to assess the grading of colitis and in detection of microscopic colitis in endoscopically silent segments. Moreover, CLE can be used in surveillance programs especially in high-risk patients. This report aims to evaluate the current data on the application of confocal endomicroscopy in clinical gastroenterology and particularly in the study of colonic mucosa in UC patients.

Role of Confocal Laser Endomicroscopy in Detection of Residual Barrett's Esophagus after Radiofrequency Ablation

Directory of Open Access Journals (Sweden)

Giorgio Diamantis

2011-01-01

Full Text Available Endoscopic endoluminal radiofrequency ablation (RFA is a novel and promising modality for Barrett's esophagus (BE treatment. Actually the only surveillance method after the ablation treatment is random biopsies throughout the whole treated area. Confocal laser endomicroscopy (CLE is a new endoscopic imaging tool that permits high-resolution microscopic examination of the gastrointestinal tract. The technology has garnered increasing attention because of its ability to provide real-time “optical” biopsy specimens, with a very high sensitivity and specificity. This paper summarize the potential application of CLE in the surveillance of the reepithelialization of BE, after endoscopic RFA.

CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR CALIBRATION, QUANTITATION AND SPECTROSCOPY

Science.gov (United States)

The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of emitted signals. The accuracy of these measurements demands that...

Confocal microscope is able to detect calcium metabolic in neuronal infection by toxoplasma gondii

Science.gov (United States)

Sensusiati, A. D.; Priya, T. K. S.; Dachlan, Y. P.

2017-05-01

Calcium metabolism plays a very important role in neurons infected by Toxoplasma. Detection of change of calcium metabolism of neuron infected by Toxoplasma and Toxoplasma requires the calculation both quantitative and qualitative method. Confocal microscope has the ability to capture the wave of the fluorescent emission of the fluorescent dyes used in the measurement of cell calcium. The purpose of this study was to prove the difference in calcium changes between infected and uninfected neurons using confocal microscopy. Neuronal culture of human-skin-derived neural stem cell were divided into 6 groups, consisting 3 uninfected groups and 3 infected groups. Among the 3 groups were 2 hours, 24 hours and 48 hours. The neuron Toxoplasma gondii ratio was 1:5. Observation of intracellular calcium of neuron and tachyzoite, evidence of necrosis, apoptosis and the expression of Hsp 70 of neuron were examined by confocal microscope. The normality of the data was analysed by Kolmogorov-Smirnov Test, differentiation test was checked by t2 Test, and ANOVAs, for correlation test was done by Pearson Correlation Test. The calcium intensity of cytosolic neuron and T. gondii was significantly different from control groups (pneurons both in quantitatively and qualitatively.

Efficacy of oral exfoliative cytology in diabetes mellitus patients: a light microscopic and confocal microscopic study.

Science.gov (United States)

Gopal, Deepika; Malathi, N; Reddy, B Thirupathi

2015-03-01

Diabetes mellitus (DM) has become a global problem. By monitoring the health status of these individuals, diabetic complications can be prevented. We aimed to analyze alterations in the morphology and cytomorphometry of buccal epithelial cells of type 2 DM patients using oral exfoliative cytology technique and determine its importance in public health screening, diagnosis and monitoring of diabetes mellitus. The study was carried out in 100 type 2 DM patients and 30 healthy individuals. Smears were taken from the right buccal mucosa and stained by the Papanicolaou technique. Staining with Acridine orange was carried out to view qualitative changes with confocal laser scanning microscope (LSM-510 Meta). The cytomorphometry was evaluated using IMAGE PRO PLUS 5.5 software with Evolution LC camera. All findings were statistically analyzed. The results showed that with increase in fasting plasma glucose levels, there is significant increase in nuclear area, decrease in cytoplasmic area, and increase in nuclear cytoplasmic ratio (p inclusion, candida and keratinization. In the present study, we found significant alterations in the cytomorphometry and cytomorphology of buccal epithelial cells of type 2 DM patients. This study supports and extends the view that these cellular changes can alert the clinician to the possibility of diabetes and aid in monitoring of diabetes throughout the lifetime of the patient.

Confocal microscope is able to detect calcium metabolic in neuronal infection by toxoplasma gondii

International Nuclear Information System (INIS)

Sensusiati, A D; Priya, T K S; Dachlan, Y P

2017-01-01

Calcium metabolism plays a very important role in neurons infected by Toxoplasma. Detection of change of calcium metabolism of neuron infected by Toxoplasma and Toxoplasma requires the calculation both quantitative and qualitative method. Confocal microscope has the ability to capture the wave of the fluorescent emission of the fluorescent dyes used in the measurement of cell calcium. The purpose of this study was to prove the difference in calcium changes between infected and uninfected neurons using confocal microscopy. Neuronal culture of human-skin-derived neural stem cell were divided into 6 groups, consisting 3 uninfected groups and 3 infected groups. Among the 3 groups were 2 hours, 24 hours and 48 hours. The neuron Toxoplasma gondii ratio was 1:5. Observation of intracellular calcium of neuron and tachyzoite, evidence of necrosis, apoptosis and the expression of Hsp 70 of neuron were examined by confocal microscope. The normality of the data was analysed by Kolmogorov-Smirnov Test, differentiation test was checked by t2 Test, and ANOVAs, for correlation test was done by Pearson Correlation Test. The calcium intensity of cytosolic neuron and T. gondii was significantly different from control groups (p<0.05). There was also significant correlation between calcium intensity with the evidence of necrosis and Hsp70 expression at 2 hours after infection. Apoptosis and necrosis were simultaneously shown with calcium contribution in this study. Confocal microscopy can be used to measure calcium changes in infected and uninfected neurons both in quantitatively and qualitatively. (paper)

A low error reconstruction method for confocal holography to determine 3-dimensional properties

Energy Technology Data Exchange (ETDEWEB)

Jacquemin, P.B., E-mail: pbjacque@nps.edu [Mechanical Engineering, University of Victoria, EOW 548,800 Finnerty Road, Victoria, BC (Canada); Herring, R.A. [Mechanical Engineering, University of Victoria, EOW 548,800 Finnerty Road, Victoria, BC (Canada)

2012-06-15

A confocal holography microscope developed at the University of Victoria uniquely combines holography with a scanning confocal microscope to non-intrusively measure fluid temperatures in three-dimensions (Herring, 1997), (Abe and Iwasaki, 1999), (Jacquemin et al., 2005). The Confocal Scanning Laser Holography (CSLH) microscope was built and tested to verify the concept of 3D temperature reconstruction from scanned holograms. The CSLH microscope used a focused laser to non-intrusively probe a heated fluid specimen. The focused beam probed the specimen instead of a collimated beam in order to obtain different phase-shift data for each scan position. A collimated beam produced the same information for scanning along the optical propagation z-axis. No rotational scanning mechanisms were used in the CSLH microscope which restricted the scan angle to the cone angle of the probe beam. Limited viewing angle scanning from a single view point window produced a challenge for tomographic 3D reconstruction. The reconstruction matrices were either singular or ill-conditioned making reconstruction with significant error or impossible. Establishing boundary conditions with a particular scanning geometry resulted in a method of reconstruction with low error referred to as 'wily'. The wily reconstruction method can be applied to microscopy situations requiring 3D imaging where there is a single viewpoint window, a probe beam with high numerical aperture, and specified boundary conditions for the specimen. The issues and progress of the wily algorithm for the CSLH microscope are reported herein. -- Highlights: Black-Right-Pointing-Pointer Evaluation of an optical confocal holography device to measure 3D temperature of a heated fluid. Black-Right-Pointing-Pointer Processing of multiple holograms containing the cumulative refractive index through the fluid. Black-Right-Pointing-Pointer Reconstruction issues due to restricting angular scanning to the numerical aperture of the

A low error reconstruction method for confocal holography to determine 3-dimensional properties

International Nuclear Information System (INIS)

Jacquemin, P.B.; Herring, R.A.

2012-01-01

A confocal holography microscope developed at the University of Victoria uniquely combines holography with a scanning confocal microscope to non-intrusively measure fluid temperatures in three-dimensions (Herring, 1997), (Abe and Iwasaki, 1999), (Jacquemin et al., 2005). The Confocal Scanning Laser Holography (CSLH) microscope was built and tested to verify the concept of 3D temperature reconstruction from scanned holograms. The CSLH microscope used a focused laser to non-intrusively probe a heated fluid specimen. The focused beam probed the specimen instead of a collimated beam in order to obtain different phase-shift data for each scan position. A collimated beam produced the same information for scanning along the optical propagation z-axis. No rotational scanning mechanisms were used in the CSLH microscope which restricted the scan angle to the cone angle of the probe beam. Limited viewing angle scanning from a single view point window produced a challenge for tomographic 3D reconstruction. The reconstruction matrices were either singular or ill-conditioned making reconstruction with significant error or impossible. Establishing boundary conditions with a particular scanning geometry resulted in a method of reconstruction with low error referred to as “wily”. The wily reconstruction method can be applied to microscopy situations requiring 3D imaging where there is a single viewpoint window, a probe beam with high numerical aperture, and specified boundary conditions for the specimen. The issues and progress of the wily algorithm for the CSLH microscope are reported herein. -- Highlights: ► Evaluation of an optical confocal holography device to measure 3D temperature of a heated fluid. ► Processing of multiple holograms containing the cumulative refractive index through the fluid. ► Reconstruction issues due to restricting angular scanning to the numerical aperture of the beam. ► Minimizing tomographic reconstruction error by defining boundary

Degeneration process of fungiform taste buds after severing the human chorda tympani nerve--observation by confocal laser scanning microscopy.

Science.gov (United States)

Saito, Takehisa; Ito, Tetsufumi; Ito, Yumi; Kato, Yuji; Manabe, Yasuhiro; Narita, Norihiko

2015-03-01

To elucidate the degeneration process of fungiform taste buds after severing the chorda tympani nerve (CTN) by confocal laser scanning microscopy in vivo. Prospective study. University hospital. Seven consecutive patients whose CTN was severed during tympanoplasty for middle ear cholesteatoma. Diagnostic. Preoperative and postoperative gustatory functions were assessed by electrogustometry (EGM). An average of 10 fungiform papillae (FP) in the midlateral region of the tongue were periodically observed, and the number of taste buds was counted using a confocal laser microscope. Among them, 2 to 3 reference FPs were selected based on the typical form of the FP or characteristic arrangements of taste pores. Observation was performed before surgery, 1 or 2 days after surgery, 2 or 3 times a week until 2 weeks after surgery, once a week between 2 and 4 weeks, and every 2 to 4 weeks thereafter until all taste buds had disappeared. EGM thresholds showed no response within 1 month after surgery in all patients. The initial change in the degeneration process was the disappearance of taste pores. The surface of taste buds became covered with epithelium. Finally, taste buds themselves atrofied and disappeared. The time course of degeneration differed depending upon individuals, each FP, and each taste bud. By employing the generalized linear mixed model under the Poisson distribution, it was calculated that all taste buds would disappear at around 50 days after surgery. Confocal laser scanning microscopy was useful for clarifying the degeneration process of fungiform taste buds.

Avances en el estudio fractográfico de fibras cerámicas de circonaerbia mediante microscopía óptica confocal

Directory of Open Access Journals (Sweden)

López-Cepero, J. M.

2005-10-01

Full Text Available Laser scanning confocal microscopy (LSCM is a microscopic technique based on an optical construction which allows the microscope to discard the light coming from unfocused zones of the sample. Whereas LSCM is extensively used in Natural Sciences (Biology, Medicine..., its use in Materials Science is almost unexplored and, in particular, there are essentially no fractographical studies using LSCM. However, its characteristics (gathering of 3D information, better than micron resolution and simple sample preparation make LSCM an ideal tool in a wide selection of fractographical problems, owing to the fast adquisition of valuable information and to the excellent sinergy with scanning electron microscopy (SEM. In the present work, the authors study an interesting system (ZrO2-5% mol Er2O3 fibers, submitted to tensile strength in hightemperature conditions in detail with LSCM. In addition to showcasing the usefulness of LSCM in fractographical studies and revealing the characteristic texture of the fracture surface in such fibers, said texture is found to closely resemble the nanometric precipitate structure which is unique to this material.

La microscopía óptica confocal (LSCM, Laser Scanning Confocal Microscopy es una técnica microscópica basada en una construcción óptica que permite eliminar la luz procedente de zonas no enfocadas de la muestra. Mientras que es de amplio uso en Ciencias de la Vida, la aplicación de LSCM a la Ciencia de Materiales no ha sido apenas explorada, siendo prácticamente inexistentes los estudios fractográficos que se apoyen en LSCM. A pesar de ello, sus características (obtención de información tridimensional, resolución por debajo de la micra y sencilla preparación de muestras la convierten en una herramienta idónea para una multitud de problemas fractográficos, debido a la obtención rápida de valiosa información y a su buena coordinación con la microscopía electrónica de barrido (SEM. En este

Bright-field scanning confocal electron microscopy using a double aberration-corrected transmission electron microscope

Energy Technology Data Exchange (ETDEWEB)

Wang, Peng; Behan, Gavin; Kirkland, Angus I. [Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH (United Kingdom); Nellist, Peter D., E-mail: peter.nellist@materials.ox.ac.uk [Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH (United Kingdom); Cosgriff, Eireann C.; D' Alfonso, Adrian J.; Morgan, Andrew J.; Allen, Leslie J. [School of Physics, University of Melbourne, Parkville, Victoria 3010 (Australia); Hashimoto, Ayako [Advanced Nano-characterization Center, National Institute for Materials Science (NIMS), 3-13 Sakura, Tsukuba 305-0003 (Japan); Takeguchi, Masaki [Advanced Nano-characterization Center, National Institute for Materials Science (NIMS), 3-13 Sakura, Tsukuba 305-0003 (Japan); High Voltage Electron Microscopy Station, NIMS, 3-13 Sakura, Tsukuba 305-0003 (Japan); Mitsuishi, Kazutaka [Advanced Nano-characterization Center, National Institute for Materials Science (NIMS), 3-13 Sakura, Tsukuba 305-0003 (Japan); Quantum Dot Research Center, NIMS, 3-13 Sakura, Tsukuba 305-0003 (Japan); Shimojo, Masayuki [High Voltage Electron Microscopy Station, NIMS, 3-13 Sakura, Tsukuba 305-0003 (Japan); Advanced Science Research Laboratory, Saitama Institute of Technology, 1690 Fusaiji, Fukaya 369-0293 (Japan)

2011-06-15

Scanning confocal electron microscopy (SCEM) offers a mechanism for three-dimensional imaging of materials, which makes use of the reduced depth of field in an aberration-corrected transmission electron microscope. The simplest configuration of SCEM is the bright-field mode. In this paper we present experimental data and simulations showing the form of bright-field SCEM images. We show that the depth dependence of the three-dimensional image can be explained in terms of two-dimensional images formed in the detector plane. For a crystalline sample, this so-called probe image is shown to be similar to a conventional diffraction pattern. Experimental results and simulations show how the diffracted probes in this image are elongated in thicker crystals and the use of this elongation to estimate sample thickness is explored. -- Research Highlights: {yields} The confocal probe image in a scanning confocal electron microscopy image reveals information about the thickness and height of the crystalline layer. {yields} The form of the contrast in a three-dimensional bright-field scanning confocal electron microscopy image can be explained in terms of the confocal probe image. {yields} Despite the complicated form of the contrast in bright-field scanning confocal electron microscopy, we see that depth information is transferred on a 10 nm scale.

Parallel detection experiment of fluorescence confocal microscopy using DMD.

Science.gov (United States)

Wang, Qingqing; Zheng, Jihong; Wang, Kangni; Gui, Kun; Guo, Hanming; Zhuang, Songlin

2016-05-01

Parallel detection of fluorescence confocal microscopy (PDFCM) system based on Digital Micromirror Device (DMD) is reported in this paper in order to realize simultaneous multi-channel imaging and improve detection speed. DMD is added into PDFCM system, working to take replace of the single traditional pinhole in the confocal system, which divides the laser source into multiple excitation beams. The PDFCM imaging system based on DMD is experimentally set up. The multi-channel image of fluorescence signal of potato cells sample is detected by parallel lateral scanning in order to verify the feasibility of introducing the DMD into fluorescence confocal microscope. In addition, for the purpose of characterizing the microscope, the depth response curve is also acquired. The experimental result shows that in contrast to conventional microscopy, the DMD-based PDFCM system has higher axial resolution and faster detection speed, which may bring some potential benefits in the biology and medicine analysis. SCANNING 38:234-239, 2016. © 2015 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

Spatially resolved analyses of uranium species using a coupled system made up of confocal laser-scanning microscopy (CLSM) and laser induced fluorescence spectroscopy (LIFS); Ortsaufgeloeste Analyse von Uranspezies mittels einem Gekoppelten System aus Konfokaler Laser-Scanning Mikroskopie (CLSM) und Laser Induzierter Fluoreszenzspektroskopie (LIFS)

Energy Technology Data Exchange (ETDEWEB)

Brockmann, S. [Verein fuer Kernverfahrenstechnik und Analytik Rossendorf e.V. (VKTA), Dresden (Germany); Grossmann, K.; Arnold, T. [Helmholtz-Zentrum Dresden-Rossendorf e.V. (Germany). Inst. fuer Ressourcenoekologie

2014-01-15

The fluorescent properties of uranium when excited by UV light are used increasingly for spectroscope analyses of uranium species within watery samples. Here, alongside the fluorescent properties of the hexavalent oxidation phases, the tetra and pentavalent oxidation phases also play an increasingly important role. The detection of fluorescent emission spectrums on solid and biological samples using (time-resolved) laser induced fluorescence spectroscopy (TRLFS or LIFS respectively) has, however, the disadvantage that no statements regarding the spatial localisation of the uranium can be made. However, particularly in complex, biological samples, such statements on the localisation of the uranium enrichment in the sample are desired, in order to e.g. be able to distinguish between intra and extra-cellular uranium bonds. The fluorescent properties of uranium (VI) compounds and minerals can also be used to detect their localisation within complex samples. So the application of fluorescent microscopic methods represents one possibility to localise and visualise uranium precipitates and enrichments in biological samples, such as biofilms or cells. The confocal laser-scanning microscopy (CLSM) is especially well suited to this purpose. Coupling confocal laser-scanning microscopy (CLSM) with laser induced fluorescence spectroscopy (LIFS) makes it possible to localise and visualise fluorescent signals spatially and three-dimensionally, while at the same time being able to detect spatially resolved, fluorescent-spectroscopic data. This technology is characterised by relatively low detection limits from up to 1.10{sup -6} M for uranium (VI) compounds within the confocal volume. (orig.)

Observation of regenerated fungiform taste buds after severing the chorda tympani nerve using confocal laser scanning microscopy in vivo.

Science.gov (United States)

Saito, Takehisa; Ito, Tetsufumi; Kato, Yuji; Yamada, Takechiyo; Manabe, Yasuhiro; Narita, Norihiko

2014-03-01

To evaluate whether regenerated fungiform taste buds after severing the chorda tympani nerve can be detected by confocal laser scanning microscopy in vivo. Retrospective study. University hospital. Six patients with a normal gustatory function (Group 1), 9 patients with taste function recovery after severing the CTN (Group 2), and 5 patients without taste function recovery (Group 3) were included. In Groups 2 and 3, canal wall up (closed) tympanoplasty or canal wall down with canal reconstruction tympanoplasty was performed in all patients. Diagnostic. The severed nerves were readapted or approximated on the temporalis muscle fascia used to reconstruct the eardrum during surgery. Preoperative and postoperative gustatory functions were assessed using electrogustometry. Twelve to 260 months after severing the CTN, the surface of the midlateral region of the tongue was observed with a confocal laser microscope. EGM thresholds showed no response 1 month after surgery in all patients of Groups 2 and 3. In Group 2, EGM thresholds showed recovery 1 to 2 years after surgery and before confocal microscopy (-1.3 ± 6.5 dB). There was a significant difference between Group 1 (-5.7 ± 2.0 dB; p taste buds were observed in each FP, and 55 (79.7%) of 69 FP contained at least 1 taste bud. The mean number of taste bud per papilla was 3.7 ± 3.6. In patients with a recovered taste function (Group 2), 0 to 8 taste buds were observed in each FP. In this group, 54 (56.2%) of 94 FP contained at least 1 taste bud. The mean number of taste bud per papilla was 2.0 ± 2.2 (p taste bud was observed. Regenerated fungiform taste bud could be observed in vivo using confocal laser scanning microscopy, indicating that regenerated taste bud can be detected without biopsy.

Diffractive elements performance in chromatic confocal microscopy

International Nuclear Information System (INIS)

Garzon, J; Duque, D; Alean, A; Toledo, M; Meneses, J; Gharbi, T

2011-01-01

The Confocal Laser Scanning Microscopy (CLSM) has been widely used in the semiconductor industry and biomedicine because of its depth discrimination capability. Subsequent to this technique has been developed in recent years Chromatic Confocal Microscopy. This method retains the same principle of confocal and offers the added advantage of removing the axial movement of the moving system. This advantage is usually accomplished with an optical element that generates a longitudinal chromatic aberration and a coding system that relates the axial position of each point of the sample with the wavelength that is focused on each. The present paper shows the performance of compact chromatic confocal microscope when some different diffractive elements are used for generation of longitudinal chromatic aberration. Diffractive elements, according to the process and manufacturing parameters, may have different diffraction efficiency and focus a specific wavelength in a specific focal position. The performance assessment is carried out with various light sources which exhibit an incoherent behaviour and a broad spectral width.

A simple but precise method for quantitative measurement of the quality of the laser focus in a scanning optical microscope.

Science.gov (United States)

Trägårdh, J; Macrae, K; Travis, C; Amor, R; Norris, G; Wilson, S H; Oppo, G-L; McConnell, G

2015-07-01

We report a method for characterizing the focussing laser beam exiting the objective in a laser scanning microscope. This method provides the size of the optical focus, the divergence of the beam, the ellipticity and the astigmatism. We use a microscopic-scale knife edge in the form of a simple transmission electron microscopy grid attached to a glass microscope slide, and a light-collecting optical fibre and photodiode underneath the specimen. By scanning the laser spot from a reflective to a transmitting part of the grid, a beam profile in the form of an error function can be obtained and by repeating this with the knife edge at different axial positions relative to the beam waist, the divergence and astigmatism of the postobjective laser beam can be obtained. The measured divergence can be used to quantify how much of the full numerical aperture of the lens is used in practice. We present data of the beam radius, beam divergence, ellipticity and astigmatism obtained with low (0.15, 0.7) and high (1.3) numerical aperture lenses and lasers commonly used in confocal and multiphoton laser scanning microscopy. Our knife-edge method has several advantages over alternative knife-edge methods used in microscopy including that the knife edge is easy to prepare, that the beam can be characterized also directly under a cover slip, as necessary to reduce spherical aberrations for objectives designed to be used with a cover slip, and it is suitable for use with commercial laser scanning microscopes where access to the laser beam can be limited. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Transient gels in colloid-polymer mixtures studied with fluorescence confocal scanning laser microscopy

NARCIS (Netherlands)

Verhaegh, N.A.M.; Asnaghi, D.; Lekkerkerker, H.N.W.

1999-01-01

We study the structure and the time evolution of transient gels formed in colloid-polymer mixtures, by means of uorescence Confocal Scanning Laser Microscopy (CSLM). This technique is used in conjunction with novel colloidal silica particles containing a uorescent core. The confocal micrographs

Insight into the Microbial Multicellular Lifestyle via Flow-Cell Technology and Confocal Microscopy

DEFF Research Database (Denmark)

Pamp, Sünje Johanna; Sternberg, Claus; Tolker-Nielsen, Tim

2009-01-01

, industry, and human health. Accordingly a number of biofilm model systems, molecular tools, microscopic techniques, and image analysis programs have been employed for the study of biofilms under controlled and reproducible conditions. Studies using confocal laser scanning microscopy (CLSM) of biofilms...

Miniature in vivo MEMS-based line-scanned dual-axis confocal microscope for point-of-care pathology

Science.gov (United States)

Yin, C.; Glaser, A.K.; Leigh, S. Y.; Chen, Y.; Wei, L.; Pillai, P. C. S.; Rosenberg, M. C.; Abeytunge, S.; Peterson, G.; Glazowski, C.; Sanai, N.; Mandella, M. J.; Rajadhyaksha, M.; Liu, J. T. C.

2016-01-01

There is a need for miniature optical-sectioning microscopes to enable in vivo interrogation of tissues as a real-time and noninvasive alternative to gold-standard histopathology. Such devices could have a transformative impact for the early detection of cancer as well as for guiding tumor-resection procedures. Miniature confocal microscopes have been developed by various researchers and corporations to enable optical sectioning of highly scattering tissues, all of which have necessitated various trade-offs in size, speed, depth selectivity, field of view, resolution, image contrast, and sensitivity. In this study, a miniature line-scanned (LS) dual-axis confocal (DAC) microscope, with a 12-mm diameter distal tip, has been developed for clinical point-of-care pathology. The dual-axis architecture has demonstrated an advantage over the conventional single-axis confocal configuration for reducing background noise from out-of-focus and multiply scattered light. The use of line scanning enables fast frame rates (16 frames/sec is demonstrated here, but faster rates are possible), which mitigates motion artifacts of a hand-held device during clinical use. We have developed a method to actively align the illumination and collection beams in a DAC microscope through the use of a pair of rotatable alignment mirrors. Incorporation of a custom objective lens, with a small form factor for in vivo clinical use, enables our device to achieve an optical-sectioning thickness and lateral resolution of 2.0 and 1.1 microns respectively. Validation measurements with reflective targets, as well as in vivo and ex vivo images of tissues, demonstrate the clinical potential of this high-speed optical-sectioning microscopy device. PMID:26977337

A confocal optical microscope for detection of single impurities in a bulk crystal at cryogenic temperatures.

Science.gov (United States)

Karlsson, Jenny; Rippe, Lars; Kröll, Stefan

2016-03-01

A compact sample-scanning confocal optical microscope for detection of single impurities below the surface of a bulk crystal at cryogenic temperatures is described. The sample, lens, and scanners are mounted inside a helium bath cryostat and have a footprint of only 19 × 19 mm. Wide field imaging and confocal imaging using a Blu-ray lens immersed in liquid helium are demonstrated with excitation at 370 nm. A spatial resolution of 300 nm and a detection efficiency of 1.6% were achieved.

WE-FG-BRA-04: A Portable Confocal Microscope to Image Live Cell Damage Response Induced by Therapeutic Radiation

Energy Technology Data Exchange (ETDEWEB)

McFadden, C; Flint, D; Grosshans, D; Sawakuchi, G [The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Sadetaporn, D [The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Rice University, Houston, TX (United States); Asaithamby, A [UT Southwestern Medical Center, Dallas, TX (United States)

2016-06-15

Purpose: To construct a custom and portable fluorescence confocal laser-scanning microscope (FCLSM) that can be placed in the path of therapeutic radiation beams to study real-time radiation-induced damage response in live cells. Methods: We designed and constructed a portable FCLSM with three laser diodes for excitation (405, 488, and 635 nm). An objective lens focuses the excitation light and collects fluorescence from the sample. A pair of galvanometer mirrors scans/collects the laser beam/fluorescence along the focal plane (x/y-directions). A stepper motor stage scans in the axial direction and positions the x/y of the image field. Barrier filters and dichroic mirrors are used to route the spectral emission bands to the appropriate photodetector. An avalanche photodiode collects near-infrared fluorescence; a photodiode collects back-reflected 635 nm light; and a photomultiplier tube collects green fluorescence in the range of eGFP/eYFP. A 200-µm diameter pinhole was used to implement the confocal geometry for near-infrared and red channels and a 150-µm diameter pinhole for the green channel. Data acquisition and system control were achieved using a high-throughput data acquisition card. In-house software developed in LabVIEW was used to control the hardware, collect data from the photodetectors and reconstruct the confocal images. Results: 6 frames/s can be acquired for a 25 µm{sup 2} (128×128 pixels) field of view, visualizing the entire volume of the cell nucleus (∼10 µm depth) in <10 s. To demonstrate the usefulness of our FCLSM, we imaged gold nanoshells in live cells, radiation-induced damage in fibrosarcoma cells expressing eGFP tagged to a DNA repair protein, and neurons expressing eGFP. The system can also image particle tracks in fluorescent nuclear track detectors. Conclusion: We developed a versatile and portable FCLSM that allows radiobiology studies in live cells exposed to therapeutic radiation. The FCLSM can be placed in any vertical beam

WE-FG-BRA-04: A Portable Confocal Microscope to Image Live Cell Damage Response Induced by Therapeutic Radiation

International Nuclear Information System (INIS)

McFadden, C; Flint, D; Grosshans, D; Sawakuchi, G; Sadetaporn, D; Asaithamby, A

2016-01-01

Purpose: To construct a custom and portable fluorescence confocal laser-scanning microscope (FCLSM) that can be placed in the path of therapeutic radiation beams to study real-time radiation-induced damage response in live cells. Methods: We designed and constructed a portable FCLSM with three laser diodes for excitation (405, 488, and 635 nm). An objective lens focuses the excitation light and collects fluorescence from the sample. A pair of galvanometer mirrors scans/collects the laser beam/fluorescence along the focal plane (x/y-directions). A stepper motor stage scans in the axial direction and positions the x/y of the image field. Barrier filters and dichroic mirrors are used to route the spectral emission bands to the appropriate photodetector. An avalanche photodiode collects near-infrared fluorescence; a photodiode collects back-reflected 635 nm light; and a photomultiplier tube collects green fluorescence in the range of eGFP/eYFP. A 200-µm diameter pinhole was used to implement the confocal geometry for near-infrared and red channels and a 150-µm diameter pinhole for the green channel. Data acquisition and system control were achieved using a high-throughput data acquisition card. In-house software developed in LabVIEW was used to control the hardware, collect data from the photodetectors and reconstruct the confocal images. Results: 6 frames/s can be acquired for a 25 µm 2 (128×128 pixels) field of view, visualizing the entire volume of the cell nucleus (∼10 µm depth) in <10 s. To demonstrate the usefulness of our FCLSM, we imaged gold nanoshells in live cells, radiation-induced damage in fibrosarcoma cells expressing eGFP tagged to a DNA repair protein, and neurons expressing eGFP. The system can also image particle tracks in fluorescent nuclear track detectors. Conclusion: We developed a versatile and portable FCLSM that allows radiobiology studies in live cells exposed to therapeutic radiation. The FCLSM can be placed in any vertical beam line

Electrophoretic Detection and Confocal Microscopic Imaging of Tyrosine Nitrated Proteins in Plant Tissue.

Science.gov (United States)

Arora, Dhara; Singh, Neha; Bhatla, Satish C

2018-01-01

Tyrosine nitrated proteins can be detected in plant cells electrophoretically and their distribution can be monitored by confocal laser scanning microscopy (CLSM) imaging. One-dimensional polyacrylamide gel electrophoresis (1D PAGE) followed by Western blotting using polyclonal antibody against 3-nitrotyrosine residues enables detection of tyrosine nitrated proteins in plant cells. Here we describe detection of tyrosine nitrated proteins in the homogenates derived from sunflower (Helianthus annuus L.) seedling cotyledons. Total soluble proteins obtained from tissue homogenates are resolved using vertical gel electrophoresis followed by their electrophoretic transfer on to a microporous membrane support for immunodetection. Spatial distribution of tyrosine nitrated proteins can be visualized using an antibody against 3-nitrotyrosine residues. Immunofluorescent localization is performed by cutting 7 μm thick wax sections of tissue followed by incubation in primary anti-nitrotyrosine antibody (dilution 1:200) and secondary Cy-3 labeled anti-rabbit IgG antibody (dilution 1:1500). Confocal laser scanning microscopy analysis is undertaken using argon lasers (ex: 530-550 nm and em: 570 nm) at pinhole 1. Modulation in the abundance and spatial localization of tyrosine nitrated proteins in plant tissues can be monitored using these techniques.

Two-Photon Fluorescence Microscope for Microgravity Research

Science.gov (United States)

Fischer, David G.; Zimmerli, Gregory A.; Asipauskas, Marius

2005-01-01

A two-photon fluorescence microscope has been developed for the study of biophysical phenomena. Two-photon microscopy is a novel form of laser-based scanning microscopy that enables three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon optical microscopy, two-photon microscopy utilizes the simultaneous nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption, so an ultra-fast pulsed laser source is typically employed. On the other hand, the critical energy threshold for two-photon absorption leads to fluorophore excitation that is intrinsically localized to the focal volume. Consequently, two-photon microscopy enables optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction (relative to one-photon optical microscopy) in photon-induced damage because of the longer excitation wavelength. This reduction is especially advantageous for in vivo studies. Relative to confocal microscopy, there is also a reduction in background fluorescence, and, because of a reduction in Rayleigh scattering, there is a 4 increase of penetration depth. The prohibitive cost of a commercial two-photon fluorescence-microscope system, as well as a need for modularity, has led to the construction of a custom-built system (see Figure 1). This system includes a coherent mode-locked titanium: sapphire laser emitting 120-fs-duration pulses at a repetition rate of 80 MHz. The pulsed laser has an average output power of 800 mW and a wavelength tuning range of 700 to 980 nm, enabling the excitation of a variety of targeted fluorophores. The output from the laser is attenuated, spatially filtered, and then directed into a confocal scanning head that has been modified to provide for side entry of the laser beam. The laser output coupler has been replaced with a dichroic filter that reflects the

Super-resolution for everybody: An image processing workflow to obtain high-resolution images with a standard confocal microscope.

Science.gov (United States)

Lam, France; Cladière, Damien; Guillaume, Cyndélia; Wassmann, Katja; Bolte, Susanne

2017-02-15

In the presented work we aimed at improving confocal imaging to obtain highest possible resolution in thick biological samples, such as the mouse oocyte. We therefore developed an image processing workflow that allows improving the lateral and axial resolution of a standard confocal microscope. Our workflow comprises refractive index matching, the optimization of microscope hardware parameters and image restoration by deconvolution. We compare two different deconvolution algorithms, evaluate the necessity of denoising and establish the optimal image restoration procedure. We validate our workflow by imaging sub resolution fluorescent beads and measuring the maximum lateral and axial resolution of the confocal system. Subsequently, we apply the parameters to the imaging and data restoration of fluorescently labelled meiotic spindles of mouse oocytes. We measure a resolution increase of approximately 2-fold in the lateral and 3-fold in the axial direction throughout a depth of 60μm. This demonstrates that with our optimized workflow we reach a resolution that is comparable to 3D-SIM-imaging, but with better depth penetration for confocal images of beads and the biological sample. Copyright © 2016 Elsevier Inc. All rights reserved.

Efficacy of 4 Irrigation Protocols in Killing Bacteria Colonized in Dentinal Tubules Examined by a Novel Confocal Laser Scanning Microscope Analysis.

Science.gov (United States)

Azim, Adham A; Aksel, Hacer; Zhuang, Tingting; Mashtare, Terry; Babu, Jegdish P; Huang, George T-J

2016-06-01

The aim of this study was to determine the efficiency of 4 irrigation systems in eliminating bacteria in root canals, particularly in dentinal tubules. Roots of human teeth were prepared to 25/04, autoclaved, and inoculated with Enterococcus faecalis for 3 weeks. Canals were then disinfected by (1) standard needle irrigation, (2) sonically agitating with EndoActivator, (3) XP Endo finisher, or (4) erbium:yttrium aluminum garnet laser (PIPS) (15 roots/group). The bacterial reduction in the canal was determined by MTT assays. For measuring live versus dead bacteria in the dentinal tubules (4 teeth/group), teeth were split open and stained with LIVE/DEAD BackLight. Coronal, middle, and apical thirds of the canal dentin were scanned by using a confocal laser scanning microscope (CLSM) to determine the ratio of dead/total bacteria in the dentinal tubules at various depths. All 4 irrigation protocols significantly eliminated bacteria in the canal, ranging from 89.6% to 98.2% reduction (P bacteria in the coronal, middle, and apical segments at 50-μm depth. On the other hand, PIPS had the greatest bacterial killing efficiency at the 150-μm depth in all 3 root segments. XP Endo appears to be more efficient than other 3 techniques in disinfecting the main canal space and up to 50 μm deep into the dentinal tubules. PIPS appears to be most effective in killing the bacteria deep in the dentinal tubules. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

An FFT-based Method for Attenuation Correction in Fluorescence Confocal Microscopy

NARCIS (Netherlands)

Roerdink, J.B.T.M.; Bakker, M.

1993-01-01

A problem in three-dimensional imaging by a confocal scanning laser microscope (CSLM) in the (epi)fluorescence mode is the darkening of the deeper layers due to absorption and scattering of both the excitation and the fluorescence light. In this paper we propose a new method to correct for these

Optomechatronics Design and Control for Confocal Laser Scanning Microscopy

NARCIS (Netherlands)

Yoo, H.W.

2015-01-01

Confocal laser scanning microscopy (CLSM) is considered as one of the major advancements in microscopy in the last century and is widely accepted as a 3D fluorescence imaging tool for biological studies. For the emerging biological questions CLSM requires fast imaging to detect rapid biological

Site-specific confocal fluorescence imaging of biological microstructures in a turbid medium

International Nuclear Information System (INIS)

Saloma, Caesar; Palmes-Saloma, Cynthia; Kondoh, Hisato

1998-01-01

Normally transparent biological structures in a turbid medium are imaged using a laser confocal microscope and multiwavelength site-specific fluorescence labelling. The spatial filtering capability of the detector pinhole in the confocal microscope limits the number of scattered fluorescent photons that reach the photodetector. Simultaneous application of different fluorescent markers on the same sample site minimizes photobleaching by reducing the excitation time for each marker. A high-contrast grey-level image is also produced by summing confocal images of the same site taken at different fluorescence wavelengths. Monte Carlo simulations are performed to obtain the quantitative behaviour of confocal fluorescence imaging in turbid media. Confocal images of the following samples were also obtained: (i) 15 μm diameter fluorescent spheres placed 1.16 mm deep beneath an aqueous suspension of 0.0823 μm diameter polystyrene latex spheres, and (ii) hindbrain of a whole-mount mouse embryo (age 10 days) that was stained to fluoresce at 515 nm and 580 nm peak wavelengths. Expression of RNA transcripts of a gene within the embryo hindbrain was detected by a fluorescence-based whole-mount in situ hybridization procedure that we recently tested. (author)

Nanoscale Energy-Filtered Scanning Confocal Electron Microscopy Using a Double-Aberration-Corrected Transmission Electron Microscope

International Nuclear Information System (INIS)

Wang Peng; Behan, Gavin; Kirkland, Angus I.; Nellist, Peter D.; Takeguchi, Masaki; Hashimoto, Ayako; Mitsuishi, Kazutaka; Shimojo, Masayuki

2010-01-01

We demonstrate that a transmission electron microscope fitted with two spherical-aberration correctors can be operated as an energy-filtered scanning confocal electron microscope. A method for establishing this mode is described and initial results showing 3D chemical mapping with nanoscale sensitivity to height and thickness changes in a carbon film are presented. Importantly, uncorrected chromatic aberration does not limit the depth resolution of this technique and moreover performs an energy-filtering role, which is explained in terms of a combined depth and energy-loss response function.

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.

Spinning-disk confocal microscopy: present technology and future trends.

Science.gov (United States)

Oreopoulos, John; Berman, Richard; Browne, Mark

2014-01-01

Live-cell imaging requires not only high temporal resolution but also illumination powers low enough to minimize photodamage. Traditional single-point laser scanning confocal microscopy (LSCM) is generally limited by both the relatively slow speed at which it can acquire optical sections by serial raster scanning (a few Hz) and the higher potential for phototoxicity. These limitations have driven the development of rapid, parallel forms of confocal microscopy, the most popular of which is the spinning-disk confocal microscope (SDCM). Here, we briefly introduce the SDCM technique, discuss its strengths and weaknesses against LSCM, and update the reader on some recent developments in SDCM technology that improve its performance and expand its utility for life science research now and in the future. © 2014 Elsevier Inc. All rights reserved.

Assessing delivery and quantifying efficacy of small interfering ribonucleic acid therapeutics in the skin using a dual-axis confocal microscope

Science.gov (United States)

Ra, Hyejun; Gonzalez-Gonzalez, Emilio; Smith, Bryan R.; Gambhir, Sanjiv S.; Kino, Gordon S.; Solgaard, Olav; Kaspar, Roger L.; Contag, Christopher H.

2010-05-01

Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.

InGaP - CREATE portal | LSDB Archive [Life Science Database Archive metadata

Lifescience Database Archive (English)

Full Text Available : Fixed mouse brain sections were fluorescent-labeled and observed by confocal laser scanning microscope usi...ted GFP-fusion KIAA/mKIAA in HEK293 cells were observed by fluorescent microscope...ope (neocortex 1) Observation by Confocal laser scanning microscope (neocortex 1) C...onfocal laser scanning microscope (neocortex 2) Observation by Confocal laser scanning microscope (neocortex... 2) Confocal laser scanning microscope (hippocampus) Observation by Confocal laser scanning microscope

Confocal laser feedback tomography for skin cancer detection.

Science.gov (United States)

Mowla, Alireza; Du, Benjamin Wensheng; Taimre, Thomas; Bertling, Karl; Wilson, Stephen; Soyer, H Peter; Rakić, Aleksandar D

2017-09-01

Tomographic imaging of soft tissue such as skin has a potential role in cancer detection. The penetration of infrared wavelengths makes a confocal approach based on laser feedback interferometry feasible. We present a compact system using a semiconductor laser as both transmitter and receiver. Numerical and physical models based on the known optical properties of keratinocyte cancers were developed. We validated the technique on three phantoms containing macro-structural changes in optical properties. Experimental results were in agreement with numerical simulations and structural changes were evident which would permit discrimination of healthy tissue and tumour. Furthermore, cancer type discrimination was also able to be visualized using this imaging technique.

3D imaging of cement-based materials at submicron resolution by combining laser scanning confocal microscopy with serial sectioning.

Science.gov (United States)

Yio, M H N; Mac, M J; Wong, H S; Buenfeld, N R

2015-05-01

In this paper, we present a new method to reconstruct large volumes of nontransparent porous materials at submicron resolution. The proposed method combines fluorescence laser scanning confocal microscopy with serial sectioning to produce a series of overlapping confocal z-stacks, which are then aligned and stitched based on phase correlation. The method can be extended in the XY plane to further increase the overall image volume. Resolution of the reconstructed image volume does not degrade with increase in sample size. We have used the method to image cementitious materials, hardened cement paste and concrete and the results obtained show that the method is reliable. Possible applications of the method such as three-dimensional characterization of the pores and microcracks in hardened concrete, three-dimensional particle shape characterization of cementitious materials and three-dimensional characterization of other porous materials such as rocks and bioceramics are discussed. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging.

Science.gov (United States)

Zhao, Ming; Li, Yu; Peng, Leilei

2014-05-05

We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community.

Confocal laser scanning microscopy in study of bone calcification

International Nuclear Information System (INIS)

Nishikawa, Tetsunari; Kokubu, Mayu; Kato, Hirohito; Imai, Koichi; Tanaka, Akio

2012-01-01

Highlights: ► High-magnification images with depth selection, and thin sections were observed using CLSM. ► The direction and velocity of calcification of the bone was observed by administration of 2 fluorescent dyes. ► In dog femora grafted with coral blocks, newly-formed bone was observed in the coral block space with a rough surface. ► Twelve weeks after dental implant was grafted in dog femora, the space between screws was filled with newly-formed bones. - Abstract: Bone regeneration in mandible and maxillae after extraction of teeth or tumor resection and the use of rough surface implants in bone induction must be investigated to elucidate the mechanism of calcification. The calcified tissues are subjected to chemical decalcification or physical grinding to observe their microscopic features with light microscopy and transmission electron microscopy where the microscopic tissue morphology is significantly altered. We investigated the usefulness of confocal laser scanning microscopy (CLSM) for this purpose. After staggering the time of administration of calcein and alizarin red to experimental rats and dogs, rat alveolar bone and dog femur grafted with coral as scaffold or dental implants were observed with CLSM. In rat alveolar bone, the calcification of newly-formed bone and net-like canaliculi was observed at the mesial bone from the roots progressed at the rate of 15 μm/day. In dog femur grafted with coral, newly-formed bones along the space of coral were observed in an orderly manner. In dog femur with dental implants, after 8 weeks, newly-formed bone proceeded along the rough surface of the implants. CLSM produced high-magnification images of newly-formed bone and thin sections were not needed.

Analysis of polymer grafted inside the porous hydrogel using confocal laser scanning microscopy

Directory of Open Access Journals (Sweden)

2007-04-01

Full Text Available Graft polymerization of glycidyl methacrylate onto the pore surface of polyacrylamide macroporous gel was implemented in DMSO-aqueous solution using diperiodatocuprate(III complexes as an initiator. The grafting densities up to 410% were achieved. The graft polymerization was confirmed by gravimetrical methods and FTIR. The graft polymerization of polymer inside the pores of the macroporous gel resulted in increased flow resistance through the gel matrix. The distribution of grafted polymer on the gel pore surface material was studied by scanning electron microscopy (SEM and confocal laser scanning microscopy (CLSM. CLSM is an alternative method for studying morphology of gel surface with grafted polymer having the advantages over the SEM allowing to investigate the distribution of grafted polymer inside the hydrogel in a native hydrated state. The microscopic techniques demonstrated uneven distribution of the grafted polymer inside the gel pores as a result of initiating the graft polymerization by insoluble initiator deposited on the pore surface.

Scanning laser microscope for imaging nanostructured superconductors

International Nuclear Information System (INIS)

Ishida, Takekazu; Arai, Kohei; Akita, Yukio; Miyanari, Mitsunori; Minami, Yusuke; Yotsuya, Tsutomu; Kato, Masaru; Satoh, Kazuo; Uno, Mayumi; Shimakage, Hisashi; Miki, Shigehito; Wang, Zhen

2010-01-01

The nanofabrication of superconductors yields various interesting features in superconducting properties. A variety of different imaging techniques have been developed for probing the local superconducting profiles. A scanning pulsed laser microscope has been developed by the combination of the XYZ piezo-driven stages and an optical fiber with an aspheric focusing lens. The scanning laser microscope is used to understand the position-dependent properties of a superconducting MgB 2 stripline of length 100 μm and width of 3 μm under constant bias current. Our results show that the superconducting stripline can clearly be seen in the contour image of the scanning laser microscope on the signal voltage. It is suggested from the observed image that the inhomogeneity is relevant in specifying the operating conditions such as detection efficiency of the sensor.

Scanning laser microscope for imaging nanostructured superconductors

Science.gov (United States)

Ishida, Takekazu; Arai, Kohei; Akita, Yukio; Miyanari, Mitsunori; Minami, Yusuke; Yotsuya, Tsutomu; Kato, Masaru; Satoh, Kazuo; Uno, Mayumi; Shimakage, Hisashi; Miki, Shigehito; Wang, Zhen

2010-10-01

The nanofabrication of superconductors yields various interesting features in superconducting properties. A variety of different imaging techniques have been developed for probing the local superconducting profiles. A scanning pulsed laser microscope has been developed by the combination of the XYZ piezo-driven stages and an optical fiber with an aspheric focusing lens. The scanning laser microscope is used to understand the position-dependent properties of a superconducting MgB 2 stripline of length 100 μm and width of 3 μm under constant bias current. Our results show that the superconducting stripline can clearly be seen in the contour image of the scanning laser microscope on the signal voltage. It is suggested from the observed image that the inhomogeneity is relevant in specifying the operating conditions such as detection efficiency of the sensor.

(LMRG): Microscope Resolution, Objective Quality, Spectral Accuracy and Spectral Un-mixing

Science.gov (United States)

Bayles, Carol J.; Cole, Richard W.; Eason, Brady; Girard, Anne-Marie; Jinadasa, Tushare; Martin, Karen; McNamara, George; Opansky, Cynthia; Schulz, Katherine; Thibault, Marc; Brown, Claire M.

2012-01-01

The second study by the LMRG focuses on measuring confocal laser scanning microscope (CLSM) resolution, objective lens quality, spectral imaging accuracy and spectral un-mixing. Affordable test samples for each aspect of the study were designed, prepared and sent to 116 labs from 23 countries across the globe. Detailed protocols were designed for the three tests and customized for most of the major confocal instruments being used by the study participants. One protocol developed for measuring resolution and objective quality was recently published in Nature Protocols (Cole, R. W., T. Jinadasa, et al. (2011). Nature Protocols 6(12): 1929–1941). The first study involved 3D imaging of sub-resolution fluorescent microspheres to determine the microscope point spread function. Results of the resolution studies as well as point spread function quality (i.e. objective lens quality) from 140 different objective lenses will be presented. The second study of spectral accuracy looked at the reflection of the laser excitation lines into the spectral detection in order to determine the accuracy of these systems to report back the accurate laser emission wavelengths. Results will be presented from 42 different spectral confocal systems. Finally, samples with double orange beads (orange core and orange coating) were imaged spectrally and the imaging software was used to un-mix fluorescence signals from the two orange dyes. Results from 26 different confocal systems will be summarized. Time will be left to discuss possibilities for the next LMRG study.

Confocal fluorescence microscopy for minimal-invasive tumor diagnosis

International Nuclear Information System (INIS)

Zenzinger, M.; Bille, J.

2000-01-01

The goal of the project ''stereotactic laser-neurosurgery'' is the development of a system for careful and minimal-invasive resection of brain tumors with ultrashort laser pulses through a thin probe. A confocal laser-scanning-microscope is integrated in the probe. In this paper, the simulation of its optical properties by a laboratory setup and the expansion by the ability for fluorescence microscopy are reported. For a valuation of the imaging properties, the point-spread-function in three dimensions and the axial depth-transfer-function were measured and thus, among other things, the resolving power and the capacity for depth discrimination were analysed. The microscope will enable intra-operative detection of tumor cells by the method of immunofluorescence. As a first model of the application in the brain, cell cultures, that fluorescein-labelled antibodies were bound to specifically, were used in this work. Due to the fluorescence signal, it was possible to detect and identify clearly the areas that had been marked in this manner, proving the suitability of the setup for minimal-invasive tumor diagnosis. (orig.)

Multicolor probe-based confocal laser endomicroscopy: a new world for in vivo and real-time cellular imaging

Science.gov (United States)

Vercauteren, Tom; Doussoux, François; Cazaux, Matthieu; Schmid, Guillaume; Linard, Nicolas; Durin, Marie-Amélie; Gharbi, Hédi; Lacombe, François

2013-03-01

Since its inception in the field of in vivo imaging, endomicroscopy through optical fiber bundles, or probe-based Confocal Laser Endomicroscopy (pCLE), has extensively proven the benefit of in situ and real-time examination of living tissues at the microscopic scale. By continuously increasing image quality, reducing invasiveness and improving system ergonomics, Mauna Kea Technologies has turned pCLE not only into an irreplaceable research instrument for small animal imaging, but also into an accurate clinical decision making tool with applications as diverse as gastrointestinal endoscopy, pulmonology and urology. The current implementation of pCLE relies on a single fluorescence spectral band making different sources of in vivo information challenging to distinguish. Extending the pCLE approach to multi-color endomicroscopy therefore appears as a natural plan. Coupling simultaneous multi-laser excitation with minimally invasive, microscopic resolution, thin and flexible optics, allows the fusion of complementary and valuable biological information, thus paving the way to a combination of morphological and functional imaging. This paper will detail the architecture of a new system, Cellvizio Dual Band, capable of video rate in vivo and in situ multi-spectral fluorescence imaging with a microscopic resolution. In its standard configuration, the system simultaneously operates at 488 and 660 nm, where it automatically performs the necessary spectral, photometric and geometric calibrations to provide unambiguously co-registered images in real-time. The main hardware and software features, including calibration procedures and sub-micron registration algorithms, will be presented as well as a panorama of its current applications, illustrated with recent results in the field of pre-clinical imaging.

Multifocus confocal Raman microspectroscopy for fast multimode vibrational imaging of living cells.

Science.gov (United States)

Okuno, Masanari; Hamaguchi, Hiro-o

2010-12-15

We have developed a multifocus confocal Raman microspectroscopic system for the fast multimode vibrational imaging of living cells. It consists of an inverted microscope equipped with a microlens array, a pinhole array, a fiber bundle, and a multichannel Raman spectrometer. Forty-eight Raman spectra from 48 foci under the microscope are simultaneously obtained by using multifocus excitation and image-compression techniques. The multifocus confocal configuration suppresses the background generated from the cover glass and the cell culturing medium so that high-contrast images are obtainable with a short accumulation time. The system enables us to obtain multimode (10 different vibrational modes) vibrational images of living cells in tens of seconds with only 1 mW laser power at one focal point. This image acquisition time is more than 10 times faster than that in conventional single-focus Raman microspectroscopy.

Confocal laser scanning microscopy in study of bone calcification

Energy Technology Data Exchange (ETDEWEB)

Nishikawa, Tetsunari, E-mail: tetsu-n@cc.osaka-dent.ac.jp [Department of Oral Pathology, Osaka Dental University, Osaka (Japan); Kokubu, Mayu; Kato, Hirohito [Department of Oral Pathology, Osaka Dental University, Osaka (Japan); Imai, Koichi [Department of Biomaterials, Osaka Dental University, Osaka (Japan); Tanaka, Akio [Department of Oral Pathology, Osaka Dental University, Osaka (Japan)

2012-12-01

Highlights: Black-Right-Pointing-Pointer High-magnification images with depth selection, and thin sections were observed using CLSM. Black-Right-Pointing-Pointer The direction and velocity of calcification of the bone was observed by administration of 2 fluorescent dyes. Black-Right-Pointing-Pointer In dog femora grafted with coral blocks, newly-formed bone was observed in the coral block space with a rough surface. Black-Right-Pointing-Pointer Twelve weeks after dental implant was grafted in dog femora, the space between screws was filled with newly-formed bones. - Abstract: Bone regeneration in mandible and maxillae after extraction of teeth or tumor resection and the use of rough surface implants in bone induction must be investigated to elucidate the mechanism of calcification. The calcified tissues are subjected to chemical decalcification or physical grinding to observe their microscopic features with light microscopy and transmission electron microscopy where the microscopic tissue morphology is significantly altered. We investigated the usefulness of confocal laser scanning microscopy (CLSM) for this purpose. After staggering the time of administration of calcein and alizarin red to experimental rats and dogs, rat alveolar bone and dog femur grafted with coral as scaffold or dental implants were observed with CLSM. In rat alveolar bone, the calcification of newly-formed bone and net-like canaliculi was observed at the mesial bone from the roots progressed at the rate of 15 {mu}m/day. In dog femur grafted with coral, newly-formed bones along the space of coral were observed in an orderly manner. In dog femur with dental implants, after 8 weeks, newly-formed bone proceeded along the rough surface of the implants. CLSM produced high-magnification images of newly-formed bone and thin sections were not needed.

Smartphone confocal microscopy for imaging cellular structures in human skin in vivo.

Science.gov (United States)

Freeman, Esther E; Semeere, Aggrey; Osman, Hany; Peterson, Gary; Rajadhyaksha, Milind; González, Salvador; Martin, Jeffery N; Anderson, R Rox; Tearney, Guillermo J; Kang, Dongkyun

2018-04-01

We report development of a low-cost smartphone confocal microscope and its first demonstration of in vivo human skin imaging. The smartphone confocal microscope uses a slit aperture and diffraction grating to conduct two-dimensional confocal imaging without using any beam scanning devices. Lateral and axial resolutions of the smartphone confocal microscope were measured as 2 and 5 µm, respectively. In vivo confocal images of human skin revealed characteristic cellular structures, including spinous and basal keratinocytes and papillary dermis. Results suggest that the smartphone confocal microscope has a potential to examine cellular details in vivo and may help disease diagnosis in resource-poor settings, where conducting standard histopathologic analysis is challenging.

Multimodal backside imaging of a microcontroller using confocal laser scanning and optical-beam-induced current imaging

Science.gov (United States)

Finkeldey, Markus; Göring, Lena; Schellenberg, Falk; Brenner, Carsten; Gerhardt, Nils C.; Hofmann, Martin

2017-02-01

Microscopy imaging with a single technology is usually restricted to a single contrast mechanism. Multimodal imaging is a promising technique to improve the structural information that could be obtained about a device under test (DUT). Due to the different contrast mechanisms of laser scanning microscopy (LSM), confocal laser scanning microscopy (CLSM) and optical beam induced current microscopy (OBICM), a combination could improve the detection of structures in integrated circuits (ICs) and helps to reveal their layout. While OBIC imaging is sensitive to the changes between differently doped areas and to semiconductor-metal transitions, CLSM imaging is mostly sensitive to changes in absorption and reflection. In this work we present the implementation of OBIC imaging into a CLSM. We show first results using industry standard Atmel microcontrollers (MCUs) with a feature size of about 250nm as DUTs. Analyzing these types of microcontrollers helps to improve in the field of side-channel attacks to find hardware Trojans, possible spots for laser fault attacks and for reverse engineering. For the experimental results the DUT is placed on a custom circuit board that allows us to measure the current while imaging it in our in-house built stage scanning microscope using a near infrared (NIR) laser diode as light source. The DUT is thinned and polished, allowing backside imaging through the Si-substrate. We demonstrate the possibilities using this optical setup by evaluating OBIC, LSM and CLSM images above and below the threshold of the laser source.

Optical depth sectioning in the aberration-corrected scanning transmission and scanning confocal electron microscope

International Nuclear Information System (INIS)

Behan, G; Nellist, P D

2008-01-01

The use of spherical aberration correctors in the scanning transmission electron microscope (STEM) has the effect of reducing the depth of field of the microscope, making three-dimensional imaging of a specimen possible by optical sectioning. Depth resolution can be improved further by placing aberration correctors and lenses pre and post specimen to achieve an imaging mode known as scanning confocal electron microscopy (SCEM). We present the calculated incoherent point spread functions (PSF) and optical transfer functions (OTF) of a STEM and SCEM. The OTF for a STEM is shown to have a missing cone region which results in severe blurring along the optic axis, which can be especially severe for extended objects. We also present strategies for reconstruction of experimental data, such as three-dimensional deconvolution of the point spread function.

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

International Nuclear Information System (INIS)

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

2008-01-01

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

Femtosecond laser subsurface scleral treatment in cadaver human sclera and evaluation using two-photon and confocal microscopy

Science.gov (United States)

Sun, Hui; Fan, Zhongwei; Yan, Ying; Lian, Fuqiang; Kurtz, Ron; Juhasz, Tibor

2016-03-01

Glaucoma is the second-leading cause of blindness worldwide and is often associated with elevated intraocular pressure (IOP). Partial-thickness drainage channels can be created with femtosecond laser in the translucent sclera for the potential treatment of glaucoma. We demonstrate the creation of partial-thickness subsurface drainage channels with the femtosecond laser in the cadaver human eyeballs and describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in cadaver human eyes. A femtosecond laser operating at a wavelength of 1700 nm was scanned along a rectangular raster pattern to create the partial thickness subsurface drainage channels in the sclera of cadaver human eyes. Analysis of the dimensions and location of these channels is important in understanding their effects. We describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in cadaver human eyes. High-resolution images, hundreds of microns deep in the sclera, were obtained to allow determination of the shape and dimension of such partial thickness subsurface scleral channels. Our studies suggest that the confocal and two-photon microscopy can be used to investigate femtosecond-laser created partial-thickness drainage channels in the sclera of cadaver human eyes.

Visualization of carbon nanotubes dispersion in composite by using confocal laser scanning microscopy

Czech Academy of Sciences Publication Activity Database

Ilčíková, M.; Danko, M.; Doroshenko, M.; Best, A.; Mrlík, M.; Csomorová, K.; Šlouf, Miroslav; Chorvát Jr., D.; Koynov, K.; Mosnáček, J.

2016-01-01

Roč. 79, June (2016), s. 187-197 ISSN 0014-3057 Institutional support: RVO:61389013 Keywords : confocal laser scanning microscopy * composites * carbon nanotubes dispersion Subject RIV: CD - Macromolecular Chemistry Impact factor: 3.531, year: 2016

Apoplastic pH in corn root gravitropism: a laser scanning confocal microscopy measurement

International Nuclear Information System (INIS)

Taylor, D.P.; Slattery, J.; Leopold, A.C.

1996-01-01

The ability to measure the pH of the apoplast in situ is of special interest as a test of the cell wall acidification theory. Optical sectioning of living seedlings of corn roots using the laser scanning confocal microscope (LSCM) permits us to make pH measurements in living tissue. The pH of the apoplast of corn roots was measured by this method after infiltration with CI-NERF, a pH-sensitive dye, along with Texas Red Dextran 3000, a pH-insensitive dye, as an internal standard. In the elongation zone of corn roots, the mean apoplastic pH was 4.9. Upon gravitropic stimulation, the pH on the convex side of actively bending roots was 4.5. The lowering of the apoplastic pH by 0.4 units appears to be sufficient to account for the increased growth on that side. This technique provides site-specific evidence for the acid growth theory of cell elongation. The LSCM permits measurements of the pH of living tissues, and has a sensitivity of approximately 0.2 pH units. (author)

Atomic force microscopy and confocal laser scanning microscopy on the cytoskeleton of permeabilised and embedded cells

International Nuclear Information System (INIS)

Meller, Karl; Theiss, Carsten

2006-01-01

We describe a technical method of cell permeabilisation and embedding to study the organisation and distribution of intracellular proteins with aid of atomic force microscopy and confocal laser scanning microscopy in identical areas. While confocal laser scanning microscopy is useful for the identification of certain proteins subsequent labelling with markers or antibodies, atomic force microscopy allows the observation of macromolecular structures in fixed and living cells. To demonstrate the field of application of this preparatory technique, cells were permeabilised, fixed, and the actin cytoskeleton was stained with phalloidin-rhodamine. Confocal laser scanning microscopy was used to show the organisation of these microfilaments, e.g. geodesic dome structures. Thereafter, cells were embedded in Durcupan water-soluble resin, followed by UV-polymerisation of resin at 4 o C. This procedure allowed intracellular visualisation of the cell nucleus or cytoskeletal elements by atomic force microscopy, for instance to analyse the globular organisation of actin filaments. Therefore, this method offers a great potential to combine both microscopy techniques in order to understand and interpret intracellular protein relations, for example, the biochemical and morphological interaction of the cytoskeleton

Análisis fractográfico de fibras de circona y de zafiro mediante microscopía óptica confocal

Directory of Open Access Journals (Sweden)

López-Cepero, J. M.

2005-08-01

Full Text Available Fractography is a very useful tool to learn about the mechanisms controlling the fracture process. In this work, the fractographical uses of laser scanning confocal microscopy (LSCM are shown. LSCM is a widely used technique in Biology and similar disciplines, but its use in Materials Science is not yet as explored. However, it is an ideal technique for fractographical studies, since by using it the three-dimensional profile of the fracture surface can be obtained. From this profile, it is possible to extract information, like the roughness of the fracture surface, which would be very difficult to obtain from other studies. In this paper, LSCM is applied to the study of the fracture surface in ceramic fibers submitted to tensile stress, making the interest of the technique evident due to features such as easy sample preparation, gathering of real 3D information, and good SEM-LSCM synergy.

La fractografía en materiales resulta de gran utilidad para la caracterización de los mecanismos que dominan la rotura. En el presente artículo se investigan las aplicaciones fractográficas de la microscopía óptica confocal o LSCM (Laser Scanning Confocal Microscopy. Dicha técnica es de amplio uso en Biología y disciplinas afines, pero su empleo en Ciencia de Materiales está poco explorado. Sin embargo, resulta ideal para el estudio fractográfico, ya que permite obtener reconstrucciones del perfil tridimensional de la superficie de fractura. De este perfil tridimensional puede extraerse información —la rugosidad de la superficie, por ejemplo— muy difícil de obtener a partir de otro tipo de estudios. En este trabajo se aplica LSCM a la caracterización de superficies de fractura en fibras cerámicas sometidas a tracción y se pone de manifiesto el interés de la técnica, debido a características tales como la preparación sencilla de muestras, la obtención de información tridimensional real y la buena coordinación SEM-LSCM.

Attempt of correlative observation of morphological synaptic connectivity by combining confocal laser-scanning microscope and FIB-SEM for immunohistochemical staining technique.

Science.gov (United States)

Sonomura, Takahiro; Furuta, Takahiro; Nakatani, Ikuko; Yamamoto, Yo; Honma, Satoru; Kaneko, Takeshi

2014-11-01

Ten years have passed since a serial block-face scanning electron microscopy (SBF-SEM) method was developed [1]. In this innovative method, samples were automatically sectioned with an ultramicrotome placed inside a scanning electron microscope column, and the block surfaces were imaged one after another by SEM to capture back-scattered electrons. The contrast-inverted images obtained by the SBF-SEM were very similar to those acquired using conventional TEM. SFB-SEM has made easy to acquire image stacks of the transmission electron microscopy (TEM) in the mesoscale, which is taken with the confocal laser-scanning microcopy(CF-LSM).Furthermore, serial-section SEM has been combined with the focused ion beam (FIB) milling method [2]. FIB-incorporated SEM (FIB-SEM) has enabled the acquisition of three-dimensional images with a higher z-axis resolution com- pared to ultramicrotome-equipped SEM.We tried immunocytochemistry for FIB-SEM and correlated this immunoreactivity with that in CF-LSM. Dendrites of neurons in the rat neostriatum were visualized using a recombinant viral vector. Moreover, the thalamostriatal afferent terminals were immunolabeled with Cy5 fluorescence for vesicular glutamate transporter 2 (VGluT2). After detection of the sites of terminals apposed to the dendrites by using CF-LSM, GFP and VGluT2 immunoreactivities were further developed for EM by using immunogold/silver enhancement and immunoperoxidase/diaminobenzidine (DAB) methods, respectively.We showed that conventional immuno-cytochemical staining for TEM was applicable to FIB-SEM. Furthermore, several synaptic contacts, which were thought to exist on the basis of CF-LSM findings, were confirmed with FIB-SEM, revealing the usefulness of the combined method of CF-LSM and FIB-SEM. © 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.

Simple method for sub-diffraction resolution imaging of cellular structures on standard confocal microscopes by three-photon absorption of quantum dots.

Directory of Open Access Journals (Sweden)

Anje Sporbert

Full Text Available This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts.

Polarized differential-phase laser scanning microscope

International Nuclear Information System (INIS)

Chou Chien; Lyu, C.-W.; Peng, L.-C.

2001-01-01

A polarized differential-phase laser scanning microscope, which combines a polarized optical heterodyne Mach-Zehnder interferometer and a differential amplifier to scan the topographic image of a surface, is proposed. In the experiment the differential amplifier, which acts as a PM-AM converter, in the experiment, converting phase modulation (PM) into amplitude modulation (AM). Then a novel, to our knowledge, phase demodulator was proposed and implemented for the differential-phase laser scanning microscope. An optical grating (1800 lp/mm) was imaged. The lateral and the depth resolutions of the imaging system were 0.5 μm and 1 nm, respectively. The detection accuracy, which was limited by the reflectivity variation of the test surface, is discussed

Confocal detection of Rayleigh scattering for residual stress measurement in chemically tempered glass

Energy Technology Data Exchange (ETDEWEB)

Hödemann, S., E-mail: siim.hodemann@ut.ee; Möls, P.; Kiisk, V.; Saar, R.; Kikas, J. [Institute of Physics, University of Tartu, Wilhelm Ostwald st., Tartu 50411 (Estonia); Murata, T. [Nippon Electric Glass Co., 7-1 Seiran 2-chome, Otsu-shi, Shiga 520-8639 (Japan)

2015-12-28

A new optical method is presented for evaluation of the stress profile in chemically tempered (chemically strengthened) glass based on confocal detection of scattered laser beam. Theoretically, a lateral resolution of 0.2 μm and a depth resolution of 0.6 μm could be achieved by using a confocal microscope with high-NA immersion objective. The stress profile in the 250 μm thick surface layer of chemically tempered lithium aluminosilicate glass was measured with a high spatial resolution to illustrate the capability of the method. The confocal method is validated using transmission photoelastic and Na{sup +} ion concentration profile measurement. Compositional influence on the stress-optic coefficient is calculated and discussed. Our method opens up new possibilities for three-dimensional scattered light tomography of mechanical imaging in birefringent materials.

Musculature of Notholca acuminata (Rotifera : Ploima : Brachionidae) revealed by confocal scanning laser microscopy

DEFF Research Database (Denmark)

Sørensen, M.V.; Funch, P.; Hooge, M.

2003-01-01

The body-wall and visceral musculature of Notholca acuminata was visualized using phalloidin-linked fluorescent dye under confocal laser scanning microscopy. The body-wall musculature includes dorsal, lateral, and ventral pairs of longitudinally oriented body retractor muscles, two pairs of head...

Confocal laser scanning microscopy to estimate nanoparticles’ human skin penetration in vitro

Directory of Open Access Journals (Sweden)

Zou Y

2017-10-01

Full Text Available Ying Zou,1,2,* Anna Celli,2,3,* Hanjiang Zhu,2,* Akram Elmahdy,2 Yachao Cao,2 Xiaoying Hui,2 Howard Maibach2 1Skin & Cosmetic Research Department, Shanghai Skin Disease Hospital, Shanghai, People’s Republic of China; 2Department of Dermatology, School of Medicine, University of California San Francisco, San Francisco, CA, USA; 3San Francisco Veterans Medical Center, San Francisco, CA, USA *These authors contributed equally to this work Objective: With rapid development of nanotechnology, there is increasing interest in nanoparticle (NP application and its safety and efficacy on human skin. In this study, we utilized confocal laser scanning microscopy to estimate NP skin penetration.Methods: Three different-sized polystyrene NPs marked with red fluorescence were applied to human skin, and Calcium Green 5N was used as a counterstain. Dimethyl sulfoxide (DMSO and ethanol were used as alternative vehicles for NPs. Tape stripping was utilized as a barrier-damaged skin model. Skin biopsies dosed with NPs were incubated at 4°C or 37°C for 24 hours and imaged using confocal laser scanning microscopy.Results: NPs were localized in the stratum corneum (SC and hair follicles without penetrating the epidermis/dermis. Barrier alteration with tape stripping and change in incubation temperature did not induce deeper penetration. DMSO enhanced NP SC penetration but ethanol did not.Conclusion: Except with DMSO vehicle, these hydrolyzed polystyrene NPs did not penetrate intact or barrier-damaged human “viable” epidermis. For further clinical relevance, in vivo human skin studies and more sensitive analytic chemical methodology are suggested. Keywords: nanoparticles, skin penetration, stratum corneum, confocal laser scanning microscopy, tape stripping

Mode-mismatched confocal thermal-lens microscope with collimated probe beam

Energy Technology Data Exchange (ETDEWEB)

Cabrera, Humberto, E-mail: hcabrera@ictp.it [SPIE-ICTP Anchor Research Laboratory, International Centre for Theoretical Physics (ICTP), Strada Costiera 11, Trieste (Italy); Centro Multidisciplinartio de Ciencias, Instituto Venezolano de Investigaciones Científicas (IVIC), Mérida 5101 (Venezuela, Bolivarian Republic of); Korte, Dorota; Franko, Mladen [Laboratory for Environmental Research, University of Nova Gorica, Vipavska 13, 5000 Nova Gorica (Slovenia)

2015-05-15

We report a thermal lens microscope (TLM) based on an optimized mode-mismatched configuration. It takes advantage of the coaxial counter propagating tightly focused excitation and collimated probe beams, instead of both focused at the sample, as it is in currently known TLM setups. A simple mathematical model that takes into account the main features of the instrument is presented. The confocal detection scheme and the introduction of highly collimated probe beam allow enhancing the versatility, limit of detection (LOD), and sensitivity of the instrument. The theory is experimentally verified measuring ethanol’s absorption coefficient at 532.8 nm. Additionally, the presented technique is applied for detection of ultra-trace amounts of Cr(III) in liquid solution. The achieved LOD is 1.3 ppb, which represents 20-fold enhancement compared to transmission mode spectrometric techniques and a 7.5-fold improvement compared to previously reported methods for Cr(III) based on thermal lens effect.

Microscope self-calibration based on micro laser line imaging and soft computing algorithms

Science.gov (United States)

Apolinar Muñoz Rodríguez, J.

2018-06-01

A technique to perform microscope self-calibration via micro laser line and soft computing algorithms is presented. In this technique, the microscope vision parameters are computed by means of soft computing algorithms based on laser line projection. To implement the self-calibration, a microscope vision system is constructed by means of a CCD camera and a 38 μm laser line. From this arrangement, the microscope vision parameters are represented via Bezier approximation networks, which are accomplished through the laser line position. In this procedure, a genetic algorithm determines the microscope vision parameters by means of laser line imaging. Also, the approximation networks compute the three-dimensional vision by means of the laser line position. Additionally, the soft computing algorithms re-calibrate the vision parameters when the microscope vision system is modified during the vision task. The proposed self-calibration improves accuracy of the traditional microscope calibration, which is accomplished via external references to the microscope system. The capability of the self-calibration based on soft computing algorithms is determined by means of the calibration accuracy and the micro-scale measurement error. This contribution is corroborated by an evaluation based on the accuracy of the traditional microscope calibration.

Relationship between gustatory function and average number of taste buds per fungiform papilla measured by confocal laser scanning microscopy in humans.

Science.gov (United States)

Saito, Takehisa; Ito, Tetsufumi; Ito, Yumi; Manabe, Yasuhiro; Sano, Kazuo

2017-02-01

The aim of this study was to elucidate the relationship between the gustatory function and average number of taste buds per fungiform papilla (FP) in humans. Systemically healthy volunteers (n = 211), pre-operative patients with chronic otitis media (n = 79), and postoperative patients, with or without a chorda tympani nerve (CTN) severed during middle ear surgery (n = 63), were included. Confocal laser scanning microscopy was employed to observe fungiform taste buds because it allows many FP to be observed non-invasively in a short period of time. Taste buds in an average of 10 FP in the midlateral region of the tongue were counted. In total, 3,849 FP were observed in 353 subjects. The gustatory function was measured by electrogustometry (EGM). An inverse relationship was found between the gustatory function and average number of fungiform taste buds per papilla. The healthy volunteers showed a lower EGM threshold (better gustatory function) and had more taste buds than did the patients with otitis media, and the patients with otitis media showed a lower EGM threshold and had more taste buds than did postoperative patients, reflecting the severity of damage to the CTN. It was concluded that the confocal laser scanning microscope is a very useful tool for using to observe a large number of taste buds non-invasively. © 2017 Eur J Oral Sci.

Re-scan confocal microscopy (RCM) improves the resolution of confocal microscopy and increases the sensitivity

NARCIS (Netherlands)

de Luca, Giulia; Breedijk, Ronald; Hoebe, Ron; Stallinga, Sjoerd; Manders, Erik

2017-01-01

Re-scan confocal microscopy (RCM) is a new super-resolution technique based on a standard confocal microscope extended with a re-scan unit in the detection path that projects the emitted light onto a sensitive camera. In this paper the fundamental properties of RCM, lateral resolution, axial

Re-scan confocal microscopy (RCM) improves the resolution of confocal microscopy and increases the sensitivity

NARCIS (Netherlands)

De Luca, G.; Breedijk, R.; Hoebe, R.; Stallinga, S.; Manders, E.

Re-scan confocal microscopy (RCM) is a new super-resolution technique based on a standard confocal microscope extended with a re-scan unit in the detection path that projects the emitted light onto a sensitive camera. In this paper the fundamental properties of RCM, lateral resolution, axial

Transverse mode selection in a monolithic microchip laser

CSIR Research Space (South Africa)

Naidoo, Darryl

2011-11-01

Full Text Available selection in a monolithic microchip laser Darryl Naidooa,b, Thomas Godinc, Michael Fromagerc, Emmanuel Cagniotc, Nicolas Passillyd, Andrew Forbesa,b and Kamel A?t-Ameurc1 a:CSIR National Laser Centre, P. O. Box 395, Pretoria 0001, South Africa b.... Lett. 77 (2000) 34-36. [14] W. Zhao, J. Tan and L. Qui, ?Improvement of confocal microscope performance by shaped annular beam and heterodyne confocal techniques,? Optik 116 (2005) 111-117. [15] T. Shiina, K. Yoshida, M. Ito and Y. Okamura, ?Long...

Re-scan confocal microscopy: scanning twice for better resolution.

Science.gov (United States)

De Luca, Giulia M R; Breedijk, Ronald M P; Brandt, Rick A J; Zeelenberg, Christiaan H C; de Jong, Babette E; Timmermans, Wendy; Azar, Leila Nahidi; Hoebe, Ron A; Stallinga, Sjoerd; Manders, Erik M M

2013-01-01

We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and high-sensitivity is required.

Toward real-time virtual biopsy of oral lesions using confocal laser endomicroscopy interfaced with embedded computing.

Science.gov (United States)

Thong, Patricia S P; Tandjung, Stephanus S; Movania, Muhammad Mobeen; Chiew, Wei-Ming; Olivo, Malini; Bhuvaneswari, Ramaswamy; Seah, Hock-Soon; Lin, Feng; Qian, Kemao; Soo, Khee-Chee

2012-05-01

Oral lesions are conventionally diagnosed using white light endoscopy and histopathology. This can pose a challenge because the lesions may be difficult to visualise under white light illumination. Confocal laser endomicroscopy can be used for confocal fluorescence imaging of surface and subsurface cellular and tissue structures. To move toward real-time "virtual" biopsy of oral lesions, we interfaced an embedded computing system to a confocal laser endomicroscope to achieve a prototype three-dimensional (3-D) fluorescence imaging system. A field-programmable gated array computing platform was programmed to enable synchronization of cross-sectional image grabbing and Z-depth scanning, automate the acquisition of confocal image stacks and perform volume rendering. Fluorescence imaging of the human and murine oral cavities was carried out using the fluorescent dyes fluorescein sodium and hypericin. Volume rendering of cellular and tissue structures from the oral cavity demonstrate the potential of the system for 3-D fluorescence visualization of the oral cavity in real-time. We aim toward achieving a real-time virtual biopsy technique that can complement current diagnostic techniques and aid in targeted biopsy for better clinical outcomes.

Molecular confocal laser endomicroscopy: a novel technique for in vivo cellular characterization of gastrointestinal lesions.

Science.gov (United States)

Karstensen, John Gásdal; Klausen, Pia Helene; Saftoiu, Adrian; Vilmann, Peter

2014-06-28

While flexible endoscopy is essential for macroscopic evaluation, confocal laser endomicroscopy (CLE) has recently emerged as an endoscopic method enabling visualization at a cellular level. Two systems are currently available, one based on miniprobes that can be inserted via a conventional endoscope or via a needle guided by endoscopic ultrasound. The second system has a confocal microscope integrated into the distal part of an endoscope. By adding molecular probes like fluorescein conjugated antibodies or fluorescent peptides to this procedure (either topically or systemically administered during on-going endoscopy), a novel world of molecular evaluation opens up. The method of molecular CLE could potentially be used for estimating the expression of important receptors in carcinomas, subsequently resulting in immediate individualization of treatment regimens, but also for improving the diagnostic accuracy of endoscopic procedures by identifying otherwise invisible mucosal lesions. Furthermore, studies have shown that fluorescein labelled drugs can be used to estimate the affinity of the drug to a target organ, which probably can be correlated to the efficacy of the drug. However, several of the studies in this research field have been conducted in animal facilities or in vitro, while only a limited number of trials have actually been carried out in vivo. Therefore, safety issues still needs further evaluations. This review will present an overview of the implications and pitfalls, as well as future challenges of molecular CLE in gastrointestinal diseases.

Digital differential confocal microscopy based on spatial shift transformation.

Science.gov (United States)

Liu, J; Wang, Y; Liu, C; Wilson, T; Wang, H; Tan, J

2014-11-01

Differential confocal microscopy is a particularly powerful surface profilometry technique in industrial metrology due to its high axial sensitivity and insensitivity to noise. However, the practical implementation of the technique requires the accurate positioning of point detectors in three-dimensions. We describe a simple alternative based on spatial transformation of a through-focus series of images obtained from a homemade beam scanning confocal microscope. This digital differential confocal microscopy approach is described and compared with the traditional Differential confocal microscopy approach. The ease of use of the digital differential confocal microscopy system is illustrated by performing measurements on a 3D standard specimen. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

Aorta Fluorescence Imaging by Using Confocal Microscopy

OpenAIRE

Wang, Chun-Yang; Tsai, Jui-che; Chuang, Ching-Cheng; Hsieh, Yao-Sheng; Sun, Chia-Wei

2011-01-01

The activated leukocyte attacked the vascular endothelium and the associated increase in VEcadherin number was observed in experiments. The confocal microscopic system with a prism-based wavelength filter was used for multiwavelength fluorescence measurement. Multiwavelength fluorescence imaging based on the VEcadherin within the aorta segment of a rat was achieved. The confocal microscopic system capable of fluorescence detection of cardiovascular tissue is a useful tool for measuring the bi...

In vitro confocal imaging of the rabbit cornea.

Science.gov (United States)

Masters, B R; Paddock, S

1990-05-01

We were able to observe in vitro the fine structure of the rabbit cornea using a laser scanning confocal microscope, especially in the regions between Descemet's membrane and the epithelial basal lamina. We observed submicrometre filaments throughout the stroma with high concentrations adjacent to Descemet's membrane, and found extensive interconnecting processes between stromal keratocytes. There are numerous regions containing nerve plexuses in the stroma. We found a deeply convoluted basal lamina adjacent to the epithelium, and observed regions containing junctions between endothelial cells in fluorescent images of rabbit corneas stained with the actin-specific compound fluorescein phalloidin.

Simultaneous Confocal Scanning Laser Ophthalmoscopy Combined with High-Resolution Spectral-Domain Optical Coherence Tomography: A Review

Directory of Open Access Journals (Sweden)

Verônica Castro Lima

2011-01-01

Full Text Available We aimed to evaluate technical aspects and the clinical relevance of a simultaneous confocal scanning laser ophthalmoscope and a high-speed, high-resolution, spectral-domain optical coherence tomography (SDOCT device for retinal imaging. The principle of confocal scanning laser imaging provides a high resolution of retinal and choroidal vasculature with low light exposure. Enhanced contrast, details, and image sharpness are generated using confocality. The real-time SDOCT provides a new level of accuracy for assessment of the angiographic and morphological correlation. The combined system allows for simultaneous recordings of topographic and tomographic images with accurate correlation between them. Also it can provide simultaneous multimodal imaging of retinal pathologies, such as fluorescein and indocyanine green angiographies, infrared and blue reflectance (red-free images, fundus autofluorescence images, and OCT scans (Spectralis HRA + OCT; Heidelberg Engineering, Heidelberg, Germany. The combination of various macular diagnostic tools can lead to a better understanding and improved knowledge of macular diseases.

Confocal laser scanning microscopy to estimate nanoparticles' human skin penetration in vitro.

Science.gov (United States)

Zou, Ying; Celli, Anna; Zhu, Hanjiang; Elmahdy, Akram; Cao, Yachao; Hui, Xiaoying; Maibach, Howard

2017-01-01

With rapid development of nanotechnology, there is increasing interest in nanoparticle (NP) application and its safety and efficacy on human skin. In this study, we utilized confocal laser scanning microscopy to estimate NP skin penetration. Three different-sized polystyrene NPs marked with red fluorescence were applied to human skin, and Calcium Green 5N was used as a counterstain. Dimethyl sulfoxide (DMSO) and ethanol were used as alternative vehicles for NPs. Tape stripping was utilized as a barrier-damaged skin model. Skin biopsies dosed with NPs were incubated at 4°C or 37°C for 24 hours and imaged using confocal laser scanning microscopy. NPs were localized in the stratum corneum (SC) and hair follicles without penetrating the epidermis/dermis. Barrier alteration with tape stripping and change in incubation temperature did not induce deeper penetration. DMSO enhanced NP SC penetration but ethanol did not. Except with DMSO vehicle, these hydrolyzed polystyrene NPs did not penetrate intact or barrier-damaged human "viable" epidermis. For further clinical relevance, in vivo human skin studies and more sensitive analytic chemical methodology are suggested.

Probe-based confocal laser endomicroscopy (pCLE) - a new imaging technique for in situ localization of spermatozoa.

Science.gov (United States)

Trottmann, Matthias; Stepp, Herbert; Sroka, Ronald; Heide, Michael; Liedl, Bernhard; Reese, Sven; Becker, Armin J; Stief, Christian G; Kölle, Sabine

2015-05-01

In azoospermic patients, spermatozoa are routinely obtained by testicular sperm extraction (TESE). However, success rates of this technique are moderate, because the site of excision of testicular tissue is determined arbitrarily. Therefore the aim of this study was to establish probe-based laser endomicroscopy (pCLE) a noval biomedical imaging technique, which provides the opportunity of non-invasive, real-time visualisation of tissue at histological resolution. Using pCLE we clearly visualized longitudinal and horizontal views of the tubuli seminiferi contorti and localized vital spermatozoa. Obtained images and real-time videos were subsequently compared with confocal laser scanning microscopy (CLSM) of spermatozoa and tissues, respectively. Comparative visualization of single native Confocal laser scanning microscopy (CLSM, left) and probe-based laser endomicroscopy (pCLE, right) using Pro Flex(TM) UltraMini O after staining with acriflavine. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A high-resolution combined scanning laser and widefield polarizing microscope for imaging at temperatures from 4 K to 300 K.

Science.gov (United States)

Lange, M; Guénon, S; Lever, F; Kleiner, R; Koelle, D

2017-12-01

Polarized light microscopy, as a contrast-enhancing technique for optically anisotropic materials, is a method well suited for the investigation of a wide variety of effects in solid-state physics, as, for example, birefringence in crystals or the magneto-optical Kerr effect (MOKE). We present a microscopy setup that combines a widefield microscope and a confocal scanning laser microscope with polarization-sensitive detectors. By using a high numerical aperture objective, a spatial resolution of about 240 nm at a wavelength of 405 nm is achieved. The sample is mounted on a 4 He continuous flow cryostat providing a temperature range between 4 K and 300 K, and electromagnets are used to apply magnetic fields of up to 800 mT with variable in-plane orientation and 20 mT with out-of-plane orientation. Typical applications of the polarizing microscope are the imaging of the in-plane and out-of-plane magnetization via the longitudinal and polar MOKE, imaging of magnetic flux structures in superconductors covered with a magneto-optical indicator film via the Faraday effect, or imaging of structural features, such as twin-walls in tetragonal SrTiO 3 . The scanning laser microscope furthermore offers the possibility to gain local information on electric transport properties of a sample by detecting the beam-induced voltage change across a current-biased sample. This combination of magnetic, structural, and electric imaging capabilities makes the microscope a viable tool for research in the fields of oxide electronics, spintronics, magnetism, and superconductivity.

A high-resolution combined scanning laser and widefield polarizing microscope for imaging at temperatures from 4 K to 300 K

Science.gov (United States)

Lange, M.; Guénon, S.; Lever, F.; Kleiner, R.; Koelle, D.

2017-12-01

Polarized light microscopy, as a contrast-enhancing technique for optically anisotropic materials, is a method well suited for the investigation of a wide variety of effects in solid-state physics, as, for example, birefringence in crystals or the magneto-optical Kerr effect (MOKE). We present a microscopy setup that combines a widefield microscope and a confocal scanning laser microscope with polarization-sensitive detectors. By using a high numerical aperture objective, a spatial resolution of about 240 nm at a wavelength of 405 nm is achieved. The sample is mounted on a 4He continuous flow cryostat providing a temperature range between 4 K and 300 K, and electromagnets are used to apply magnetic fields of up to 800 mT with variable in-plane orientation and 20 mT with out-of-plane orientation. Typical applications of the polarizing microscope are the imaging of the in-plane and out-of-plane magnetization via the longitudinal and polar MOKE, imaging of magnetic flux structures in superconductors covered with a magneto-optical indicator film via the Faraday effect, or imaging of structural features, such as twin-walls in tetragonal SrTiO3. The scanning laser microscope furthermore offers the possibility to gain local information on electric transport properties of a sample by detecting the beam-induced voltage change across a current-biased sample. This combination of magnetic, structural, and electric imaging capabilities makes the microscope a viable tool for research in the fields of oxide electronics, spintronics, magnetism, and superconductivity.

Chondrocytes provide a model for in-situ confocal microscopy and 3D reconstructions

Science.gov (United States)

Hirsch, Michelle S.; Svoboda, Kathy K. H.

1994-04-01

Hyaline cartilage is composed of chondrocytes that reside in lacunae surrounded by extracellular matrix molecules. Microscopic and histochemical features of cartilage have been studied with many techniques. Many of these techniques can be time consuming and may alter natural cartilage characteristics. In addition, the orientation and order of sectioned tissue must be maintained to create 3D reconstructions. We show that confocal laser scanning microscopy may replace traditional methods for studying cartilage.

Enhancement of fluorescence confocal scanning microscopy lateral resolution by use of structured illumination

International Nuclear Information System (INIS)

Kim, Taejoong; Gweon, DaeGab; Lee, Jun-Hee

2009-01-01

Confocal microscopy is an optical imaging technique used to reconstruct three-dimensional images without physical sectioning. As with other optical microscopes, the lateral resolution of the confocal microscope cannot surpass the diffraction limit. This paper presents a novel imaging system, structured illumination confocal scanning microscopy (SICSM), that uses structured illumination to improve the lateral resolution of the confocal microscope. The SICSM can easily be implemented by introducing a structured illumination generating optics to conventional line-scanning fluorescence confocal microscopy. In this paper, we report our analysis of the lateral and axial resolutions of the SICSM by use of mathematical imaging theory. Numerical simulation results show that the lateral resolution of the SICSM is 1.43-fold better than that of the confocal microscope. In the axial direction, however, the resolution of the SICSM is ∼15% poorer than that of the confocal microscope. This deterioration arises because of a decrease in the axial cut-off frequency caused by the process of generating structured illumination. We propose the use of imaging conditions under which a compromise between the axial and lateral resolutions is chosen. Finally, we show simulated images of diversely shaped test objects to demonstrate the lateral and axial resolution performance of the SICSM

Automatic segmentation of cell nuclei from confocal laser scanning microscopy images

International Nuclear Information System (INIS)

Kelemen, A.; Reist, H.W.

1997-01-01

A newly developed experimental method combines the possibility of irradiating more than a thousand cells simultaneous with an efficient colony-forming ability and with the capability of localizing a particle track through a cell nucleus together with the assessment of the energy transfer by digital superposition of the image containing the track with that of the cells. To assess the amount of energy deposition by particles traversing the cell nucleus the intersection lengths of the particle tracks have to be known. Intersection lengths can be obtained by determining the 3D surface contours of the irradiated cell nuclei. Confocal laser scanning microscopy using specific DNA fluorescent dye offers a possible way for the determination of the 3D shape of individual nuclei. Unfortunately, such experiments cannot be performed on living cells. One solution to this problem can be provided by building a statistical model of the shape of the nuclei of the exposed cells. In order to build such a statistical model, a large number of cell nuclei have to be identified and segmented from confocal laser scanning microscopy images. The present paper describes a method to perform this 3D segmentation in an automatic manner in order to create a solid basis for the statistical model. (author) 2 figs., 4 refs

Automated Microscopy: Macro Language Controlling a Confocal Microscope and its External Illumination: Adaptation for Photosynthetic Organisms

Czech Academy of Sciences Publication Activity Database

Steinbach, Gabor; Kaňa, Radek

2016-01-01

Roč. 22, č. 2 (2016), s. 258-263 ISSN 1431-9276 R&D Projects: GA ČR GAP501/12/0304; GA MŠk EE2.3.30.0059; GA MŠk ED2.1.00/03.0110; GA MŠk(CZ) LO1416 Institutional support: RVO:61388971 Keywords : automated microscopy * remote controlled microscopy * confocal microscopy Subject RIV: BH - Optics, Masers, Lasers Impact factor: 1.891, year: 2016

Confocal laser scanning microscopy to estimate nanoparticles’ human skin penetration in vitro

Science.gov (United States)

Elmahdy, Akram; Cao, Yachao; Hui, Xiaoying; Maibach, Howard

2017-01-01

Objective With rapid development of nanotechnology, there is increasing interest in nanoparticle (NP) application and its safety and efficacy on human skin. In this study, we utilized confocal laser scanning microscopy to estimate NP skin penetration. Methods Three different-sized polystyrene NPs marked with red fluorescence were applied to human skin, and Calcium Green 5N was used as a counterstain. Dimethyl sulfoxide (DMSO) and ethanol were used as alternative vehicles for NPs. Tape stripping was utilized as a barrier-damaged skin model. Skin biopsies dosed with NPs were incubated at 4°C or 37°C for 24 hours and imaged using confocal laser scanning microscopy. Results NPs were localized in the stratum corneum (SC) and hair follicles without penetrating the epidermis/dermis. Barrier alteration with tape stripping and change in incubation temperature did not induce deeper penetration. DMSO enhanced NP SC penetration but ethanol did not. Conclusion Except with DMSO vehicle, these hydrolyzed polystyrene NPs did not penetrate intact or barrier-damaged human “viable” epidermis. For further clinical relevance, in vivo human skin studies and more sensitive analytic chemical methodology are suggested. PMID:29184403

Laser confocal measurement system for curvature radius of lenses based on grating ruler

Science.gov (United States)

Tian, Jiwei; Wang, Yun; Zhou, Nan; Zhao, Weirui; Zhao, Weiqian

2015-02-01

In the modern optical measurement field, the radius of curvature (ROC) is one of the fundamental parameters of optical lens. Its measurement accuracy directly affects the other optical parameters, such as focal length, aberration and so on, which significantly affect the overall performance of the optical system. To meet the demand of measurement instruments for radius of curvature (ROC) with high accuracy in the market, we develop a laser confocal radius measurement system with grating ruler. The system uses the peak point of the confocal intensity curve to precisely identify the cat-eye and confocal positions and then measure the distance between these two positions by using the grating ruler, thereby achieving the high-precision measurement for the ROC. The system has advantages of high focusing sensitivity and anti-environment disturbance ability. And the preliminary theoretical analysis and experiments show that the measuring repeatability can be up to 0.8 um, which can provide an effective way for the accurate measurement of ROC.

Ex Vivo Confocal Spectroscopy of Autofluorescence in Age-Related Macular Degeneration.

Directory of Open Access Journals (Sweden)

Joel Kaluzny

Full Text Available We investigated the autofluorescence (AF signature of the microscopic features of retina with age-related macular degeneration (AMD using 488 nm excitation.The globes of four donors with AMD and four age-matched controls were embedded in paraffin and sectioned through the macula. Sections were excited using a 488 nm argon laser, and the AF emission was captured using a laser scanning confocal microscope (496-610 nm, 6 nm resolution. The data cubes were then analyzed to compare peak emission spectra between the AMD and the controls. Microscopic features, including individual lipofuscin and melanolipofuscin granules, Bruch's Membrane, as well macroscopic features, were considered.Overall, the AMD eyes showed a trend of blue-shifted emission peaks compared with the controls. These differences were statistically significant when considering the emission of the combined RPE/Bruch's Membrane across all the tissue cross-sections (p = 0.02.The AF signatures of ex vivo AMD RPE/BrM show blue-shifted emission spectra (488 nm excitation compared with the control tissue. The magnitude of these differences is small (~4 nm and highlights the potential challenges of detecting these subtle spectral differences in vivo.

High incidence of rainbow glare after femtosecond laser assisted-LASIK using the upgraded FS200 femtosecond laser.

Science.gov (United States)

Zhang, Yu; Chen, Yue-Guo

2018-03-05

To compare the incidence of rainbow glare (RG) after femtosecond laser assisted-LASIK (FS-LASIK) using the upgraded FS200 femtosecond laser with different flap cut parameter settings. A consecutive series of 129 patients (255 eyes) who underwent FS-LASIK for correcting myopia and/or astigmatism using upgraded WaveLight FS200 femtosecond laser with the original settings was included in group A. Another consecutive series of 129 patients (255 eyes) who underwent FS-LASIK using upgraded WaveLight FS200 femtosecond laser with flap cut parameter settings changed (decreased pulse energy, spot and line separation) was included in group B. The incidence and fading time of RG, confocal microscopic image and postoperative clinical results were compared between the two groups. There were no differences between the two groups in age, baseline refraction, excimer laser ablation depth, postoperative uncorrected visual acuity and refraction. The incidence rate of RG in group A (35/255, 13.73%) was significantly higher than that in group B (4/255, 1.57%) (P  0.05).The confocal microscopic images showed wider laser spot spacing in group A than group B. The incidence of RG was significantly correlated with age and grouping (P laser with original flap cut parameter settings could increase the incidence of RG. The narrower grating size and lower pulse energy could ameliorate this side effect.

Real time detection of antibody-antigen interaction using a laser scanning confocal imaging-surface plasmon resonance system

International Nuclear Information System (INIS)

Zhang Hong-Yan; Yang Li-Quan; Ning Ting-Yin; Liu Wei-Min; Sun Jia-Yu; Wang Peng-Fei; Meng Lan; Nie Jia-Cai

2012-01-01

A laser scanning confocal imaging-surface plasmon resonance (LSCI-SPR) instrument integrated with a wavelength-dependent surface plasmon resonance (SPR) sensor and a laser scanning confocal microscopy (LSCM) is built to detect the bonding process of human IgG and fluorescent-labeled affinity purified antibodies in real time. The shifts of resonant wavelength at different reaction time stages are obtained by SPR, corresponding well with the changes of the fluorescence intensity collected by using LSCM. The instrument shows the merits of the combination and complementation of the SPR and LSCM, with such advantages as quantificational analysis, high spatial resolution and real time monitor, which are of great importance for practical applications in biosensor and life science. (general)

Confocal scanning microscopy

DEFF Research Database (Denmark)

Bariani, Paolo

This report is based on a metrological investigation on confocal microscopy technique carried out by Uffe Rolf Arlø Theilade and Paolo Bariani. The purpose of the experimental activity was twofold a metrological instrument characterization and application to assessment of rough PP injection moulded...... replicated topography. Confocal microscopy is seen to be a promising technique in metrology of microstructures. Some limitations with respect to surface metrology were found during the experiments. The experiments were carried out using a Zeiss LSM 5 Pascal microscope owned by the Danish Polymer Centre...

Method and apparatus for a high-resolution three dimensional confocal scanning transmission electron microscope

Science.gov (United States)

de Jonge, Niels [Oak Ridge, TN

2010-08-17

A confocal scanning transmission electron microscope which includes an electron illumination device providing an incident electron beam propagating in a direction defining a propagation axis, and a precision specimen scanning stage positioned along the propagation axis and movable in at least one direction transverse to the propagation axis. The precision specimen scanning stage is configured for positioning a specimen relative to the incident electron beam. A projector lens receives a transmitted electron beam transmitted through at least part of the specimen and focuses this transmitted beam onto an image plane, where the transmitted beam results from the specimen being illuminated by the incident electron beam. A detection system is placed approximately in the image plane.

3D reconstruction and characterization of laser induced craters by in situ optical microscopy

Energy Technology Data Exchange (ETDEWEB)

Casal, A.; Cerrato, R.; Mateo, M.P.; Nicolas, G., E-mail: gines@udc.es

2016-06-30

Highlights: • Evolution of the laser induced crater and ablation features by in situ homemade optical microscope. • Performance comparison between confocal microscope for material characterization and homemade optical microscope. • Coupled system of laser ablation setup with a low cost optical microscope. - Abstract: A low-cost optical microscope was developed and coupled to an irradiation system in order to study the induced effects on material during a multipulse regime by an in situ visual inspection of the surface, in particular of the spot generated at different pulses. In the case of laser ablation, a reconstruction of the crater in 3D was made from the images of the sample surface taken during the irradiation process, and the subsequent profiles of ablated material were extracted. The implementation of this homemade optical device gives an added value to the irradiation system, providing information about morphology evolution of irradiated area when successive pulses are applied. In particular, the determination of ablation rates in real time can be especially useful for a better understanding and controlling of the ablation process in applications where removal of material is involved, such as laser cleaning and in-depth characterization of multilayered samples and diffusion processes. The validation of the developed microscope was made by a comparison with a commercial confocal microscope configured for the characterization of materials where similar results of crater depth and diameter were obtained for both systems.

3D reconstruction and characterization of laser induced craters by in situ optical microscopy

International Nuclear Information System (INIS)

Casal, A.; Cerrato, R.; Mateo, M.P.; Nicolas, G.

2016-01-01

Highlights: • Evolution of the laser induced crater and ablation features by in situ homemade optical microscope. • Performance comparison between confocal microscope for material characterization and homemade optical microscope. • Coupled system of laser ablation setup with a low cost optical microscope. - Abstract: A low-cost optical microscope was developed and coupled to an irradiation system in order to study the induced effects on material during a multipulse regime by an in situ visual inspection of the surface, in particular of the spot generated at different pulses. In the case of laser ablation, a reconstruction of the crater in 3D was made from the images of the sample surface taken during the irradiation process, and the subsequent profiles of ablated material were extracted. The implementation of this homemade optical device gives an added value to the irradiation system, providing information about morphology evolution of irradiated area when successive pulses are applied. In particular, the determination of ablation rates in real time can be especially useful for a better understanding and controlling of the ablation process in applications where removal of material is involved, such as laser cleaning and in-depth characterization of multilayered samples and diffusion processes. The validation of the developed microscope was made by a comparison with a commercial confocal microscope configured for the characterization of materials where similar results of crater depth and diameter were obtained for both systems.

In-situ investigation of thermal instabilities and solid state dewetting in polycrystalline platinum thin films via confocal laser microscopy

Energy Technology Data Exchange (ETDEWEB)

Jahangir, S.; Cheng, Xuan; Huang, H. H.; Nagarajan, V. [School of Materials Science and Engineering, University of New South Wales, Sydney 2052 (Australia); Ihlefeld, J. [Electronic, Optical, and Nanomaterials Department, Sandia National Laboratories, Albuquerque, New Mexico 87185 (United States)

2014-10-28

Solid state dewetting and the subsequent morphological changes for platinum thin films grown on zinc oxide (ZnO) buffered (001) silicon substrates (Pt/ZnO/SiO{sub 2}/(001)Si system) is investigated under vacuum conditions via a custom-designed confocal laser microscope coupled with a laser heating system. Live imaging of thin film dewetting under a range of heating and quenching vacuum ambients reveals events including hillock formation, hole formation, and hole growth that lead to formation of a network of Pt ligaments, break up of Pt ligaments to individual islands and subsequent Pt islands shape reformation, in chronological fashion. These findings are corroborated by ex-situ materials characterization and quantitative electron microscopy analysis. A secondary hole formation via blistering before film rupture is revealed to be the critical stage, after which a rapid dewetting catastrophe occurs. This process is instantaneous and cannot be captured by ex-situ methods. Finally, an intermetallic phase forms at 900 °C and alters the morphology of Pt islands, suggesting a practical limit to the thermal environments that may be used for these platinized silicon wafers in vacuum conditions.

In-situ investigation of thermal instabilities and solid state dewetting in polycrystalline platinum thin films via confocal laser microscopy

International Nuclear Information System (INIS)

Jahangir, S.; Cheng, Xuan; Huang, H. H.; Nagarajan, V.; Ihlefeld, J.

2014-01-01

Solid state dewetting and the subsequent morphological changes for platinum thin films grown on zinc oxide (ZnO) buffered (001) silicon substrates (Pt/ZnO/SiO 2 /(001)Si system) is investigated under vacuum conditions via a custom-designed confocal laser microscope coupled with a laser heating system. Live imaging of thin film dewetting under a range of heating and quenching vacuum ambients reveals events including hillock formation, hole formation, and hole growth that lead to formation of a network of Pt ligaments, break up of Pt ligaments to individual islands and subsequent Pt islands shape reformation, in chronological fashion. These findings are corroborated by ex-situ materials characterization and quantitative electron microscopy analysis. A secondary hole formation via blistering before film rupture is revealed to be the critical stage, after which a rapid dewetting catastrophe occurs. This process is instantaneous and cannot be captured by ex-situ methods. Finally, an intermetallic phase forms at 900 °C and alters the morphology of Pt islands, suggesting a practical limit to the thermal environments that may be used for these platinized silicon wafers in vacuum conditions.

Model wavefront sensor for adaptive confocal microscopy

Science.gov (United States)

Booth, Martin J.; Neil, Mark A. A.; Wilson, Tony

2000-05-01

A confocal microscope permits 3D imaging of volume objects by the inclusion of a pinhole in the detector path which eliminates out of focus light. This configuration is however very sensitive to aberrations induced by the specimen or the optical system and would therefore benefit from an adaptive optics approach. We present a wavefront sensor capable of measuring directly the Zernike components of an aberrated wavefront and show that it is particularly applicable to the confocal microscope since only those wavefronts originating in the focal region contribute to the measured aberration.

In vivo confocal Raman spectroscopy of the human cornea.

Science.gov (United States)

Bauer, N J; Hendrikse, F; March, W F

1999-07-01

To investigate the feasibility of a confocal Raman spectroscopic technique for the noninvasive assessment of corneal hydration in vivo in two legally blind subjects. A laser beam (632.8 nm; 15 mJ) was maintained on the cornea by using a microscope objective lens (x25 magnification, NA = 0.5, f = 10 mm) both for focusing the incident light as well as collecting the Raman backscattered light, in a 180 degrees backscatter configuration. An optical fiber, acting as the confocal pinhole for elimination of light from out-of-focus places, was coupled to a spectrometer that dispersed the collected light onto a sensitive array detector for rapid spectral data acquisition over a range from 2,890 to 3,590/cm(-1). Raman spectra were recorded from the anterior 100-150 microm of the cornea over a period before and after topical application of a mild dehydrating solution. The ratio between the amplitudes of the signals at 3,400/cm(-1) (OH-vibrational mode of water) and 2,940/cm(-1) (CH-vibrational mode of proteins) was used as a measure for corneal hydration. High signal-to-noise ratio (SNR = 25) Raman spectra were obtained from the human corneas by using 15 mJ of laser light energy. Qualitative changes in the hydration of the anteriormost part of the corneas could be observed as a result of the dehydrating agent. With adequate improvements in system safety, confocal Raman spectroscopy could potentially be applied clinically as a noninvasive tool for the assessment of corneal hydration in vivo.

Characterization of particle deformation during compression measured by confocal laser scanning microscopy.

Science.gov (United States)

Guo, H X; Heinämäki, J; Yliruusi, J

1999-09-20

Direct compression of riboflavin sodium phosphate tablets was studied by confocal laser scanning microscopy (CLSM). The technique is non-invasive and generates three-dimensional (3D) images. Tablets of 1% riboflavin sodium phosphate with two grades of microcrystalline cellulose (MCC) were individually compressed at compression forces of 1.0 and 26.8 kN. The behaviour and deformation of drug particles on the upper and lower surfaces of the tablets were studied under compression forces. Even at the lower compression force, distinct recrystallized areas in the riboflavin sodium phosphate particles were observed in both Avicel PH-101 and Avicel PH-102 tablets. At the higher compression force, the recrystallization of riboflavin sodium phosphate was more extensive on the upper surface of the Avicel PH-102 tablet than the Avicel PH-101 tablet. The plastic deformation properties of both MCC grades reduced the fragmentation of riboflavin sodium phosphate particles. When compressed with MCC, riboflavin sodium phosphate behaved as a plastic material. The riboflavin sodium phosphate particles were more tightly bound on the upper surface of the tablet than on the lower surface, and this could also be clearly distinguished by CLSM. Drug deformation could not be visualized by other techniques. Confocal laser scanning microscopy provides valuable information on the internal mechanisms of direct compression of tablets.

Quantitative measurements with x-ray microscopes in laser-fusion experiments

International Nuclear Information System (INIS)

Marshall, F.J.; Su, Q.

1995-01-01

X-ray imaging of laser-fusion target implosions has been performed on the University of Rochester's OMEGA laser system by means of grazing-incidence optical imaging with Kirkpatrick--Baez (KB) microscopes. High spatial resolution imaging (∼5 μm) of hard x-ray emission (up to ∼7 keV) has been achieved. New grazing-incidence optics are currently being fabricated for the OMEGA Upgrade experimental laser-fusion facility. Projected performance indicates that resolution may be increased to ∼2 μm at the center of the field of view and sensitivity extended to ∼8 keV. Uses of KB microscopes on the OMEGA Upgrade will include hard x-ray imaging, grating-dispersed imaged spectroscopy, and framed imaging. A novel technique for monochromatic imaging with KB microscopes has also been demonstrated enabling images of target emission in a narrow energy band (10 to 20 eV) to be obtained

3D confocal imaging in CUBIC-cleared mouse heart

Energy Technology Data Exchange (ETDEWEB)

Nehrhoff, I.; Bocancea, D.; Vaquero, J.; Vaquero, J.J.; Lorrio, M.T.; Ripoll, J.; Desco, M.; Gomez-Gaviro, M.V.

2016-07-01

Acquiring high resolution 3D images of the heart enables the ability to study heart diseases more in detail. Here, the CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) clearing protocol was adapted for thick mouse heart sections to increase the penetration depth of the confocal microscope lasers into the tissue. The adapted CUBIC clearing of the heart lets the antibody penetrate deeper into the tissue by a factor of five. The here shown protocol enables deep 3D highresolution image acquisition in the heart. This allows a much more accurate assessment of the cellular and structural changes that underlie heart diseases. (Author)

3D confocal imaging in CUBIC-cleared mouse heart

International Nuclear Information System (INIS)

Nehrhoff, I.; Bocancea, D.; Vaquero, J.; Vaquero, J.J.; Lorrio, M.T.; Ripoll, J.; Desco, M.; Gomez-Gaviro, M.V.

2016-01-01

Acquiring high resolution 3D images of the heart enables the ability to study heart diseases more in detail. Here, the CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) clearing protocol was adapted for thick mouse heart sections to increase the penetration depth of the confocal microscope lasers into the tissue. The adapted CUBIC clearing of the heart lets the antibody penetrate deeper into the tissue by a factor of five. The here shown protocol enables deep 3D highresolution image acquisition in the heart. This allows a much more accurate assessment of the cellular and structural changes that underlie heart diseases. (Author)

Confocal microscopic observation of structural changes in glass-ionomer cements and tooth interfaces.

Science.gov (United States)

Watson, T F; Pagliari, D; Sidhu, S K; Naasan, M A

1998-03-01

This study aimed to develop techniques to allow dynamic imaging of a cavity before, during and after placement of glass-ionomer restorative materials. Cavities were cut in recently extracted third molars and the teeth longitudinally sectioned. Each hemisected tooth surface was placed in green modelling compound at 90 to the optical axis of the microscope. The cavity surface was imaged using a video rate confocal microscope in conjunction with an internally focusable microscope objective. The sample on the stage was pushed up to the objective lens which 'clamped' the cover glass onto it. Water, glycerine or oil was placed below the coverglass, with oil above. Internal tooth structures were imaged by changing the internal focus of the objective. The restorative material was then placed into the cavity. Video images were stored either onto video tape or digitally, using a frame grabber, computer and mass memory storage. Software controls produced time-lapse recordings of the interface over time. Preliminary experiments have examined the placement and early maturation of conventional glass-ionomer cements and a syringeable resin-modified glass-ionomer cement. Initial contact of the cement matrix and glass particles was visible as the plastic material rolled past the enamel and dentine, before making a bond. Evidence for water movement from the dentine into the cement has also been seen. After curing, the early dimensional changes in the cements due to water flux were apparent using the time-lapse facility. This new technique enables examination of developing tooth/restoration interfaces and the tracking of movement in materials.

Quantitative assessment of wound-healing process as a response to laser-induced micro-injuries

Science.gov (United States)

Liu, Yang; Bargo, Paulo; Kollias, Nikiforos

2009-02-01

Currently, most investigations of wound healing rely on invasive biopsy followed by histology and immunohistochemistry staining. There is a great need to develop non-invasive techniques for in vivo diagnostic, clinical and scientific evaluation. Here, we performed a comprehensive investigation on the dynamic wound healing process as a response to laser-induced microinjuries using non-invasive imaging techniques such as reflectance laser-scanning confocal microscopy and video microscopy. Eight healthy subjects ranging from Fitzpatrick skin type II-VI with age from 27 to 57 years were recruited. The volar forearm of each subject was treated with a laser device that generates an array of microbeams with an infrared wavelength. The microscopic changes of epidermal cells and collagen during the wound healing process were assessed non-invasively using confocal microscopy. We also developed a quantitative method to evaluate the dynamic wound healing process at the microscopic level in three areas of interest: (1) treated micro-wounding zone, (2) surrounding collateral damage zone and (3) normal area. The depth-dependent intensity profile derived from reflectance confocal microscope images clearly distinguishes the three areas of interest and quantitatively measures the cellular structure-associated changes. A progressive change in depth-dependent intensity profiles in subjects with different ages parallels the clinical observation of wound healing rate. The quantitative analysis developed in this study may find broad applications in assessing the skin response to treatment at a microscopic level.

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

OpenAIRE

Heintz, Keely A.; Mayerich, David; Slater, John H.

2017-01-01

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope...

Confocal laser scanning microscopy in vivo for diagnosing melanocytic skin neoplasms

Directory of Open Access Journals (Sweden)

A. A. Kubanova

2014-01-01

Full Text Available The authors discuss the use of confocal laser scanning microscopy in vivo (CLSM for diagnosing melanocytic skin neoplasms and its value for early diagnostics of melanoma. CLSM is an innovation noninvasive visual examination method for real-time multiple and painless examinations of the patient’s skin without injuring the skin integument. The method ensures early diagnostics of skin melanomas with high sensitivity and specificity, which makes it possible to use CLSM for screening melanocytic skin neoplasms for the sake of the early onset of treatment to save patient life and health.

Theoretical investigation of confocal microscopy using an elliptically polarized cylindrical vector laser beam: Visualization of quantum emitters near interfaces

Science.gov (United States)

Boichenko, Stepan

2018-04-01

We theoretically study laser-scanning confocal fluorescence microscopy using elliptically polarized cylindrical vector excitation light as a tool for visualization of arbitrarily oriented single quantum dipole emitters located (1) near planar surfaces enhancing fluorescence, (2) in a thin supported polymer film, (3) in a freestanding polymer film, and (4) in a dielectric planar microcavity. It is shown analytically that by using a tightly focused azimuthally polarized beam, it is possible to exclude completely the orientational dependence of the image intensity maximum of a quantum emitter that absorbs light as a pair of incoherent independent linear dipoles. For linear dipole quantum emitters, the orientational independence degree higher than 0.9 can normally be achieved (this quantity equal to 1 corresponds to completely excluded orientational dependence) if the collection efficiency of the microscope objective and the emitter's total quantum yield are not strongly orientationally dependent. Thus, the visualization of arbitrarily oriented single quantum emitters by means of the studied technique can be performed quite efficiently.

Study of laser-imploded core plasmas with an advanced Kirkpatrick endash Baez x-ray microscope

International Nuclear Information System (INIS)

Kodama, R.; Shiraga, H.; Miyanaga, M.; Matsushita, T.; Nakai, M.; Azechi, H.; Mima, K.; Kato, Y.

1997-01-01

We developed an advanced Kirkpatrick endash Baez (AKB) x-ray microscope which consisted of two hyperbolic mirrors and two elliptic mirrors. The spatial resolution of approx-lt 3 μm was realized over ∼1 mm diam. This AKB microscope was used for x-ray imaging in laser fusion experiments. Laser absorption nonuniformity with a large wave number on a spherical solid target or a plane slab target was estimated by measurements of x-ray emission from the target surface with the microscope. The x-ray images of the imploded core plasmas were also obtained with the AKB microscope, changing the laser focus condition and the laser energy balance. copyright 1997 American Institute of Physics

Multi-wavelength study of PPDs using an OPO tunable pulse laser microscope system

International Nuclear Information System (INIS)

Yoshimura, Koji; Nakamura, Isamu

2012-01-01

We have developed a new pulsed laser microscope system whose wavelength is continuously tunable from 410 nm to 2200 nm by using an optical parametric oscillator (OPO) laser system. The laser spot can be focused to ∼2μm diameter, small enough to measure pixel-by-pixel performance of PPDs (pixelated photon detectors). Using multi-wavelength laser light, we plan to probe PPDs at various depths, thanks to their different penetration lengths in the silicon layer. In this paper, details of the commissioning of the laser microscope system and pilot measurements on a PPD at several wavelengths will be presented.

Multi-wavelength study of PPDs using an OPO tunable pulse laser microscope system

Energy Technology Data Exchange (ETDEWEB)

Yoshimura, Koji, E-mail: koji.yoshimura@kek.jp [High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801 (Japan); Nakamura, Isamu [High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801 (Japan)

2012-12-11

We have developed a new pulsed laser microscope system whose wavelength is continuously tunable from 410 nm to 2200 nm by using an optical parametric oscillator (OPO) laser system. The laser spot can be focused to {approx}2{mu}m diameter, small enough to measure pixel-by-pixel performance of PPDs (pixelated photon detectors). Using multi-wavelength laser light, we plan to probe PPDs at various depths, thanks to their different penetration lengths in the silicon layer. In this paper, details of the commissioning of the laser microscope system and pilot measurements on a PPD at several wavelengths will be presented.

Histochemical study of trans-polyisoprene accumulation by spectral confocal laser scanning microscopy and a specific dye showing fluorescence solvatochromism in the rubber-producing plant, Eucommia ulmoides Oliver.

Science.gov (United States)

Nakazawa, Yoshihisa; Takeda, Tsuyoshi; Suzuki, Nobuaki; Hayashi, Tatsushi; Harada, Yoko; Bamba, Takeshi; Kobayashi, Akio

2013-09-01

A microscopic technique combining spectral confocal laser scanning microscopy with a lipophilic fluorescent dye, Nile red, which can emit trans-polyisoprene specific fluorescence, was developed, and unmixed images of synthesized trans-polyisoprene in situ in Eucommia ulmoides were successfully obtained. The images showed that trans-polyisoprene was initially synthesized as granules in non-articulated laticifers that changed shape to fibers during laticifer maturation. Non-articulated laticifers are developed from single laticiferous cells, which are differentiated from surrounding parenchyma cells in the cambium. Therefore, these observations suggested that trans-polyisoprene biosynthesis first started in laticifer cells as granules and then the granules accumulated and fused in the inner space of the laticifers over time. Finally, laticifers were filled with the synthesized trans-polyisoprene, which formed a fibrous structure fitting the laticifers shape. Both trans- and cis-polyisoprene are among the most important polymers naturally produced by plants, and this microscopic technique combined with histological study should provide useful information in the fields of plant histology, bioindustry and phytochemistry.

Bright-field scanning confocal electron microscopy using a double aberration-corrected transmission electron microscope.

Science.gov (United States)

Wang, Peng; Behan, Gavin; Kirkland, Angus I; Nellist, Peter D; Cosgriff, Eireann C; D'Alfonso, Adrian J; Morgan, Andrew J; Allen, Leslie J; Hashimoto, Ayako; Takeguchi, Masaki; Mitsuishi, Kazutaka; Shimojo, Masayuki

2011-06-01

Scanning confocal electron microscopy (SCEM) offers a mechanism for three-dimensional imaging of materials, which makes use of the reduced depth of field in an aberration-corrected transmission electron microscope. The simplest configuration of SCEM is the bright-field mode. In this paper we present experimental data and simulations showing the form of bright-field SCEM images. We show that the depth dependence of the three-dimensional image can be explained in terms of two-dimensional images formed in the detector plane. For a crystalline sample, this so-called probe image is shown to be similar to a conventional diffraction pattern. Experimental results and simulations show how the diffracted probes in this image are elongated in thicker crystals and the use of this elongation to estimate sample thickness is explored. Copyright © 2010 Elsevier B.V. All rights reserved.

Embryological study of Herminium monorchis (Orchidaceae) using confocal scanning laser microscopy

International Nuclear Information System (INIS)

Fredrikson, M.

1990-01-01

The embryology of Herminium monorchis (Orchidaceae) was studied using confocal scanning laser microscopy (CSLM), a new technique for embryological studies. This technique may contribute new information to plant embryology. Herminium monorchis has a monosporic embryo sac development. The mature embryo sac is 8-nucleate. Two integuments, both 2-layered, are formed, but only the inner takes part in formation of the micropyle. Double fertilization takes place. The primary endosperm nucleus does not divide, but remains alive at least at the 3-celled stage of embryo development. The three antipodals do not show any sign of degeneration at this stage. (author)

Recommendations for the design and the installation of large laser scanning microscopy systems

Science.gov (United States)

Helm, P. Johannes

2012-03-01

Laser Scanning Microscopy (LSM) has since the inventions of the Confocal Scanning Laser Microscope (CLSM) and the Multi Photon Laser Scanning Microscope (MPLSM) developed into an essential tool in contemporary life science and material science. The market provides an increasing number of turn-key and hands-off commercial LSM systems, un-problematic to purchase, set up and integrate even into minor research groups. However, the successful definition, financing, acquisition, installation and effective use of one or more large laser scanning microscopy systems, possibly of core facility character, often requires major efforts by senior staff members of large academic or industrial units. Here, a set of recommendations is presented, which are helpful during the process of establishing large systems for confocal or non-linear laser scanning microscopy as an effective operational resource in the scientific or industrial production process. Besides the description of technical difficulties and possible pitfalls, the article also illuminates some seemingly "less scientific" processes, i.e. the definition of specific laboratory demands, advertisement of the intention to purchase one or more large systems, evaluation of quotations, establishment of contracts and preparation of the local environment and laboratory infrastructure.

Investigation of phosphatidylcholine enhancing FITC-insulin across buccal mucosa by confocal laser scanning microscopy

Science.gov (United States)

Tian, Weiqun; Su, Li; Zeng, Shaoqun; Luo, Qingming; Gao, Qiuhua; Xu, Huibi

2002-04-01

The aim was to characterize the transport of fluorescein isothiocyanate (FITC)-labeled dextran and insulin with different resoluble compounds for peptides and proteins through buccal mucosa. The penetration rate of insulin molecules through porcine buccal mucosa (a nonkeratinized epithelium, comparable to human buccal mucosa) was investigated by measuring transbuccal fluxes and by analyzing the distribution of the fluorescent probe in the rabbit buccal mucosa epithelium, using confocal laser scanning microscopy for visualizing permeation pathways. The confocal images of the distribution pattern of FITC-dextran and FITC-insulin showed that the paracellular route is the major pathway of FITC-dextran through buccal mucosa epithelium, the intra-cellular route is the major pathway of FITC-insulin through buccal mucosa epithelium. The permeation rate can be increased by co-administration of soybean phosphatidylcholine (SPC).

Confocal laser endomicroscopy for diagnosis of Barrett´s esophagus

Directory of Open Access Journals (Sweden)

Helmut eNeumann

2012-05-01

Full Text Available Barrett´s esophagus (BE is established as a premalignant condition in the distal esophagus. Current surveillance guidelines recommend random biopsies every 1-2 cm at intervals of 3-5 years. Advanced endoscopic imaging of BE underwent several technical revolutions within the last decade including broad-field (red-flag techniques (e.g. chromoendoscopy and small-field techniques with confocal laser endomicroscopy (CLE at the forefront. In this review we will focus on advanced endoscopic imaging using CLE for the diagnosis and characterization of BE and associated neoplasia. In addition, we will critically discuss the technique of CLE and provide some tricks and hints for the daily routine practice of CLE for diagnosis of BE.

Naural Responses to Injury: Prevention, Protection, and Repair. Volume 8. Vision, Laser Eye Injury, and Infectious Diseases

National Research Council Canada - National Science Library

Bazan, Nicolas

1997-01-01

Four specific aims were proposed in the original grant application. Develop a new confocal microscope that can be used in living eyes to understand the earliest stages of trauma, laser injuries, and disease...

Fiber laser-microscope system for femtosecond photodisruption of biological samples.

Science.gov (United States)

Yavaş, Seydi; Erdogan, Mutlu; Gürel, Kutan; Ilday, F Ömer; Eldeniz, Y Burak; Tazebay, Uygar H

2012-03-01

We report on the development of a ultrafast fiber laser-microscope system for femtosecond photodisruption of biological targets. A mode-locked Yb-fiber laser oscillator generates few-nJ pulses at 32.7 MHz repetition rate, amplified up to ∼125 nJ at 1030 nm. Following dechirping in a grating compressor, ∼240 fs-long pulses are delivered to the sample through a diffraction-limited microscope, which allows real-time imaging and control. The laser can generate arbitrary pulse patterns, formed by two acousto-optic modulators (AOM) controlled by a custom-developed field-programmable gate array (FPGA) controller. This capability opens the route to fine optimization of the ablation processes and management of thermal effects. Sample position, exposure time and imaging are all computerized. The capability of the system to perform femtosecond photodisruption is demonstrated through experiments on tissue and individual cells.

3D Imaging of Porous Media Using Laser Scanning Confocal Microscopy with Application to Microscale Transport Processes

Energy Technology Data Exchange (ETDEWEB)

Fredrich, J.T.

1999-02-10

We present advances in the application of laser scanning confocal microscopy (LSCM) to image, reconstruct, and characterize statistically the microgeometry of porous geologic and engineering materials. We discuss technical and practical aspects of this imaging technique, including both its advantages and limitations. Confocal imaging can be used to optically section a material, with sub-micron resolution possible in the lateral and axial planes. The resultant volumetric image data, consisting of fluorescence intensities for typically {approximately}50 million voxels in XYZ space, can be used to reconstruct the three-dimensional structure of the two-phase medium. We present several examples of this application, including studying pore geometry in sandstone, characterizing brittle failure processes in low-porosity rock deformed under triaxial loading conditions in the laboratory, and analyzing the microstructure of porous ceramic insulations. We then describe approaches to extract statistical microgeometric descriptions from volumetric image data, and present results derived from confocal volumetric data sets. Finally, we develop the use of confocal image data to automatically generate a three-dimensional mesh for numerical pore-scale flow simulations.

4Pi-confocal microscopy of live cells

Science.gov (United States)

Bahlmann, Karsten; Jakobs, Stefan; Hell, Stefan W.

2002-06-01

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3 fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live cell microscopy.

Porosity of natural stone and use of confocal laser scanning microscopy on calcitic marble aged in laboratory

Directory of Open Access Journals (Sweden)

Ana Mladenovič

2008-06-01

Full Text Available Porosity is one of the key characteristics of natural stone, which influences ondurability as well as functionality of stone as building material. Further, deterioration processes themselves are also characterized by change of porosity. Different direct and indirect techniques can be used for porosity determination. In the following paper overview of these methods, as well as their advantages and disadvantages, is given. Confocal laser scanning microscopy (CLSM is indirect (microscopic technique. Despite its numerous advantages, among which 3D visualizationof pore structure is of major importance, this technique is less known in the area of building materials. An example how CLSM can be applied for qualitative and quantitative evaluation of porosity of calcitic polygonal granoblastic marble is given in this paper. Studied marble has been, despite of its poor durability, often used as building material, especially in the case of claddings. It is shown that thermal hydric factors of deterioration can influence porosity significantly,especially formation of intergranular cracks.This kind of deterioration can be successfully evaluated with use of CLSM method, if samples are suitable prepared and if suitable image analysis tools are developed.

Development of an add-on kit for scanning confocal microscopy (Conference Presentation)

Science.gov (United States)

Guo, Kaikai; Zheng, Guoan

2017-03-01

Scanning confocal microscopy is a standard choice for many fluorescence imaging applications in basic biomedical research. It is able to produce optically sectioned images and provide acquisition versatility to address many samples and application demands. However, scanning a focused point across the specimen limits the speed of image acquisition. As a result, scanning confocal microscope only works well with stationary samples. Researchers have performed parallel confocal scanning using digital-micromirror-device (DMD), which was used to project a scanning multi-point pattern across the sample. The DMD based parallel confocal systems increase the imaging speed while maintaining the optical sectioning ability. In this paper, we report the development of an add-on kit for high-speed and low-cost confocal microscopy. By adapting this add-on kit to an existing regular microscope, one can convert it into a confocal microscope without significant hardware modifications. Compared with current DMD-based implementations, the reported approach is able to recover multiple layers along the z axis simultaneously. It may find applications in wafer inspection and 3D metrology of semiconductor circuit. The dissemination of the proposed add-on kit under $1000 budget could also lead to new types of experimental designs for biological research labs, e.g., cytology analysis in cell culture experiments, genetic studies on multicellular organisms, pharmaceutical drug profiling, RNA interference studies, investigation of microbial communities in environmental systems, and etc.

Coherent anti-Stokes Raman scattering spectroscope/microscope based on a widely tunable laser source

Science.gov (United States)

Dementjev, A.; Gulbinas, V.; Serbenta, A.; Kaucikas, M.; Niaura, G.

2010-03-01

We present a coherent anti-Stokes Raman scattering (CARS) microscope based on a robust and simple laser source. A picosecond laser operating in a cavity dumping regime at the 1 MHz repetition rate was used to pump a traveling wave optical parametric generator, which serves as a two-color excitation light source for the CARS microscope. We demonstrate the ability of the presented CARS microscope to measure CARS spectra and images by using several detection schemes.

Line-scanning tomographic optical microscope with isotropic transfer function

International Nuclear Information System (INIS)

Gajdátsy, Gábor; Dudás, László; Erdélyi, Miklós; Szabó, Gábor

2010-01-01

An imaging method and optical system, referred to as a line-scanning tomographic optical microscope (LSTOM) using a combination of line-scanning technique and CT reconstruction principle, is proposed and studied theoretically and experimentally. In our implementation a narrow focus line is scanned over the sample and the reflected light is measured in a confocal arrangement. One such scan is equivalent to a transverse projection in tomography. Repeating the scanning procedure in several directions, a number of transverse projections are recorded from which the image can be obtained using conventional CT reconstruction algorithms. The resolution of the image is independent of the spatial dimensions and structure of the applied detector; furthermore, the transfer function of the system is isotropic. The imaging performance of the implemented confocal LSTOM was compared with a point-scanning confocal microscope, based on recorded images. These images demonstrate that the resolution of the confocal LSTOM exceeds (by 15%) the resolution limit of a point-scanning confocal microscope

In situ laser processing in a scanning electron microscope

Energy Technology Data Exchange (ETDEWEB)

Roberts, Nicholas A.; Magel, Gregory A.; Hartfield, Cheryl D.; Moore, Thomas M.; Fowlkes, Jason D.; Rack, Philip D. [Department of Materials Science and Engineering, University of Tennessee, Knoxville, Tennessee 37996 (United States) and Omniprobe, Inc., an Oxford Instruments Company, 10410 Miller Rd., Dallas, Texas 75238 (United States); Omniprobe, Inc., an Oxford Instruments Company, 10410 Miller Rd., Dallas, Texas 75238 (United States); Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (United States); Department of Materials Science and Engineering, University of Tennessee, Knoxville, Tennessee 37996 (United States) and Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (United States)

2012-07-15

Laser delivery probes using multimode fiber optic delivery and bulk focusing optics have been constructed and used for performing materials processing experiments within scanning electron microscope/focused ion beam instruments. Controlling the current driving a 915-nm semiconductor diode laser module enables continuous or pulsed operation down to sub-microsecond durations, and with spot sizes on the order of 50 {mu}m diameter, achieving irradiances at a sample surface exceeding 1 MW/cm{sup 2}. Localized laser heating has been used to demonstrate laser chemical vapor deposition of Pt, surface melting of silicon, enhanced purity, and resistivity via laser annealing of Au deposits formed by electron beam induced deposition, and in situ secondary electron imaging of laser induced dewetting of Au metal films on SiO{sub x}.

Scanning differential polarization microscope: Its use to image linear and circular differential scattering

International Nuclear Information System (INIS)

Mickols, W.; Maestre, M.F.

1988-01-01

A differential polarization microscope that couples the sensitivity of single-beam measurement of circular dichroism and circular differential scattering with the simultaneous measurement of linear dichroism and linear differential scattering has been developed. The microscope uses a scanning microscope stage and single-point illumination to give the very shallow depth of field found in confocal microscopy. This microscope can operate in the confocal mode as well as in the near confocal condition that can allow one to program the coherence and spatial resolution of the microscope. This microscope has been used to study the change in the structure of chromatin during the development of sperm in Drosophila

Analysis of mitochondrial mechanical dynamics using a confocal fluorescence microscope with a bent optical fibre.

Science.gov (United States)

Li, Yongbo; Honda, Satoshi; Iwami, Kentaro; Ohta, Yoshihiro; Umeda, Norihiro

2015-11-01

The cells in the cardiovascular system are constantly subjected to mechanical forces created by blood flow and the beating heart. The effect of forces on cells has been extensively investigated, but their effect on cellular organelles such as mitochondria remains unclear. We examined the impact of nano-Newton forces on mitochondria using a bent optical fibre (BOF) with a flat-ended tip (diameter exceeding 2 μm) and a confocal fluorescence microscope. By indenting a single mitochondrion with the BOF tip, we found that the mitochondrial elastic modulus was proportional to the (-1/2) power of the mitochondrial radius in the 9.6-115 kPa range. We stained the mitochondria with a potential-metric dye (TMRE) and measured the changes in TMRE fluorescence intensity. We confirmed that more active mitochondria exhibit a higher frequency of repetitive transient depolarization. The same trend was observed at forces lower than 50 nN. We further showed that the depolarization frequency of mitochondria decreases under an extremely large force (nearly 100 nN). We conclude that mitochondrial function is affected by physical environmental factors, such as external forces at the nano-Newton level. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Optical feedback effects on terahertz quantum cascade lasers: modelling and applications

Science.gov (United States)

Rakić, Aleksandar D.; Lim, Yah Leng; Taimre, Thomas; Agnew, Gary; Qi, Xiaoqiong; Bertling, Karl; Han, She; Wilson, Stephen J.; Kundu, Iman; Grier, Andrew; Ikonić, Zoran; Valavanis, Alexander; Demić, Aleksandar; Keeley, James; Li, Lianhe H.; Linfield, Edmund H.; Davies, A. Giles; Harrison, Paul; Ferguson, Blake; Walker, Graeme; Prow, Tarl; Indjin, Dragan; Soyer, H. Peter

2016-11-01

Terahertz (THz) quantum cascade lasers (QCLs) are compact sources of radiation in the 1-5 THz range with significant potential for applications in sensing and imaging. Laser feedback interferometry (LFI) with THz QCLs is a technique utilizing the sensitivity of the QCL to the radiation reflected back into the laser cavity from an external target. We will discuss modelling techniques and explore the applications of LFI in biological tissue imaging and will show that the confocal nature of the QCL in LFI systems, with their innate capacity for depth sectioning, makes them suitable for skin diagnostics with the well-known advantages of more conventional confocal microscopes. A demonstration of discrimination of neoplasia from healthy tissue using a THz, LFI-based system in the context of melanoma is presented using a transgenic mouse model.

Fluorescence confocal microscopy for pathologists.

Science.gov (United States)

Ragazzi, Moira; Piana, Simonetta; Longo, Caterina; Castagnetti, Fabio; Foroni, Monica; Ferrari, Guglielmo; Gardini, Giorgio; Pellacani, Giovanni

2014-03-01

Confocal microscopy is a non-invasive method of optical imaging that may provide microscopic images of untreated tissue that correspond almost perfectly to hematoxylin- and eosin-stained slides. Nowadays, following two confocal imaging systems are available: (1) reflectance confocal microscopy, based on the natural differences in refractive indices of subcellular structures within the tissues; (2) fluorescence confocal microscopy, based on the use of fluorochromes, such as acridine orange, to increase the contrast epithelium-stroma. In clinical practice to date, confocal microscopy has been used with the goal of obviating the need for excision biopsies, thereby reducing the need for pathological examination. The aim of our study was to test fluorescence confocal microscopy on different types of surgical specimens, specifically breast, lymph node, thyroid, and colon. The confocal images were correlated to the corresponding histological sections in order to provide a morphologic parallel and to highlight current limitations and possible applications of this technology for surgical pathology practice. As a result, neoplastic tissues were easily distinguishable from normal structures and reactive processes such as fibrosis; the use of fluorescence enhanced contrast and image quality in confocal microscopy without compromising final histologic evaluation. Finally, the fluorescence confocal microscopy images of the adipose tissue were as accurate as those of conventional histology and were devoid of the frozen-section-related artefacts that can compromise intraoperative evaluation. Despite some limitations mainly related to black/white images, which require training in imaging interpretation, this study confirms that fluorescence confocal microscopy may represent an alternative to frozen sections in the assessment of margin status in selected settings or when the conservation of the specimen is crucial. This is the first study to employ fluorescent confocal microscopy on

Potential of confocal laser scanning microscopy for non-invasive diagnostics of malignant epithelial skin tumors in the course of dermatoheliosis progression

Directory of Open Access Journals (Sweden)

E. S. Snarskaya

2016-01-01

Full Text Available Most cases of malignant epithelial skin neoplasms including actinic keratosis and basal cell carcinoma, which are characterized by the most complicated course and numerous clinical and morphological options, involve dermatoheliosis progression. The risk of actinic keratosis transformation into basal cell carcinoma varies from 0.1% to 20% and up to 80% in cases of multiple AK lesion foci. A non-invasive method known as reflectance confocal laser scanning microscopy is the most promising one for the purposes of early diagnostics of signs pointing at epithelial skin neoplasm development and makes it possible to monitor the tumor in progress in vivo to diagnose the presence of a pool of squamous cells on a timely basis. The confocal laser scanning microscopy method provides high-contrast images of for any horizontal-oriented morphologic structures in the epidermis and upper dermis with a resolution comparable to those characteristic of traditional optical microscopy of skin tissue samples. According to our data obtained as a result of studying dynamic changes and morphologic structures in actinic keratosis foci (50 cases using the confocal laser scanning microscopy method, we discovered a number of morphologic features, and their further analysis will distinguish the signs of progressing carcinogenesis in case of dermatoheliosis.

Microscopia confocal en operados de queratoplastia perforante Confocal microscopy in patients operated from penetrating keratoplasty

Directory of Open Access Journals (Sweden)

Zulema Gómez Castillo

2009-06-01

Full Text Available La microscopia confocal es un examen exploratorio, práctico y poco invasivo que permite conocer las características microscópicas del tejido corneal después del trasplante, por lo que constituye una herramienta muy útil en el manejo de los pacientes operados de queratoplastia. El presente trabajo tiene como finalidad describir las características del tejido corneal en pacientes operados de este tipo de trasplante, mediante la microscopia confocal in vivo. MÉTODOS: Se realizó un estudio descriptivo, de corte transversal, en 40 ojos de 40 pacientes operados de queratoplastia perforante, en el Servicio de Córnea del Instituto Cubano de Oftalmología "Ramón Pando Ferrer", de marzo de 2006 a marzo de 2007. Se confeccionó una historia clínica oftalmológica y se les realizó a todos el examen de microscopia confocal en el injerto corneal con el microscopio confocal CONFOSCAN 4. RESULTADOS: La queratopatía bullosa pseudofáquica fue la afección más frecuente previa a la cirugía y estuvo presente en el 77,5 % de los pacientes. En el 72,5 % de los intervenidos se encontró una disminución del grosor corneal. El epitelio presentó alteraciones en el 62,5 % de los pacientes. Todos presentaron afectación de la forma y el tamaño celular endotelial. En el 82,5 % de los pacientes se observó ausencia de plexos nerviosos. CONCLUSIONES: La microscopia confocal como nueva ciencia en el campo de la oftalmología, favorece el seguimiento evolutivo de las queratoplastias perforantes y con esto no solo a prevenir la aparición de posibles complicaciones, sino además de garantizar el éxito de la cirugía y la función refractiva de la córnea.Confocal microscopy is a practical, exploratory and less invassive examination that allows finding out the microscopic characteristics of the corneal tissue after transplantation, so it is a very useful tool for the management of patients operated from keratoplasty. The present paper was aimed at describing

In vitro studies of Rickettsia-host cell interactions: Confocal laser scanning microscopy of Rickettsia helvetica-infected eukaryotic cell lines.

Science.gov (United States)

Speck, Stephanie; Kern, Tanja; Aistleitner, Karin; Dilcher, Meik; Dobler, Gerhard; Essbauer, Sandra

2018-02-01

Rickettsia (R.) helvetica is the most prevalent rickettsia found in Ixodes ricinus ticks in Germany. Several studies reported antibodies against R. helvetica up to 12.5% in humans investigated, however, fulminant clinical cases are rare indicating a rather low pathogenicity compared to other rickettsiae. We investigated growth characteristics of R. helvetica isolate AS819 in two different eukaryotic cell lines with focus on ultra-structural changes of host cells during infection determined by confocal laser scanning microscopy. Further investigations included partially sequencing of rickA, sca4 and sca2 genes, which have been reported to encode proteins involved in cell-to-cell spread and virulence in some rickettsiae. R. helvetica grew constantly but slowly in both cell lines used. Confocal laser scanning microscopy revealed that the dissemination of R. helvetica AS819 in both cell lines was rather mediated by cell break-down and bacterial release than cell-to-cell spread. The cytoskeleton of both investigated eukaryotic cell lines was not altered. R. helvetica possesses rickA, but its expression is not sufficient to promote actin-based motility as demonstrated by confocal laser scanning microscopy. Hypothetical Sca2 and Sca4 proteins were deduced from nucleotide gene sequences but the predicted amino acid sequences were disrupted or truncated compared to other rickettsiae most likely resulting in non-functional proteins. Taken together, these results might give a first hint to the underlying causes of the reduced virulence and pathogenicity of R. helvetica.

Cryogenic scanning laser microscopy. Investigation of large BSCCO mesas and development of a polarizing microscope

International Nuclear Information System (INIS)

Guenon, Stefan Alexander

2011-01-01

confirmed that the frequency of the emitted radiation and the bias voltage is determined by the Josephson relation for a wide range of different base temperatures. This way other mechanisms, causing THz radiation, rather than the Josephson effect can be excluded. Concerning the second part: Originally it was planned to extend the low-temperature scanning laser microscope with the facility of polarizing microscopy. The idea was to combine the LTSLM voltage imaging with the possibility of magneto-optical imaging. But it soon turned out that a new design would be necessary. A laser scanning polarizing microscope has certain advantages in comparison with a conventional polarizing microscope: Very high illumination intensities can be reached easily, the resolution can be improved by the factor 1.4 if a confocal optical design is used, and the serial signal processing facilitates the optimization of the signal-to-noise ratio. In addition, it is usually not necessary to remove the contrast of non-magnetic origin by subtracting an image of the uniform magnetized sample from the image of interest. In this thesis a design for a cryogenic scanning polarizing microscope (CSPM) is discussed in detail, tests and first results of the system are presented, and an outlook is given how two proceed with this project.

Optical sectioning using a digital Fresnel incoherent-holography-based confocal imaging system

OpenAIRE

Kelner, Roy; Katz, Barak; Rosen, Joseph

2014-01-01

We propose a new type of confocal microscope using Fresnel incoherent correlation holography (FINCH). Presented here is a confocal configuration of FINCH using a phase pinhole and point illumination that is able to suppress out-of-focus information from the recorded hologram and hence combine the super-resolution capabilities of FINCH with the sectioning capabilities of confocal microscopy.

The fractional laser-induced coagulation zone characterized over time by laser scanning confocal microscopy-A proof of concept study.

Science.gov (United States)

Banzhaf, Christina A; Lin, Lynlee L; Dang, Nhung; Freeman, Michael; Haedersdal, Merete; Prow, Tarl W

2018-01-01

Ablative fractional laser (AFXL) is an acknowledged technique to increase uptake of topical agents in skin. Micro thermal ablation zones (MAZs) consist of ablated vertical channels surrounded by a coagulation zone (CZ). Laser scanning confocal microscopy (LSCM) images individual MAZs at 733 nm (reflectance confocal microscopy (RCM)). Further, LSCM can image sodium fluorescein (NaF) fluorescence with 488 nm excitation (fluorescence confocal microcopy (FCM)), a small hydrophilic test molecule (370 MW, log P -1.52), which may simulate uptake, bio-distribution and kinetics of small hydrophilic drugs. To explore LSCM for combined investigations of CZ thickness and uptake, bio-distribution and kinetics of NaF in AFXL-exposed skin. Excised human abdominal skin samples were exposed to AFXL (15 mJ/microbeam, 2% density) and NaF gel (1000 μg/ml, 10 μl/cm2) in six repetitions, including untreated control samples. CZ thickness and spatiotemporal fluorescence intensities (FI) were quantified up to four hours after NaF application by RCM and FCM. Test sites were scanned to a depth of 200 μm, quantifying thickness of skin compartments (stratum corneum, epidermis, upper dermis), individual CZ thicknesses and FI in CZ and surrounding skin. RCM images established skin morphology to a depth of 200 μm. The CZ thickness measurements were feasible to a depth of 50 μm, and remained unchanged over time at 50 μm (P > 0.5). FI were detected to a depth of 160 μm and remained constant in CZ up to four hours after NaF application (15 minutes: 79 AU (73-92 AU), 60 minutes: 72 AU (58-82 AU), four hours: 78 AU (71-90 AU), P > 0.1). In surrounding skin, FI increased significantly over time, but remained lower than FI in CZ (15 minutes: 21 AU (17-22 AU), 60 minutes: 21 AU (19-26 AU), four hours: 42 (31- 48 AU), P = 0.03). AFXL-processed skin generated higher FI compared to non-laser processed skin in epidermis and upper dermis at 60 minutes and four hours

X-ray generation by femtosecond laser pulses and its application to soft X-ray imaging microscope

International Nuclear Information System (INIS)

Ikeda, Kenichi; Kotaki, Hideyuki; Nakajima, Kazuhisa

2002-01-01

We have developed laser-produced plasma X-ray sources using femtosecond laser pulses at 10Hz repetition rate in a table-top size in order to investigate basic mechanism of X-ray emission from laser-matter interactions and its application to a X-ray microscope. In a soft X-ray region over 5 nm wavelength, laser-plasma X-ray emission from a solid target achieved an intense flux of photons of the order of 1011 photons/rad per pulse with duration of a few 100 ps, which is intense enough to make a clear imaging in a short time exposure. As an application of laser-produced plasma X-ray source, we have developed a soft X-ray imaging microscope operating in the wavelength range around 14 nm. The microscope consists of a cylindrically ellipsoidal condenser mirror and a Schwarzshird objective mirror with highly-reflective multilayers. We report preliminary results of performance tests of the soft X-ray imaging microscope with a compact laser-produced plasma X-ray source

Confocal Raman Microscopy

CERN Document Server

Dieing, Thomas; Toporski, Jan

2011-01-01

Confocal Raman Microscopy is a relatively new technique that allows chemical imaging without specific sample preparation. By integrating a sensitive Raman spectrometer within a state-of-the-art microscope, Raman microscopy with a spatial resolution down to 200nm laterally and 500nm vertically can be achieved using visible light excitation. Recent developments in detector and computer technology as well as optimized instrument design have reduced integration times of Raman spectra by orders of magnitude, so that complete images consisting of tens of thousands of Raman spectra can be acquired in seconds or minutes rather than hours, which used to be standard just one decade ago. The purpose of this book is to provide the reader a comprehensive overview of the rapidly developing field of Confocal Raman Microscopy and its applications.

Parameters in selective laser melting for processing metallic powders

Science.gov (United States)

Kurzynowski, Tomasz; Chlebus, Edward; Kuźnicka, Bogumiła; Reiner, Jacek

2012-03-01

The paper presents results of studies on Selective Laser Melting. SLM is an additive manufacturing technology which may be used to process almost all metallic materials in the form of powder. Types of energy emission sources, mainly fiber lasers and/or Nd:YAG laser with similar characteristics and the wavelength of 1,06 - 1,08 microns, are provided primarily for processing metallic powder materials with high absorption of laser radiation. The paper presents results of selected variable parameters (laser power, scanning time, scanning strategy) and fixed parameters such as the protective atmosphere (argon, nitrogen, helium), temperature, type and shape of the powder material. The thematic scope is very broad, so the work was focused on optimizing the process of selective laser micrometallurgy for producing fully dense parts. The density is closely linked with other two conditions: discontinuity of the microstructure (microcracks) and stability (repeatability) of the process. Materials used for the research were stainless steel 316L (AISI), tool steel H13 (AISI), and titanium alloy Ti6Al7Nb (ISO 5832-11). Studies were performed with a scanning electron microscope, a light microscopes, a confocal microscope and a μCT scanner.

SU-F-T-665: Confocal Microscopy Imaging of Cell Cycle Distribution in Cells Treated with Pegylated Gold Nanoshells

Energy Technology Data Exchange (ETDEWEB)

Sadetaporn, D [Rice University, Houston, TX (United States); The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Flint, D; McFadden, C; Sawakuchi, G [The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Asaithamby, A [UT Southwestern Medical Center, Dallas, TX (United States)

2016-06-15

Purpose: To use confocal microscopy to distinguish cells in different phases of the cell cycle before and after treatment with pegylated gold nanoshells (PEG-AuNSs). Methods: Transfected fibrosarcoma cells (HT1080-EYFP-53BP1-FUCCI) were cultured in T-25 flasks and seeded in glass bottom dishes. These cells express the fluorescent probe AmCyan during the G2/S phases of the cell cycle, mCherry during the G1 phase, and EYFP tagged to the DNA repair protein 53BP1. After allowing cells 4 h to adhere to dishes, PEG-AuNS (Nanospectra Biosciences, Houston, TX) at a concentration of 0.15 OD were administered. At time points of 8, 16 and 24 h following treatment, the PEG-AuNS-treated and control samples were washed with phosphate buffered saline (PBS) and fixed using 4% paraformaldehyde in PBS. Samples were imaged with an Olympus FV1200 confocal microscope using 473, 543, and 641 nm excitation lasers. We used band-pass filters to select AmCyan and mCherry fluorescence. Reflection from the 641 nm laser was used to detect PEG-AuNSs. Z-stack images were analyzed to assess cell cycle distribution through fluorescent probe expression. Live cells were imaged after PEG-AuNS treatment using a confocal microscope with a stage top CO2 incubator. Results: We were able to obtain high-resolution images of cells with internalized AuNSs. We were also able to distinguish cells in different phases of the cell cycle. Conclusion: This work demonstrates a new assay to investigate the effect of AuNSs on the cell cycle phase in live cells. Future work will employ confocal microscopy and flow cytometry to focus on effects of AuNS treatment on cell cycle distribution. This research was supported by the Sister Institution Network Fund and the Center for Radiation Oncology Research at The University of Texas MD Anderson Cancer Center and Cancer Prevention and Research Institute of Texas. Gabriel Sawakuchi has research support from Elekta Inc.

SU-F-T-665: Confocal Microscopy Imaging of Cell Cycle Distribution in Cells Treated with Pegylated Gold Nanoshells

International Nuclear Information System (INIS)

Sadetaporn, D; Flint, D; McFadden, C; Sawakuchi, G; Asaithamby, A

2016-01-01

Purpose: To use confocal microscopy to distinguish cells in different phases of the cell cycle before and after treatment with pegylated gold nanoshells (PEG-AuNSs). Methods: Transfected fibrosarcoma cells (HT1080-EYFP-53BP1-FUCCI) were cultured in T-25 flasks and seeded in glass bottom dishes. These cells express the fluorescent probe AmCyan during the G2/S phases of the cell cycle, mCherry during the G1 phase, and EYFP tagged to the DNA repair protein 53BP1. After allowing cells 4 h to adhere to dishes, PEG-AuNS (Nanospectra Biosciences, Houston, TX) at a concentration of 0.15 OD were administered. At time points of 8, 16 and 24 h following treatment, the PEG-AuNS-treated and control samples were washed with phosphate buffered saline (PBS) and fixed using 4% paraformaldehyde in PBS. Samples were imaged with an Olympus FV1200 confocal microscope using 473, 543, and 641 nm excitation lasers. We used band-pass filters to select AmCyan and mCherry fluorescence. Reflection from the 641 nm laser was used to detect PEG-AuNSs. Z-stack images were analyzed to assess cell cycle distribution through fluorescent probe expression. Live cells were imaged after PEG-AuNS treatment using a confocal microscope with a stage top CO2 incubator. Results: We were able to obtain high-resolution images of cells with internalized AuNSs. We were also able to distinguish cells in different phases of the cell cycle. Conclusion: This work demonstrates a new assay to investigate the effect of AuNSs on the cell cycle phase in live cells. Future work will employ confocal microscopy and flow cytometry to focus on effects of AuNS treatment on cell cycle distribution. This research was supported by the Sister Institution Network Fund and the Center for Radiation Oncology Research at The University of Texas MD Anderson Cancer Center and Cancer Prevention and Research Institute of Texas. Gabriel Sawakuchi has research support from Elekta Inc.

Confocal imaging of protein distributions in porous silicon optical structures

International Nuclear Information System (INIS)

De Stefano, Luca; D'Auria, Sabato

2007-01-01

The performances of porous silicon optical biosensors depend strongly on the arrangement of the biological probes into their sponge-like structures: it is well known that in this case the sensing species do not fill the pores but instead cover their internal surface. In this paper, the direct imaging of labelled proteins into different porous silicon structures by using a confocal laser microscope is reported. The distribution of the biological matter in the nanostructured material follows a Gaussian behaviour which is typical of the diffusion process in the porous media but with substantial differences between a porous silicon monolayer and a multilayer such as a Bragg mirror. Even if semi-quantitative, the results can be very useful in the design of the porous silicon based biosensing devices

Inverted follicular keratosis: dermoscopic and reflectance confocal microscopic features.

Science.gov (United States)

Armengot-Carbo, M; Abrego, A; Gonzalez, T; Alarcon, I; Alos, L; Carrera, C; Malvehy, J; Puig, S

2013-01-01

Inverted follicular keratosis (IFK) is a rare benign tumor which usually appears as a firm papule on the face. The diagnosis is generally made by histopathology because the clinical appearance is difficult to differentiate from other lesions. Dermoscopic features of IFK have not been established to date. Herein we describe the dermoscopic findings of 4 cases of IFK. Radial peripheral hairpin vessels surrounded by a whitish halo arranged around a central white-yellowish amorphous area were observed in 3 cases, and glomerular vessels were present in the central area of one of them. The fourth case also presented a central white amorphous area but showed arborizing vessels. Reflectance confocal microscopy (available in 1 case) revealed a broadened honeycomb pattern, epidermal projections and hairpin and glomerular vessels. To our knowledge this is the first case series describing the dermoscopic features of inverted follicular keratosis and the first confocal microscopy description of this entity.

Spatiotemporal closure of fractional laser-ablated channels imaged by optical coherence tomography and reflectance confocal microscopy

DEFF Research Database (Denmark)

Banzhaf, Christina A.; Wind, Bas S.; Mogensen, Mette

2016-01-01

Background and Objective Optical coherence tomography (OCT) and reflectance confocal microscopy (RCM) offer high-resolution optical imaging of the skin, which may provide benefit in the context of laser-assisted drug delivery. We aimed to characterize postoperative healing of ablative fractional...... laser (AFXL)-induced channels and dynamics in their spatiotemporal closure using in vivo OCT and RCM techniques. Study design/Materials and Methods The inner forearm of healthy subjects (n = 6) was exposed to 10,600 nm fractional CO2 laser using 5 and 25% densities, 120 μm beam diameter, 5, 15, and 25 m......J/microbeam. Treatment sites were scanned with OCT to evaluate closure of AFXL-channels and RCM to evaluate subsequent re-epithelialization. Results OCT and RCM identified laser channels in epidermis and upper dermis as black, ablated tissue defects surrounded by characteristic hyper-and hyporeflective zones. OCT imaged...

Optical sectioning using a digital Fresnel incoherent-holography-based confocal imaging system

Science.gov (United States)

Kelner, Roy; Katz, Barak; Rosen, Joseph

2015-01-01

We propose a new type of confocal microscope using Fresnel incoherent correlation holography (FINCH). Presented here is a confocal configuration of FINCH using a phase pinhole and point illumination that is able to suppress out-of-focus information from the recorded hologram and hence combine the super-resolution capabilities of FINCH with the sectioning capabilities of confocal microscopy. PMID:26413560

Near-Infrared Confocal Laser Reflectance Cytoarchitectural Imaging of the Substantia Nigra and Cerebellum in the Fresh Human Cadaver.

Science.gov (United States)

Cheyuo, Cletus; Grand, Walter; Balos, Lucia L

2017-01-01

Cytoarchitectural neuroimaging remains critical for diagnosis of many brain diseases. Fluorescent dye-enhanced, near-infrared confocal in situ cellular imaging of the brain has been reported. However, impermeability of the blood-brain barrier to most fluorescent dyes limits clinical utility of this modality. The differential degree of reflectance from brain tissue with unenhanced near-infrared imaging may represent an alternative technique for in situ cytoarchitectural neuroimaging. We assessed the utility of unenhanced near-infrared confocal laser reflectance imaging of the cytoarchitecture of the cerebellum and substantia nigra in 2 fresh human cadaver brains using a confocal near-infrared laser probe. Cellular images based on near-infrared differential reflectance were captured at depths of 20-180 μm from the brain surface. Parts of the cerebellum and substantia nigra imaged using the probe were subsequently excised and stained with hematoxylin and eosin for histologic correlation. Near-infrared reflectance imaging revealed the 3-layered cytoarchitecture of the cerebellum, with Purkinje cells appearing hyperreflectant. In the substantia nigra, neurons appeared hyporeflectant with hyperreflectant neuromelanin cytoplasmic inclusions. Cytoarchitecture of the cerebellum and substantia nigra revealed on near-infrared imaging closely correlated with the histology on hematoxylin-eosin staining. We showed that unenhanced near-infrared reflectance imaging of fresh human cadaver brain can reliably identify and distinguish neurons and detailed cytoarchitecture of the cerebellum and substantia nigra. Copyright © 2016 Elsevier Inc. All rights reserved.

Collection of trace evidence of explosive residues from the skin in a death due to a disguised letter bomb. The synergy between confocal laser scanning microscope and inductively coupled plasma atomic emission spectrometer analyses.

Science.gov (United States)

Turillazzi, Emanuela; Monaci, Fabrizio; Neri, Margherita; Pomara, Cristoforo; Riezzo, Irene; Baroni, Davide; Fineschi, Vittorio

2010-04-15

In most deaths caused by explosive, the victim's body becomes a depot for fragments of explosive materials, so contributing to the collection of trace evidence which may provide clues about the specific type of device used with explosion. Improvised explosive devices are used which contain "homemade" explosives rather than high explosives because of the relative ease with which such components can be procured. Many methods such as chromatography-mass spectrometry, scanning electron microscopy, stereomicroscopy, capillary electrophoresis are available for use in the identification of explosive residues on objects and bomb fragments. Identification and reconstruction of the distribution of explosive residues on the decedent's body may give additional hints in assessing the position of the victim in relation to the device. Traditionally these residues are retrieved by swabbing the body and clothing during the early phase, at autopsy. Gas chromatography-mass spectrometry and other analytical methods may be used to analyze the material swabbed from the victim body. The histological examination of explosive residues on skin samples collected during the autopsy may reveal significant details. The information about type, quantity and particularly about anatomical distribution of explosive residues obtained utilizing confocal laser scanning microscope (CLSM) together with inductively coupled plasma atomic emission spectrometer (ICP-AES), may provide very significant evidence in the clarification and reconstruction of the explosive-related events. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

The binding of cellulase variants to dislocations: a semi-quantitative analysis based on CLSM (confocal laser scanning microscopy) images

DEFF Research Database (Denmark)

Hidayat, Budi J.; Weisskopf, Carmen; Felby, Claus

2015-01-01

or slip planes. Here we study whether cellulases bind to dislocations to a higher extent than to the surrounding cell wall. The binding of fluorescently labelled cellobiohydrolases and endoglucanases to filter paper fibers was investigated using confocal laser scanning microscopy and a ratiometric method...

The simplicity of males: Dwarf males of four species of Osedax (Siboglinidae; Annelida) investigated by confocal laser scanning microscopy

DEFF Research Database (Denmark)

Worsaae, Katrine; Rouse, Greg W

2010-01-01

. Here, we present the first investigation of the entire muscle and nervous system in dwarf males of Osedax frankpressi, O. roseus, O. rubiplumus, and O. spiral analyzed by multistaining and confocal laser scanning microscopy. Sperm shape and spermiogenesis, the sperm duct and internal and external...

In vivo subsurface morphological and functional cellular and subcellular imaging of the gastrointestinal tract with confocal mini-microscopy

Institute of Scientific and Technical Information of China (English)

Martin Goetz; Beena Memadathil; Stefan Biesterfeld; Constantin Schneider; Sebastian Gregor; Peter R Galle; Markus F Neurath; Ralf Kiesslich

2007-01-01

AIM: To evaluate a newly developed hand-held confocal probe for in vivo microscopic imaging of the complete gastrointestinal tract in rodents.METHODS: A novel rigid confocal probe (diameter 7 mm) was designed with optical features similar to the flexible endomicroscopy system for use in humans using a 488 nm single line laser for fluorophore excitation.Light emission was detected at 505 to 750 nm. The field of view was 475 μm × 475 μm. Optical slice thickness was 7 μm with a lateral resolution of 0.7 μm. Subsurface serial images at different depths (surface to 250 μm)were generated in real time at 1024 × 1024 pixels (0.8 frames/s) by placing the probe onto the tissue in gentle,stable contact. Tissue specimens were sampled for histopathological correlation.RESULTS: The esophagus, stomach, small and large intestine and meso, liver, pancreas and gall bladder were visualised in vivo at high resolution in n = 48 mice.Real time microscopic imaging with the confocal minimicroscopy probe was easy to achieve. The different staining protocols (fluorescein, acriflavine, FITC-labelled dextran and L. esculentum lectin) each highlighted specific aspects of the tissue, and in vivo imaging correlated excellently with conventional histology. In vivo blood flow monitoring added a functional quality to morphologic imaging.CONCLUSION: Confocal microscopy is feasible in vivo allowing the visualisation of the complete GI tract at high resolution even of subsurface tissue structures.The new confocal probe design evaluated in this study is compatible with laparoscopy and significantly expands the field of possible applications to intra-abdominal organs. It allows immediate testing of new in vivo staining and application options and therefore permits rapid transfer from animal studies to clinical use in patients.

Development of laser plasma x-ray microscope for living hydrated biological specimens

International Nuclear Information System (INIS)

Kado, Masataka; Daido, Hiroyuki

2005-01-01

Investigating the structure and the function of life object performing advanced life activity becomes important. In order to investigate the life object, it is necessary to observe living specimens with high spatial resolution and high temporal resolution. Since laser plasma x-ray source has high brightness and short pulse duration, x-ray microscope with the laser plasma x-ray source makes possible to observe living specimens. Such as chromosomes, macrophages, bacterium, and so on have been observed by contact x-ray microscopy. The x-ray images obtained by indirect measurements such as the contact x-ray microscopy have difficulty to avoid artificial effect such as irregular due to developing process. Development of an x-ray microscope with laser plasma x-ray source is necessary to avoid such defects. (author)

Fiber optical laser spot microscope: A new concept for photoelectrochemical characterization of semiconductor electrodes

OpenAIRE

Carlsson, Per; Holmström, Bertil; Uosaki, Kohei; Kita, Hideaki

1988-01-01

A fiber optical laser spot microscope, which allows the simultaneous measurements of photocurrent and reflected light intensity or the measurement of laser spot photocurrent under the illumination of other light sources, has been developed to study semiconductor/electrolyte interfaces. The capability of this microscope was demonstrated on as-cleaved and Pt-treated p-InSe. The Pt treatment increased the photocurrent and improved the lateral resolution due to the increase of surface reaction ra...

Polarization-preserving confocal microscope for optical experiments in a dilution refrigerator with high magnetic field.

Science.gov (United States)

Sladkov, Maksym; Bakker, M P; Chaubal, A U; Reuter, D; Wieck, A D; van der Wal, C H

2011-04-01

We present the design and operation of a fiber-based cryogenic confocal microscope. It is designed as a compact cold-finger that fits inside the bore of a superconducting magnet, and which is a modular unit that can be easily swapped between use in a dilution refrigerator and other cryostats. We aimed at application in quantum optical experiments with electron spins in semiconductors and the design has been optimized for driving with and detection of optical fields with well-defined polarizations. This was implemented with optical access via a polarization maintaining fiber together with Voigt geometry at the cold finger, which circumvents Faraday rotations in the optical components in high magnetic fields. Our unit is versatile for use in experiments that measure photoluminescence, reflection, or transmission, as we demonstrate with a quantum optical experiment with an ensemble of donor-bound electrons in a thin GaAs film. © 2011 American Institute of Physics

Laser-induced microscopic phase-transition on an ionic liquid

International Nuclear Information System (INIS)

Iguchi, Natsuki; Datta, Alokmay; Yoshikawa, Kenichi; Ma Yue

2009-01-01

Nematic-isotropic transition is induced in a 5 μm 'droplet' within an oriented bulk of a mixture of a liquid crystalline material with a room-temperature ionic liquid, by a laser working at 532 nm with an output power of 200 mW and a beam diameter of 1 μm. No microscopic phase transition is observed either in absence of the ionic liquid or at the other wavelength of 1064 nm, available to the Nd-YAG laser. This indicates the essential role on a resonant transfer of energy to the ionic liquid from the laser radiation, which is subsequently transferred to the liquid crystal. Spectroscopy of the pure liquid crystal and ionic liquid samples confirms this concept. Spatio-temporal image of the droplet growth shows, however, that the phase transition remains confined within the microscopic domain for the first 50 s, and then spreads out rapidly. Since resonant, quantum transitions between molecular levels takes place in less than microseconds, the about seven orders of magnitude slowing down of energy transfer observed here suggests unique hierarchical dynamics including the coupling between the intra-molecular motions in the ionic liquid and the inter-molecular forces between ionic liquid and liquid crystal.

Combination of Small Molecule Microarray and Confocal Microscopy Techniques for Live Cell Staining Fluorescent Dye Discovery

Directory of Open Access Journals (Sweden)

Attila Bokros

2013-08-01

Full Text Available Discovering new fluorochromes is significantly advanced by high-throughput screening (HTS methods. In the present study a combination of small molecule microarray (SMM prescreening and confocal laser scanning microscopy (CLSM was developed in order to discover novel cell staining fluorescent dyes. Compounds with high native fluorescence were selected from a 14,585-member library and further tested on living cells under the microscope. Eleven compartment-specific, cell-permeable (or plasma membrane-targeted fluorochromes were identified. Their cytotoxicity was tested and found that between 1–10 micromolar range, they were non-toxic even during long-term incubations.

Influence of operating microscope in the sealing of cervical perforations.

Science.gov (United States)

Schmidt, Bruna Schwingel; Zaccara, Ivana Maria; Reis Só, Marcus Vinícius; Kuga, Milton Carlos; Palma-Dibb, Regina Guenka; Kopper, Patrícia Maria Poli

2016-01-01

Accidental root canal perforations are among the main complications of endodontic treatment. This study evaluated the influence of operating microscope (OM) in the marginal adaptation of mineral trioxide aggregate (MTA) (Angelus(®)) and glass ionomer (Vitremer) inserted into cervical perforations. Perforations were made in the cervical third of the buccal wall of the root canal in mandibular incisors. Next, the teeth were divided into four groups (N = 10): MG - MTA without OM; VG - Vitremer without OM; MOMG - MTA with OM; VOMG - Vitremer with OM. The perforations were sealed according to the group and the teeth were prepared for analysis by confocal laser scanning microscope. Images of perforation region (1,024×) were made and the gap presented by the materials was measured using the Image J program. LEXT OLS4100 three dimensional (3D) measuring laser microscope measured the volumetric misfit. Data of gap were analyzed by Kruskal-Wallis and Dunn's tests. Analysis of variance (ANOVA) and Tukey's tests compared the volumetric misfits. The results showed lower volume and gap in the interface dentin/material in VOMG compared to the other groups (P < 0.05). The use of OM improved the quality of cervical perforations sealed with Vitremer, being indicated in clinical situations of iatrogenic cervical perforations.

Laser speckle contrast imaging using light field microscope approach

Science.gov (United States)

Ma, Xiaohui; Wang, Anting; Ma, Fenghua; Wang, Zi; Ming, Hai

2018-01-01

In this paper, a laser speckle contrast imaging (LSCI) system using light field (LF) microscope approach is proposed. As far as we known, it is first time to combine LSCI with LF. To verify this idea, a prototype consists of a modified LF microscope imaging system and an experimental device was built. A commercially used Lytro camera was modified for microscope imaging. Hollow glass tubes with different depth fixed in glass dish were used to simulate the vessels in brain and test the performance of the system. Compared with conventional LSCI, three new functions can be realized by using our system, which include refocusing, extending the depth of field (DOF) and gathering 3D information. Experiments show that the principle is feasible and the proposed system works well.

Adipocyte size and cellular expression of caveolar proteins analyzed by confocal microscopy

DEFF Research Database (Denmark)

Hulstrøm, Veronica; Prats Gavalda, Clara; Vinten, Jørgen

2013-01-01

Caveolae are abundant in adipocytes and are involved in the regulation of lipid accumulation, which is the main volume determinant of these cells. We have developed and applied a confocal microscopic technique for measuring individual cellular expression of the caveolar proteins cavin-1 and caveo......Caveolae are abundant in adipocytes and are involved in the regulation of lipid accumulation, which is the main volume determinant of these cells. We have developed and applied a confocal microscopic technique for measuring individual cellular expression of the caveolar proteins cavin-1...

Ti-6Al-4V electron beam weld qualification using laser scanning confocal microscopy

International Nuclear Information System (INIS)

Wanjara, P.; Brochu, M.; Jahazi, M.

2005-01-01

Processing conditions for manufacturing Ti-6Al-4V components by welding using an electron beam source are known to influence the transformation microstructure in the narrow fusion and heat-affected zones of the weld region. This work examined the effect of multiple-sequence welding on the characteristics of the transformed beta microstructure, using laser scanning confocal microscopy to resolve the Widmanstaetten alpha-beta structure in the fusion zone. The evolution in the alpha interlamellar spacing and plate thickness with processing was then related to microhardness measurements in the weld region

Multimodal nonlinear microscope based on a compact fiber-format laser source

Science.gov (United States)

Crisafi, Francesco; Kumar, Vikas; Perri, Antonio; Marangoni, Marco; Cerullo, Giulio; Polli, Dario

2018-01-01

We present a multimodal non-linear optical (NLO) laser-scanning microscope, based on a compact fiber-format excitation laser and integrating coherent anti-Stokes Raman scattering (CARS), stimulated Raman scattering (SRS) and two-photon-excitation fluorescence (TPEF) on a single platform. We demonstrate its capabilities in simultaneously acquiring CARS and SRS images of a blend of 6-μm poly(methyl methacrylate) beads and 3-μm polystyrene beads. We then apply it to visualize cell walls and chloroplast of an unprocessed fresh leaf of Elodea aquatic plant via SRS and TPEF modalities, respectively. The presented NLO microscope, developed in house using off-the-shelf components, offers full accessibility to the optical path and ensures its easy re-configurability and flexibility.

Confocal Microscopy

Science.gov (United States)

Liu, Jian; Tan, Jiubin

2016-12-01

The confocal microscope is appropriate for imaging cells or the measurement of industrial artefacts. However, junior researchers and instrument users sometimes misuse imaging concepts and metrological characteristics, such as position resolution in industrial metrology and scale resolution in bio-imaging. And, metrological characteristics or influence factors in 3D measurement such as height assessment error caused by 3D coupling effect are so far not yet identified. In this book, the authors outline their practices by the working experiences on standardization and system design. This book assumes little previous knowledge of optics, but rich experience in engineering of industrial measurements, in particular with profile metrology or areal surface topography will be very helpful to understand the theoretical concerns and value of the technological advances. It should be useful for graduate students or researchers as extended reading material, as well as microscope users alongside their handbook.

Spectral imaging technique for retinal perfusion detection using confocal scanning laser ophthalmoscopy

Science.gov (United States)

Rasta, Seyed Hossein; Manivannan, Ayyakkannu; Sharp, Peter F.

2012-11-01

To evaluate retinal perfusion in the human eye, a dual-wavelength confocal scanning laser ophthalmoscope (cSLO) was developed that provides spectral imaging of the fundus using a combination of red (670 nm) and near-infrared (810 nm) wavelengths. The image of the ocular fundus was analyzed to find out if quantitative measurements of the reflectivity of tissue permit assessment of the oxygen perfusion of tissue. We explored problems that affect the reproducibility of patient measurements such as non-uniformity errors on the image. For the first time, an image processing technique was designed and used to minimize the errors of oxygen saturation measurements by illumination correction in retina wide field by increasing SNR. Retinal images were taken from healthy and diabetic retinopathy eyes using the cSLO with a confocal aperture of 100 μm. The ratio image (RI) of red/IR, as oxygen saturation (SO2) index, was calculated for normal eyes. The image correction technique improved the reproducibility of the measurements. Average RI intensity variation of healthy retina tissue was determined within a range of about 5.5%. The capability of the new technique to discriminate oxygenation levels of retinal artery and vein was successfully demonstrated and showed good promise in the diagnosis of the perfused retina.

Quality Control of Laser-Beam-Melted Parts by a Correlation Between Their Mechanical Properties and a Three-Dimensional Surface Analysis

Science.gov (United States)

Grimm, T.; Wiora, G.; Witt, G.

2017-03-01

Good correlations between three-dimensional surface analyses of laser-beam-melted parts of nickel alloy HX and their mechanical properties were found. The surface analyses were performed with a confocal microscope, which offers a more profound surface data basis than a conventional, two-dimensional tactile profilometry. This new approach results in a wide range of three-dimensional surface parameters, which were each evaluated with respect to their feasibility for quality control in additive manufacturing. As a result of an automated surface analysis process by the confocal microscope and an industrial six-axis robot, the results are an innovative approach for quality control in additive manufacturing.

Confocal laser endomicroscopy for diagnosis and histomorphologic imaging of brain tumors in vivo.

Directory of Open Access Journals (Sweden)

Sebastian Foersch

Full Text Available Early detection and evaluation of brain tumors during surgery is crucial for accurate resection. Currently cryosections during surgery are regularly performed. Confocal laser endomicroscopy (CLE is a novel technique permitting in vivo histologic imaging with miniaturized endoscopic probes at excellent resolution. Aim of the current study was to evaluate CLE for in vivo diagnosis in different types and models of intracranial neoplasia. In vivo histomorphology of healthy brains and two different C6 glioma cell line allografts was evaluated in rats. One cell line expressed EYFP, the other cell line was used for staining with fluorescent dyes (fluorescein, acriflavine, FITC-dextran and Indocyanine green. To evaluate future application in patients, fresh surgical resection specimen of human intracranial tumors (n = 15 were examined (glioblastoma multiforme, meningioma, craniopharyngioma, acoustic neurinoma, brain metastasis, medulloblastoma, epidermoid tumor. Healthy brain tissue adjacent to the samples served as control. CLE yielded high-quality histomorphology of normal brain tissue and tumors. Different fluorescent agents revealed distinct aspects of tissue and cell structure (nuclear pattern, axonal pathways, hemorrhages. CLE discrimination of neoplastic from healthy brain tissue was easy to perform based on tissue and cellular architecture and resemblance with histopathology was excellent. Confocal laser endomicroscopy allows immediate in vivo imaging of normal and neoplastic brain tissue at high resolution. The technology might be transferred to scientific and clinical application in neurosurgery and neuropathology. It may become helpful to screen for tumor free margins and to improve the surgical resection of malignant brain tumors, and opens the door to in vivo molecular imaging of tumors and other neurologic disorders.

The HVAC Challenges of Upgrading an Old Lab for High-end Light Microscopes

Science.gov (United States)

Richard, R.; Martone, P.; Callahan, L.M.

2014-01-01

The University of Rochester Medical Center forms the centerpiece of the University of Rochester's health research, teaching, patient care, and community outreach missions. Within this large facility of over 5 million square feet, demolition and remodeling of existing spaces is a constant activity. With more than $145 million in federal research funding, lab space is frequently repurposed and renovated to support this work. The URMC Medical Center Facilities Organization supporting small to medium space renovations is constantly challenged and constrained by the existing mechanical infrastructure and budgets to deliver a renovated space that functions within the equipment environmental parameters. One recent project, sponsored by the URMC Shared Resources Laboratory, demonstrates these points. The URMC Light Microscopy Shared Resource Laboratory requested renovation of a 121 sq. ft. room in a 40 year old building which would enable placement of a laser capture microdissection microscope and a Pascal 5 laser scanning confocal microscope with the instruments separated by a blackout curtain. This poster discusses the engineering approach implemented to bring an older lab into the environmental specifications needed for the proper operation of the high-end light microscopes.

Evaluation of transdermal delivery of nanoemulsions in ex vivo porcine skin using two-photon microscopy and confocal laser-scanning microscopy

Science.gov (United States)

Choi, Sanghoon; Kim, Jin Woong; Lee, Yong Joong; Delmas, Thomas; Kim, Changhwan; Park, Soyeun; Lee, Ho

2014-10-01

This study experimentally evaluates the self-targeting ability of asiaticoside-loaded nanoemulsions compared with nontargeted nanoemulsions in ex vivo experiments with porcine skin samples. Homebuilt two-photon and confocal laser-scanning microscopes were employed to noninvasively examine the transdermal delivery of two distinct nanoemulsions. Prior to the application of nanoemulsions, we noninvasively observed the morphology of porcine skin using two-photon microscopy. We have successfully visualized the distributions of the targeted and nontargeted nanoemulsions absorbed into the porcine skin samples. Asiaticoside-loaded nanoemulsions showed an improved ex vivo transdermal delivery through the stratum corneum compared with nonloaded nanoemulsions. As a secondary measure, nanoemulsions-applied samples were sliced in the depth direction with a surgical knife in order to obtain the complete depth-direction distribution profile of Nile red fluorescence. XZ images demonstrated that asiaticoside-loaded nanoemulsion penetrated deeper into the skin compared with nontargeted nanoemulsions. The basal layer boundary is clearly visible in the case of the asiaticoside-loaded skin sample. These results reaffirm the feasibility of using self-targeting ligands to improve permeation through the skin barrier for cosmetics and topical drug applications.

Confocal stereology: an efficient tool for measurement of microscopic structures

Czech Academy of Sciences Publication Activity Database

Kubínová, Lucie; Janáček, Jiří

2015-01-01

Roč. 360, č. 1 (2015), s. 13-28 ISSN 0302-766X R&D Projects: GA MŠk(CZ) LH13028 Institutional support: RVO:67985823 Keywords : 3-D images * confocal microscopy * geometrical characteristics * spatial probes * stereology Subject RIV: EA - Cell Biology Impact factor: 2.948, year: 2015

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

DEFF Research Database (Denmark)

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

2015-01-01

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

Signal and noise modeling in confocal laser scanning fluorescence microscopy.

Science.gov (United States)

Herberich, Gerlind; Windoffer, Reinhard; Leube, Rudolf E; Aach, Til

2012-01-01

Fluorescence confocal laser scanning microscopy (CLSM) has revolutionized imaging of subcellular structures in biomedical research by enabling the acquisition of 3D time-series of fluorescently-tagged proteins in living cells, hence forming the basis for an automated quantification of their morphological and dynamic characteristics. Due to the inherently weak fluorescence, CLSM images exhibit a low SNR. We present a novel model for the transfer of signal and noise in CLSM that is both theoretically sound as well as corroborated by a rigorous analysis of the pixel intensity statistics via measurement of the 3D noise power spectra, signal-dependence and distribution. Our model provides a better fit to the data than previously proposed models. Further, it forms the basis for (i) the simulation of the CLSM imaging process indispensable for the quantitative evaluation of CLSM image analysis algorithms, (ii) the application of Poisson denoising algorithms and (iii) the reconstruction of the fluorescence signal.

Concurrent Reflectance Confocal Microscopy and Laser Doppler Flowmetry to Improve Skin Cancer Imaging: A Monte Carlo Model and Experimental Validation

Directory of Open Access Journals (Sweden)

Alireza Mowla

2016-09-01

Full Text Available Optical interrogation of suspicious skin lesions is standard care in the management of skin cancer worldwide. Morphological and functional markers of malignancy are often combined to improve expert human diagnostic power. We propose the evaluation of the combination of two independent optical biomarkers of skin tumours concurrently. The morphological modality of reflectance confocal microscopy (RCM is combined with the functional modality of laser Doppler flowmetry, which is capable of quantifying tissue perfusion. To realize the idea, we propose laser feedback interferometry as an implementation of RCM, which is able to detect the Doppler signal in addition to the confocal reflectance signal. Based on the proposed technique, we study numerical models of skin tissue incorporating two optical biomarkers of malignancy: (i abnormal red blood cell velocities and concentrations and (ii anomalous optical properties manifested through tissue confocal reflectance, using Monte Carlo simulation. We also conduct a laboratory experiment on a microfluidic channel containing a dynamic turbid medium, to validate the efficacy of the technique. We quantify the performance of the technique by examining a signal to background ratio (SBR in both the numerical and experimental models, and it is shown that both simulated and experimental SBRs improve consistently using this technique. This work indicates the feasibility of an optical instrument, which may have a role in enhanced imaging of skin malignancies.

Development of confocal X-ray fluorescence (XRF) microscopy at the Cornell high energy synchrotron source

International Nuclear Information System (INIS)

Woll, A.R.; Huang, R.; Mass, J.; Bisulca, C.; Bilderback, D.H.; Gruner, S.; Gao, N.

2006-01-01

A confocal X-ray fluorescence microscope was built at the Cornell High Energy Synchrotron Source (CHESS) to obtain compositional depth profiles of historic paintings. The microscope consists of a single-bounce, borosilicate monocapillary optic to focus the incident beam onto the painting and a commercial borosilicate polycapillary lens to collect the fluorescent X-rays. The resolution of the microscope was measured by scanning a variety of thin metal films through this confocal volume while monitoring the fluorescence signal. The capabilities of the technique were then probed using test paint microstructures with up to four distinct layers, each having a thickness in the range of 10-80 microns. Results from confocal XRF were compared with those from stand-alone XRF and visible light microscopy of the paint cross-sections. A large area, high-resolution scanner is currently being built to perform 3D scans on moderately sized paintings. (orig.)

Penetration pattern of rhodamine dyes into enamel and dentin: confocal laser microscopy observation.

Science.gov (United States)

Kwon, S R; Wertz, P W; Li, Y; Chan, D C N

2012-02-01

Enamel and dentin are susceptible to extrinsic and intrinsic stains. The purposes of this study were to determine the penetration pattern of Rhodamine B and dextran-conjugated Rhodamine B into the enamel and dentin as observed by confocal laser microscopy and to relate it to the penetration pattern of hydrogen peroxide commonly used as an active ingredient in tooth-whitening agents and high-molecular-weight staining molecules. Eighteen recently extracted human maxillary anterior teeth were used. Teeth were cleaned and painted with nail varnish except for the crown area above the cemento-enamel junction (CEJ). The painted teeth were then immersed in Rhodamine B and dextran-conjugated Rhodamine B (70 000 MW) for 4, 7, 10 and 15 days. Teeth were sliced to 3 mm thickness in transverse plane and mounted on a glass slide just prior to observation with confocal laser microscopy. Rhodamine B and dextran-conjugated Rhodamine B readily penetrated into the enamel and dentin when exposed for 4 and 7 days, respectively. Rhodamine B penetrated along the interprismatic spaces of the enamel into the dentin. The penetration was accentuated in sections with existing crack lines in the enamel. Rhodamine B was readily absorbed into the dentinal tubules at the dentino-enamel junction and continued to penetrate through the dentin via the dentinal tubules into the pre-dentin. Within the limitations of this study, it is concluded that Rhodamine B and dextran-conjugated Rhodamine B when applied to the external surface of the tooth readily penetrate into the enamel and dentin via the interprismatic spaces in the enamel and dentinal tubules in the dentin, suggesting that stain molecules and bleaching agents possibly exhibit similar penetration pathways. © 2011 The Authors. ICS © 2011 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Confocal endomicroscopy: Is it time to move on?

Science.gov (United States)

Robles-Medranda, Carlos

2016-01-10

Confocal laser endomicroscopy permits in-vivo microscopy evaluation during endoscopy procedures. It can be used in all the parts of the gastrointestinal tract and includes: Esophagus, stomach, small bowel, colon, biliary tract through and endoscopic retrograde cholangiopancreatography and pancreas through needles during endoscopic ultrasound procedures. Many researches demonstrated a high correlation of results between confocal laser endomicroscopy and histopathology in the diagnosis of gastrointestinal lesions; with accuracy in about 86% to 96%. Moreover, in spite that histopathology remains the gold-standard technique for final diagnosis of any diseases; a considerable number of misdiagnosis rate could be present due to many factors such as interpretation mistakes, biopsy site inaccuracy, or number of biopsies. Theoretically; with the diagnostic accuracy rates of confocal laser endomicroscopy could help in a daily practice to improve diagnosis and treatment management of the patients. However, it is still not routinely used in the clinical practice due to many factors such as cost of the procedure, lack of codification and reimbursement in some countries, absence of standard of care indications, availability, physician image-interpretation training, medico-legal problems, and the role of the pathologist. These limitations are relative, and solutions could be found based on new researches focused to solve these barriers.

Surface wettability of silicon substrates enhanced by laser ablation

Energy Technology Data Exchange (ETDEWEB)

Tseng, Shih-Feng [National Applied Research Laboratories, Instrument Technology Research Center, Hsinchu (China); National Chiao Tung University, Department of Mechanical Engineering, Hsinchu (China); Hsiao, Wen-Tse; Huang, Kuo-Cheng; Hsiao, Sheng-Yi [National Applied Research Laboratories, Instrument Technology Research Center, Hsinchu (China); Chen, Ming-Fei [National Changhua University of Education, Department of Mechatronics Engineering, Changhua (China); Lin, Yung-Sheng [Hungkuang University, Department of Applied Cosmetology and Graduate Institute of Cosmetic Science, Taichung (China); Chou, Chang-Pin [National Chiao Tung University, Department of Mechanical Engineering, Hsinchu (China)

2010-11-15

Laser-ablation techniques have been widely applied for removing material from a solid surface using a laser-beam irradiating apparatus. This paper presents a surface-texturing technique to create rough patterns on a silicon substrate using a pulsed Nd:YAG laser system. The different degrees of microstructure and surface roughness were adjusted by the laser fluence and laser pulse duration. A scanning electron microscope (SEM) and a 3D confocal laser-scanning microscope are used to measure the surface micrograph and roughness of the patterns, respectively. The contact angle variations between droplets on the textured surface were measured using an FTA 188 video contact angle analyzer. The results indicate that increasing the values of laser fluence and laser pulse duration pushes more molten slag piled around these patterns to create micro-sized craters and leads to an increase in the crater height and surface roughness. A typical example of a droplet on a laser-textured surface shows that the droplet spreads very quickly and almost disappears within 0.5167 s, compared to a contact angle of 47.9 on an untextured surface. This processing technique can also be applied to fabricating Si solar panels to increase the absorption efficiency of light. (orig.)

Measurement of buried undercut structures in microfluidic devices by laser fluorescent confocal microscopy

International Nuclear Information System (INIS)

Li Shiguang; Liu Jing; Nguyen, Nam-Trung; Fang Zhongping; Yoon, Soon Fatt

2009-01-01

Measuring buried, undercut microstructures is a challenging task in metrology. These structures are usually characterized by measuring their cross sections after physically cutting the samples. This method is destructive and the obtained information is incomplete. The distortion due to cutting also affects the measurement accuracy. In this paper, we first apply the laser fluorescent confocal microscopy and intensity differentiation algorithm to obtain the complete three-dimensional profile of the buried, undercut structures in microfluidic devices, which are made by the soft lithography technique and bonded by the oxygen plasma method. The impact of material wettability and the refractive index (n) mismatch among the liquid, samples, cover layer, and objective on the measurement accuracy are experimentally investigated.

3D Image Analysis of Geomaterials using Confocal Microscopy

Science.gov (United States)

Mulukutla, G.; Proussevitch, A.; Sahagian, D.

2009-05-01

Confocal microscopy is one of the most significant advances in optical microscopy of the last century. It is widely used in biological sciences but its application to geomaterials lingers due to a number of technical problems. Potentially the technique can perform non-invasive testing on a laser illuminated sample that fluoresces using a unique optical sectioning capability that rejects out-of-focus light reaching the confocal aperture. Fluorescence in geomaterials is commonly induced using epoxy doped with a fluorochrome that is impregnated into the sample to enable discrimination of various features such as void space or material boundaries. However, for many geomaterials, this method cannot be used because they do not naturally fluoresce and because epoxy cannot be impregnated into inaccessible parts of the sample due to lack of permeability. As a result, the confocal images of most geomaterials that have not been pre-processed with extensive sample preparation techniques are of poor quality and lack the necessary image and edge contrast necessary to apply any commonly used segmentation techniques to conduct any quantitative study of its features such as vesicularity, internal structure, etc. In our present work, we are developing a methodology to conduct a quantitative 3D analysis of images of geomaterials collected using a confocal microscope with minimal amount of prior sample preparation and no addition of fluorescence. Two sample geomaterials, a volcanic melt sample and a crystal chip containing fluid inclusions are used to assess the feasibility of the method. A step-by-step process of image analysis includes application of image filtration to enhance the edges or material interfaces and is based on two segmentation techniques: geodesic active contours and region competition. Both techniques have been applied extensively to the analysis of medical MRI images to segment anatomical structures. Preliminary analysis suggests that there is distortion in the

Visible-to-visible four-photon ultrahigh resolution microscopic imaging with 730-nm diode laser excited nanocrystals.

Science.gov (United States)

Wang, Baoju; Zhan, Qiuqiang; Zhao, Yuxiang; Wu, Ruitao; Liu, Jing; He, Sailing

2016-01-25

Further development of multiphoton microscopic imaging is confronted with a number of limitations, including high-cost, high complexity and relatively low spatial resolution due to the long excitation wavelength. To overcome these problems, for the first time, we propose visible-to-visible four-photon ultrahigh resolution microscopic imaging by using a common cost-effective 730-nm laser diode to excite the prepared Nd(3+)-sensitized upconversion nanoparticles (Nd(3+)-UCNPs). An ordinary multiphoton scanning microscope system was built using a visible CW diode laser and the lateral imaging resolution as high as 161-nm was achieved via the four-photon upconversion process. The demonstrated large saturation excitation power for Nd(3+)-UCNPs would be more practical and facilitate the four-photon imaging in the application. A sample with fine structure was imaged to demonstrate the advantages of visible-to-visible four-photon ultrahigh resolution microscopic imaging with 730-nm diode laser excited nanocrystals. Combining the uniqueness of UCNPs, the proposed visible-to-visible four-photon imaging would be highly promising and attractive in the field of multiphoton imaging.

In-situ investigation of laser surface modifications of WC-Co hard metals inside a scanning electron microscope

Science.gov (United States)

Mueller, H.; Wetzig, K.; Schultrich, B.; Pompe, Wolfgang; Chapliev, N. I.; Konov, Vitaly I.; Pimenov, S. M.; Prokhorov, Alexander M.

1989-05-01

The investigation of laser interaction with solid surfaces and of the resulting mechanism of surface modification are of technical interest to optimize technological processes, and they are also of fundamental scientific importance. Most instructive indormation is available with the ail of the in-situ techniques. For instance, measuring of the photon emission of the irradiated surface ane the plasma torch (if it is produced) simultaneously to laser action, makes it possible to gain a global characterization of the laser-solid interaction. In order to obtain additional information about surface and structure modifications in microscopic detail , a laser and scanning electron microscope were combined in to a tandem equipment (LASEM). Inside this eqiipment the microscopic observation is carried out directly at the laser irradiated area without any displacement of the sample. In this way, the stepwise development of surface modification during multipulse irradiation is visible in microscopic details and much more reliable information about the surface modification process is obtainable in comparison to an external laser irradiation. Such kind of equipments were realized simultaneously and independently in the Institut of General Physics (Moscow) and the Central Institute of Solid State Physics and Material Research (Dresden) using a CO2 and a LTd-glass-laser, respectively. In the following the advantages and possibilities of a LASEM shall be demonstrated by some selected investigations of WC-CO hardmeta. The results were obtained in collaboration by both groups with the aid of the pulsed CO2-laser. The TEA CO2 laser was transmitted through a ZnSe-window into the sample chamber of the SEM and focused ofAo tfte sample surface. It was operated in TEM - oo mode with a repetition rate of about 1 pulse per second. A peak power density of about 160 MW/cm2 was achieved in front of the sample surface.

Implementação de um sistema de eletroforese capilar com detecção de fluorescência induzida por laser

Directory of Open Access Journals (Sweden)

Santos Marcia R.

2000-01-01

Full Text Available A capillary electrophoresis system using laser induced fluorescence detection is described. A Raman system equipped with a microscope has been used to focus the laser beam on the capillary giving a lateral resolution of 1.5 mm. The fluorescence signal of the analyte (ZnPcTS - tetrasulfonated zinc-phthalocyanine was collected by the microscope objectives and analysed by a monochromator with confocal characteristics equipped with a CCD detector. Electropherograms obtained with this system were compared to those obtained on a commercial instrument, showing that the described system presents a lower detection limit and better resolution.

Spatially selective Er/Yb-doped CaF2 crystal formation by CO2 laser exposure

International Nuclear Information System (INIS)

Kim, Dong-Seon; Lee, Jin-Ho; Lim, Ki-Soo

2014-01-01

Highlights: • Oxyfluoride glass–ceramics containing CaF 2 nanocrystals doped with Er 3+ and Yb 3+ ions were formed on the glass surface by CO 2 laser and a heat gun exposure. • Most of Er and Yb ions were distributed inside CaF 2 nanocrystals and fluorine loss was observed in the EDS element maps. • IR-to-VIS upconversion emission efficiency of laser annealed glass ceramics was much increased and compared with that of the furnace-annealed glass ceramics. • Distributed volume of the glass ceramics were estimated by a confocal fluorescence microscope imaging. - Abstract: We report the glass–ceramic precipitation on the oxyfluoride glass surface by spatially selective annealing with a CO 2 laser and a heat gun exposure. X-ray diffraction analysis showed the formation of major CaF 2 and miner Ca 2 SiO 4 nanoparticles. We observed ∼100 nm nanoparticle aggregation by tunneling electron microscopy and element distribution in glass and crystal phases. Spatial distribution of glass ceramics near the glass surface was probed by confocal fluorescence microscope by using much enhanced emission from the Er ions in the laser-treated area. Strong emissions at 365 nm excitation and visible up-conversion emissions at 980 nm excitation also indicated well incorporation of Er and Yb ions into a crystalline environment

Spatially selective Er/Yb-doped CaF2 crystal formation by CO2 laser exposure

International Nuclear Information System (INIS)

Kim, Dong-Seon; Lee, Jin-Ho; Lim, Ki-Soo

2015-01-01

Highlights: • Oxyfluoride glass–ceramics containing CaF 2 nanocrystals doped with Er 3+ and Yb 3+ ions were formed on the glass surface by CO 2 laser and a heat gun exposure. • Most of Er and Yb ions were distributed inside CaF 2 nanocrystals and fluorine loss was observed in the EDS element maps. • IR-to-VIS upconversion emission efficiency of laser annealed glass ceramics was much increased and compared with that of the furnace-annealed glass ceramics. • Distributed volume of the glass ceramics were estimated by a confocal fluorescence microscope imaging. - Abstract: We report the glass–ceramic precipitation on the oxyfluoride glass surface by spatially selective annealing with a CO 2 laser and a heat gun exposure. X-ray diffraction analysis showed the formation of major CaF 2 and miner Ca 2 SiO 4 nanoparticles. We observed ∼100 nm nanoparticle aggregation by tunneling electron microscopy and element distribution in glass and crystal phases. Spatial distribution of glass ceramics near the glass surface was probed by confocal fluorescence microscope by using much enhanced emission from the Er ions in the laser-treated area. Strong emissions at 365 nm excitation and visible up-conversion emissions at 980 nm excitation also indicated well incorporation of Er and Yb ions into a crystalline environment

Dual filtered backprojection for micro-rotation confocal microscopy

International Nuclear Information System (INIS)

Laksameethanasan, Danai; Brandt, Sami S; Renaud, Olivier; Shorte, Spencer L

2009-01-01

Micro-rotation confocal microscopy is a novel optical imaging technique which employs dielectric fields to trap and rotate individual cells to facilitate 3D fluorescence imaging using a confocal microscope. In contrast to computed tomography (CT) where an image can be modelled as parallel projection of an object, the ideal confocal image is recorded as a central slice of the object corresponding to the focal plane. In CT, the projection images and the 3D object are related by the Fourier slice theorem which states that the Fourier transform of a CT image is equal to the central slice of the Fourier transform of the 3D object. In the micro-rotation application, we have a dual form of this setting, i.e. the Fourier transform of the confocal image equals the parallel projection of the Fourier transform of the 3D object. Based on the observed duality, we present here the dual of the classical filtered back projection (FBP) algorithm and apply it in micro-rotation confocal imaging. Our experiments on real data demonstrate that the proposed method is a fast and reliable algorithm for the micro-rotation application, as FBP is for CT application

Note: Tandem Kirkpatrick-Baez microscope with sixteen channels for high-resolution laser-plasma diagnostics

Science.gov (United States)

Yi, Shengzhen; Zhang, Zhe; Huang, Qiushi; Zhang, Zhong; Wang, Zhanshan; Wei, Lai; Liu, Dongxiao; Cao, Leifeng; Gu, Yuqiu

2018-03-01

Multi-channel Kirkpatrick-Baez (KB) microscopes, which have better resolution and collection efficiency than pinhole cameras, have been widely used in laser inertial confinement fusion to diagnose time evolution of the target implosion. In this study, a tandem multi-channel KB microscope was developed to have sixteen imaging channels with the precise control of spatial resolution and image intervals. This precise control was created using a coarse assembly of mirror pairs with high-accuracy optical prisms, followed by precise adjustment in real-time x-ray imaging experiments. The multilayers coated on the KB mirrors were designed to have substantially the same reflectivity to obtain a uniform brightness of different images for laser-plasma temperature analysis. The study provides a practicable method to achieve the optimum performance of the microscope for future high-resolution applications in inertial confinement fusion experiments.

Characterization of LiF-based soft X-ray imaging detectors by confocal fluorescence microscopy

International Nuclear Information System (INIS)

Bonfigli, F; Gaudio, P; Lupelli, I; Nichelatti, E; Richetta, M; Vincenti, M A; Montereali, R M

2010-01-01

X-ray microscopy represents a powerful tool to obtain images of samples with very high spatial resolution. The main limitation of this technique is represented by the poor spatial resolution of standard imaging detectors. We proposed an innovative high-performance X-ray imaging detector based on the visible photoluminescence of colour centres in lithium fluoride. In this work, a confocal microscope in fluorescence mode was used to characterize LiF-based imaging detectors measuring CC integrated visible fluorescence signals of LiF crystals and films (grown on several kinds of substrates) irradiated by soft X-rays produced by a laser plasma source in different exposure conditions. The results are compared with the CC photoluminescence spectra measured on the same samples and discussed.

An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy

International Nuclear Information System (INIS)

Dunsby, C; Lanigan, P M P; McGinty, J; Elson, D S; Requejo-Isidro, J; Munro, I; Galletly, N; McCann, F; Treanor, B; Oenfelt, B; Davis, D M; Neil, M A A; French, P M W

2004-01-01

Fluorescence imaging is used widely in microscopy and macroscopic imaging applications for fields ranging from biomedicine to materials science. A critical component for any fluorescence imaging system is the excitation source. Traditionally, wide-field systems use filtered thermal or arc-generated white light sources, while point scanning confocal microscope systems require spatially coherent (point-like) laser sources. Unfortunately, the limited range of visible wavelengths available from conventional laser sources constrains the design and usefulness of fluorescent probes in confocal microscopy. A 'hands-off' laser-like source, electronically tunable across the visible spectrum, would be invaluable for fluorescence imaging and provide new opportunities, e.g. automated excitation fingerprinting and in situ measurement of excitation cross-sections. Yet more information can be obtained using fluorescence lifetime imaging (FLIM), which requires that the light source be pulsed or rapidly modulated. We show how a white light continuum, generated by injecting femtosecond optical radiation into a micro-structured optical fibre, coupled with a simple prism-based tunable filter arrangement, can fulfil all these roles as a continuously electronically tunable (435-1150 nm) visible ultrafast light source in confocal, wide-field and FLIM systems

  In situ identification of streptococci and other bacteria in initial dental biofilm by confocal laser scanning microscopy and fluorescence in situ hybridization

DEFF Research Database (Denmark)

Dige, Irene; Kilian, Mogens; Nilsson, Holger

2007-01-01

Confocal laser scanning microscopy (CLSM) has been employed as a method for studying intact natural biofilm. When combined with fluorescence in situ hybridization (FISH) it is possible to analyze spatial relationships and changes of specific members of microbial populations over time. The aim...

Function analysis of working integrated circuit with scanning laser microscope. Laser kenbikyo ni yoru IC no dosa kansatsu

Energy Technology Data Exchange (ETDEWEB)

Ode, T. (Lasertec Corp., Kanagawa (Japan))

1992-10-20

By scanning a laser light, the reaction of a specimen against the light is detected in some means. The optical effect can be visualized by displaying that on the CRT or the like in synchronism with the scanning. Among these, an image formed and visualized by internal photoelectric effect by light is called OBIC image, and chiefly used for evaluating and analyzing semiconductor devices. Observing this OBIC image by a high speed scanning laser microscope has been spotlighted these days as an effective means for observing the state of p-n junction of an IC in operation. This paper descries the principle, the observing method, the detecting circuit, etc. of the semiconductor observing method using a laser microscope. Further, actual examples of detecting defects of an IC by means of OBIC image are shown. As for the problem, since leak parts are displayed as negative contrast in the OBIC image to affect finding work of leak part, the necessity of improvement is pointed out. 39 refs., 11 figs.

Caustic meso-optical confocal microscope for vertical particle tracks. Proposal

International Nuclear Information System (INIS)

Soroko, L.M.

1995-01-01

The principal of the proposed caustic meso-optical microscope for vertical particle tracks in the nuclear photoemulsion is explained. The results of the experiments performed to illustrate the main features of this new meso-optical microscope are given. The proposed caustic meso-optical microscope for vertical particle tracks in the nuclear photoemulsion can be effectively used in the experimental investigation of such rare processes as ν μ - ν τ oscillations and of the Pb-Pb interactions. 2 refs., 7 figs

Tissue imaging with a stigmatic mass microscope using laser desorption/ionization

Science.gov (United States)

Awazu, Kunio; Hazama, Hisanao; Hamanaka, Tomonori; Aoki, Jun; Toyoda, Michisato; Naito, Yasuhide

2012-03-01

A novel stigmatic mass microscope using laser desorption/ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG, has been developed. Stigmatic ion images of crystal violet masked by a fine square mesh grid with a 12.7 μm pitch were clearly observed, and the estimated spatial resolution was about 3 μm in the linear mode with a 20-fold ion optical magnification. Tissue sections of a brain and eyes of a mouse stained with crystal violet and methylene blue were observed in the linear mode, and the stigmatic total ion images of crystal violet and methylene blue agreed well with the optical photomicrograph of the same sections. Especially, the fine structure in the cornea tissue was clearly observed with a spatial resolution in the range of micrometers. Although the total measurement time of the stigmatic ion image for the whole-eye section was about 59 minutes using a laser with a 10 Hz repetition rate, the measurement time could be reduced to about 35 s using a laser with a 1 kHz repetition rate and automation of measurements. The stigmatic mass microscope developed in this research should be suitable for high-spatial resolution and high-throughput imaging mass spectrometry for pathology, pharmacokinetics, and so on.

Spatially selective Er/Yb-doped CaF{sub 2} crystal formation by CO{sub 2} laser exposure

Energy Technology Data Exchange (ETDEWEB)

Kim, Dong-Seon; Lee, Jin-Ho; Lim, Ki-Soo, E-mail: kslim@chungbuk.ac.kr

2014-10-30

Highlights: • Oxyfluoride glass–ceramics containing CaF{sub 2} nanocrystals doped with Er{sup 3+} and Yb{sup 3+} ions were formed on the glass surface by CO{sub 2} laser and a heat gun exposure. • Most of Er and Yb ions were distributed inside CaF{sub 2} nanocrystals and fluorine loss was observed in the EDS element maps. • IR-to-VIS upconversion emission efficiency of laser annealed glass ceramics was much increased and compared with that of the furnace-annealed glass ceramics. • Distributed volume of the glass ceramics were estimated by a confocal fluorescence microscope imaging. - Abstract: We report the glass–ceramic precipitation on the oxyfluoride glass surface by spatially selective annealing with a CO{sub 2} laser and a heat gun exposure. X-ray diffraction analysis showed the formation of major CaF{sub 2} and miner Ca{sub 2}SiO{sub 4} nanoparticles. We observed ∼100 nm nanoparticle aggregation by tunneling electron microscopy and element distribution in glass and crystal phases. Spatial distribution of glass ceramics near the glass surface was probed by confocal fluorescence microscope by using much enhanced emission from the Er ions in the laser-treated area. Strong emissions at 365 nm excitation and visible up-conversion emissions at 980 nm excitation also indicated well incorporation of Er and Yb ions into a crystalline environment.

Spatially selective Er/Yb-doped CaF{sub 2} crystal formation by CO{sub 2} laser exposure

Energy Technology Data Exchange (ETDEWEB)

Kim, Dong-Seon; Lee, Jin-Ho; Lim, Ki-Soo, E-mail: kslim@chungbuk.ac.kr

2015-04-15

Highlights: • Oxyfluoride glass–ceramics containing CaF{sub 2} nanocrystals doped with Er{sup 3+} and Yb{sup 3+} ions were formed on the glass surface by CO{sub 2} laser and a heat gun exposure. • Most of Er and Yb ions were distributed inside CaF{sub 2} nanocrystals and fluorine loss was observed in the EDS element maps. • IR-to-VIS upconversion emission efficiency of laser annealed glass ceramics was much increased and compared with that of the furnace-annealed glass ceramics. • Distributed volume of the glass ceramics were estimated by a confocal fluorescence microscope imaging. - Abstract: We report the glass–ceramic precipitation on the oxyfluoride glass surface by spatially selective annealing with a CO{sub 2} laser and a heat gun exposure. X-ray diffraction analysis showed the formation of major CaF{sub 2} and miner Ca{sub 2}SiO{sub 4} nanoparticles. We observed ∼100 nm nanoparticle aggregation by tunneling electron microscopy and element distribution in glass and crystal phases. Spatial distribution of glass ceramics near the glass surface was probed by confocal fluorescence microscope by using much enhanced emission from the Er ions in the laser-treated area. Strong emissions at 365 nm excitation and visible up-conversion emissions at 980 nm excitation also indicated well incorporation of Er and Yb ions into a crystalline environment.

Investigation of the petrophysical properties of a porous sandstone sample using confocal scanning laser microscopy

Energy Technology Data Exchange (ETDEWEB)

Petford, N. [Kingston Univ., Centre for Earth and Environmental Science Research, Kingston (United Kingdom); Davidson, G. [University Coll., Dept. of Electronic and Electrical Engineering, London (United Kingdom); Miller, J.A. [Cambridge Univ., Dept. of Earth Sciences, Cambridge (United Kingdom)

2001-05-01

Confocal scanning laser microscopy (CSLM) is used to produce images of the two- and three-dimensional distribution and geometry of pore space in a reservoir sandstone and measure the 2D distribution of pore throat radii. Non-destructive serial sectioning of the rock using laser light at 100% illumination, combined with image thresholding and histogram equalization techniques allow the pore volume structure of the uppermost 100 {mu}m of the sample to be reconstructed. Negative imaging of the pore volume gave superior depth and feature resolution compared to positive (reflection) imaging. Artefacts encountered in applying classical Medial Axial Transforms to CSLM images include branch networks dominated by coordination numbers of 3. Skeletonization using Euclidean distance maps gives increased accuracy in the description of the pore network. Measured pore throat size distribution in the rock is strongly exponential and described by the expression y 219e{sup -0.25x} where y is the number of pore throats. (Author)

Self-mixing laser diode included in scanning microwave microscope to the control of probe nanodisplacement

Science.gov (United States)

Usanov, D. A.; Skripal, A. V.; Astakhov, E. I.; Dobdin, S. Y.

2018-04-01

The possibilities of self-mixing interferometry for measuring nanodisplacement of a probe included in a near-field scanning microwave microscope have been considered. The features of the formation of a laser interference signal at current modulation of the wavelength of laser radiation have been investigated. Experimental responses of a semiconductor laser system included in scanning microwave microscope to control nanodisplacement of the probe have been demonstrated.To register the nanodisplacement of the probe, it is proposed to use the method of determining the stationary phase of a laser interference signal by low-frequency spectrum of a semiconductor laser. The change of the amplitudes of the spectral components in the spectrum of the interference signal due to creation of the standing wave in the external resonator of the laser self-mixing system has been shown. The form of the interference signal at current modulation of the radiation wavelength was experimentally obtained when the probe moves with a step of 80 nm. The results of measuring nanodisplacements of an electromagnetic translator STANDA 8MVT40-13 have been demonstrated. Deviation of the nanodisplacement of the proposed method does not exceed 15%.

Surface profile measurement by using the integrated Linnik WLSI and confocal microscope system

Science.gov (United States)

Wang, Wei-Chung; Shen, Ming-Hsing; Hwang, Chi-Hung; Yu, Yun-Ting; Wang, Tzu-Fong

2017-06-01

The white-light scanning interferometer (WLSI) and confocal microscope (CM) are the two major optical inspection systems for measuring three-dimensional (3D) surface profile (SP) of micro specimens. Nevertheless, in practical applications, WLSI is more suitable for measuring smooth and low-slope surfaces. On the other hand, CM is more suitable for measuring uneven-reflective and low-reflective surfaces. As for aspect of surface profiles to be measured, the characteristics of WLSI and CM are also different. WLSI is generally used in semiconductor industry while CM is more popular in printed circuit board industry. In this paper, a self-assembled multi-function optical system was integrated to perform Linnik white-light scanning interferometer (Linnik WLSI) and CM. A connecting part composed of tubes, lenses and interferometer was used to conjunct finite and infinite optical systems for Linnik WLSI and CM in the self-assembled optical system. By adopting the flexibility of tubes and lenses, switching to perform two different optical measurements can be easily achieved. Furthermore, based on the shape from focus method with energy of Laplacian filter, the CM was developed to enhance the on focal information of each pixel so that the CM can provide all-in-focus image for performing the 3D SP measurement and analysis simultaneously. As for Linnik WLSI, eleven-step phase shifting algorithm was used to analyze vertical scanning signals and determine the 3D SP.

Real time diagnosis of bladder cancer with probe-based confocal laser endomicroscopy

Science.gov (United States)

Liu, Jen-Jane; Wu, Katherine; Adams, Winifred; Hsiao, Shelly T.; Mach, Kathleen E.; Beck, Andrew H.; Jensen, Kristin C.; Liao, Joseph C.

2011-02-01

Probe-based confocal laser endomicroscopy (pCLE) is an emerging technology for in vivo optical imaging of the urinary tract. Particularly for bladder cancer, real time optical biopsy of suspected lesions will likely lead to improved management of bladder cancer. With pCLE, micron scale resolution is achieved with sterilizable imaging probes (1.4 or 2.6 mm diameter), which are compatible with standard cystoscopes and resectoscopes. Based on our initial experience to date (n = 66 patients), we have demonstrated the safety profile of intravesical fluorescein administration and established objective diagnostic criteria to differentiate between normal, benign, and neoplastic urothelium. Confocal images of normal bladder showed organized layers of umbrella cells, intermediate cells, and lamina propria. Low grade bladder cancer is characterized by densely packed monomorphic cells with central fibrovascular cores, whereas high grade cancer consists of highly disorganized microarchitecture and pleomorphic cells with indistinct cell borders. Currently, we are conducting a diagnostic accuracy study of pCLE for bladder cancer diagnosis. Patients scheduled to undergo transurethral resection of bladder tumor are recruited. Patients undergo first white light cystocopy (WLC), followed by pCLE, and finally histologic confirmation of the resected tissues. The diagnostic accuracy is determined both in real time by the operative surgeon and offline after additional image processing. Using histology as the standard, the sensitivity, specificity, positive and negative predictive value of WLC and WLC + pCLE are calculated. With additional validation, pCLE may prove to be a valuable adjunct to WLC for real time diagnosis of bladder cancer.

Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging.

Science.gov (United States)

Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

2012-10-17

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0-85 μm from the surface of a coverglass. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Optical coherence tomography, scanning laser polarimetry and confocal scanning laser ophthalmoscopy in retinal nerve fiber layer measurements of glaucoma patients.

Science.gov (United States)

Fanihagh, Farsad; Kremmer, Stephan; Anastassiou, Gerasimos; Schallenberg, Maurice

2015-01-01

To determine the correlations and strength of association between different imaging systems in analyzing the retinal nerve fiber layer (RNFL) of glaucoma patients: optical coherence tomography (OCT), scanning laser polarimetry (SLP) and confocal scanning laser ophthalmoscopy (CSLO). 114 eyes of patients with moderate open angle glaucoma underwent spectral domain OCT (Topcon SD-OCT 2000 and Zeiss Cirrus HD-OCT), SLP (GDx VCC and GDx Pro) and CSLO (Heidelberg Retina Tomograph, HRT 3). Correlation coefficients were calculated between the structural parameters yielded by these examinations. The quantitative relationship between the measured RNFL thickness globally and for the four regions (superior, inferior, nasal, temporal) were evaluated with different regression models for all used imaging systems. The strongest correlation of RNFL measurements was found between devices using the same technology like GDx VCC and GDx Pro as well as Topcon OCT and Cirrus OCT. In glaucoma patients, the strongest associations (R²) were found between RNFL measurements of the two optical coherence tomography devices Topcon OCT and Cirrus OCT (R² = 0.513) and between GDx VCC and GDx Pro (R² = 0.451). The results of the OCTs and GDX Pro also had a strong quantitative relationship (Topcon OCT R² = 0.339 and Cirrus OCT R² = 0.347). GDx VCC and the OCTs showed a mild to moderate association (Topcon OCT R² = 0.207 and Cirrus OCT R² = 0.258). The confocal scanning laser ophthalmoscopy (HRT 3) had the lowest association to all other devices (Topcon OCT R² = 0.254, Cirrus OCT R² = 0.158, GDx Pro R² = 0.086 and GDx VCC R² = 0.1). The measurements of the RNFL in glaucoma patients reveal a high correlation of OCT and GDx devices because OCTs can measure all major retinal layers and SLP can detect nerve fibers allowing a comparison between the results of this devices. However, CSLO by means of HRT topography can only measure height values of the retinal surface but it cannot distinguish

Penetration and binding of monoclonal antibody in human osteosarcoma multicell spheroids. Comparison of confocal laser scanning microscopy and autoadiography

International Nuclear Information System (INIS)

Hjelstuen, M.H.; Rasch-Halvorsen, K.; Brekken, C.; Bruland, Oe.; Davies, C. de L.

1996-01-01

Penetration and binding of monoclonal antibody (MAb) in multicell osteosarcoma spheroids have been studied by autoradiography and confocal laser scanning microscopy (CLSM). Optical sectioning of the 3-dimensional spheroids was performed by CLSM. Owing to attenuation of fluorescence intensity, FITC-labelled MAb could not be detected at depths greater than 60 μm within the spheroids. The antibody uptake seen in autoradiographs and CLSM images 60 μm within the spheroids were essentially identical. MAb had reached all parts of the spheroids within 6 h. Quantitative measurements of the fluorescence intensity of FITC-labelled MAb seen in confocal images and measurements of MAb bound per cell using flow cytometry, showed that maximum uptake was reached after 6 h. The possibility to perform both quantatitive and qualitative measurements makes CLSM a promising method for studying antibody uptake in thick tissue samples. (orig.)

An invertebrate embryologist's guide to routine processing of confocal images.

Science.gov (United States)

von Dassow, George

2014-01-01

It is almost impossible to use a confocal microscope without encountering the need to transform the raw data through image processing. Adherence to a set of straightforward guidelines will help ensure that image manipulations are both credible and repeatable. Meanwhile, attention to optimal data collection parameters will greatly simplify image processing, not only for convenience but for quality and credibility as well. Here I describe how to conduct routine confocal image processing tasks, including creating 3D animations or stereo images, false coloring or merging channels, background suppression, and compressing movie files for display.

Speckle-illuminated fluorescence confocal microscopy, using a digital micro-mirror device

International Nuclear Information System (INIS)

Jiang, Shi-Hong; Walker, John G

2009-01-01

An implementation of a speckle-illuminated fluorescence confocal microscope using a digital micro-mirror device (DMD) is described. The DMD not only projects a sequence of imaged binary speckle patterns onto the specimen at a very high frame rate but also operates as a spatial light modulator to perform real-time optical data processing. Frame averaging is accomplished by CCD charge accumulation during a single exposure. The recorded time-averaged image is a confocal image plus an unwanted non-confocal image which can be removed by recording a separate image. Experimental results with image acquisition within a fraction of a second are shown. Images of a thin biological sample are also shown to demonstrate practical application of the technique

A novel cryogenic scanning laser microscope tested on Josephson tunnel junctions

DEFF Research Database (Denmark)

Holm, Jesper; Mygind, Jesper

1995-01-01

to a very localized heating induced by irradiation with 675 nm wavelength light from a semiconductor laser. The hot spot is moved by a specially designed piezoelectric scanner sweeping the tip of a single-mode optical fiber a few µm above the circuit. Depending on the scanner design the scanning area can...... be as large as 50×500 µm2 at 4.2 K. The microscope can be operated in the temperature range 2–300 K using a standard temperature controller. The central microscope body is mounted inside the vacuum can of a dip-stick-type cryoprobe. A damped spring system is used to reduce interference from extraneous...

Confocal fluorescence microscopy investigation of visible emitting defects induced by electron beam lithography in LIF films

International Nuclear Information System (INIS)

Montereali, R. M.; Bigotta, S.; Pace, A.; Piccinini, M.; Burattini, E.; Grilli, A.; Raco, A.; Giammatteo, M.; L'Aquila Univ., L'Aquila; Picozzi, P.; Santucci, S.; L'Aquila Univ., L'Aquila

2000-01-01

Low energy electron irradiation of lithium fluoride (LiF), in the form of bulk crystals and films, gives rise to the stable formation of primary F defects and aggregated color centers in a thin layer located at the surface of the investigated material. For the first time a confocal light scanning microscope (CLSM) in fluorescence mode was used to reconstruct the depth distribution of efficiently emitting laser active color centers in a stripe-like region induced by 12 and 16 keV electrons on LiF films thermally evaporated on glass. The formation of the F3+ and F2 aggregated defects appears restricted to the electron penetration and proportional to their energy depth profile, as obtained from Monte Carlo simulations [it

Chlorophyll and its degradation products in the two-spotted spider mite, Tetranychus urticae: observations using epifluorescence and confocal laser scanning microscopy.

Science.gov (United States)

Occhipinti, Andrea; Maffei, Massimo E

2013-10-01

Chlorophyll and chlorophyll degradation products were observed in the two-spotted spider mite (Tetranychus urticae) using epifluorescence microscopy (EFM) and confocal laser scanning microscopy (CLSM). A clear red fluorescence (EFM) and a fluorescence induced by a laser wavelength of 650 nm (CLSM) were observed. In the lateral caeca, in the ventriculus and in the excretory organ, a bright light blue fluorescence was observed in close association with chlorophyll by using EFM. The same material can be localized with CLSM by using a laser with a wavelength of 488 nm. By comparison with synthetic guanine, this bright fluorescence is supposed to be guanine. The presence of guanine fluorescence in the mite pellets confirms this hypothesis. A possible mechanism for guanine formation is discussed.

Morphological characteristics of the optic nerve evaluated by confocal laser tomography (HRT3) and laser polarimetry (GDx-VCC) in a normal population from the city of Barcelona.

Science.gov (United States)

Fallon, M; Pazos, M; Morilla, A; Sebastián, M A; Xancó, R; Mora, C; Calderón, B; Vega, Z; Antón, A

2015-11-01

To evaluate morphological parameters of optic disc and retinal nerve fiber layer (RNFL) examined with confocal laser tomography (HRT3) and laser polarimetry (GDx-VCC) in a normal population, and analyze correlations of these parameters with demographic variables. Cross-sectional study in the context of a glaucoma screening campaign in the primary care center of Barcelona. The individuals selected were non-hypertensive Mediterranean Caucasians with risk for glaucoma development (individuals≥60 years old or≥40 years old with family history of glaucoma or intraocular pressure or myopia>3diopter). All subjects underwent a complete ophthalmic examination, confocal laser tomography (HRT3) and scanning laser polarimetry (GDX-VCC), subjects with results within normal limits only being included. Structural parameters were analyzed along with age, refraction, and pachymetry based on the Spearman rank correlation test. A total of 224 subjects included, with a mean age of 63.4±11.1 years. Disc areas, excavation and ring area were 2.14±0.52mm(2), 0.44±0.34mm (2) and 1.69±0.38mm(2), respectively. The mean RNFL (GDX) was 55.9±6.9μm. Age was correlated with lower ring volume, highest rate of cup shape measure, largest mean and maximum cup depth, lower nerve fiber index (NFI) and RNFL (all p-values below .05). The mean values and distribution of several parameters of the papilla and the RNFL in normal Mediterranean Caucasians population are presented. A loss of thickness of the RNFL, ring thinning, and enlarged cup was observed with increased age. Copyright © 2014 Sociedad Española de Oftalmología. Published by Elsevier España, S.L.U. All rights reserved.

Conversion efficiency of implanted ions by confocal micro-luminescence mapping

International Nuclear Information System (INIS)

Deshko, Y.; Huang, Mengbing; Gorokhovsky, A.A.

2013-01-01

We report on the further development of the statistical approach to determine the conversion efficiency of implanted ions into emitting centers and present the measurement method based on the confocal micro-luminescence mapping. It involves the micro-luminescence mapping with a narrow-open confocal aperture, followed by the statistical analysis of the photoluminescence signal from an ensemble of emitting centers. The confocal mapping method has two important advantages compared to the recently discussed aperture-free method (J. Lumin. 131 (2011) 489): it is less sensitive to errors in the laser spot size and has a well defined useful area. The confocal mapping has been applied to the Xe center in diamond. The conversion efficiency has been found to be about 0.28, which is in good agreement with the results of the aperture-free method. - Highlights: ► Conversion efficiency of implanted ions into emitting centers – statistical approach. ► Micro-luminescence mapping with open and narrow confocal aperture – comparison. ► Advantages of the confocal micro-luminescence mapping. ► Confocal micro-luminescence mapping has been applied to the Xe center in diamond. ► The conversion efficiency has been found to be about 0.28.

RELIABILITY OF CONFOCAL MICROSCOPY SPECTRAL IMAGING SYSTEMS: USE OF MULTISPECTRAL BEADS

Science.gov (United States)

Background: There is a need for a standardized, impartial calibration, and validation protocol on confocal spectral imaging (CSI) microscope systems. To achieve this goal, it is necessary to have testing tools to provide a reproducible way to evaluate instrument performance. ...

In situ detection of the Zn(2+) release process of ZnO NPs in tumour cells by confocal laser scanning fluorescence microscopy.

Science.gov (United States)

Song, Wenshuang; Tang, Xiaoling; Li, Yong; Sun, Yang; Kong, Jilie; Qingguang, Ren

2016-08-01

The use of zinc oxide (ZnO) nanoparticles (NPs) for cancer is not yet clear for human clinical applications, which is primarily due to the lack of a better understanding of the action mechanisms and cellular consequences of the direct exposure of cells to these NPs. In this work, the authors have selected zinquin ethyl ester, a Zn(2+)-specific fluorescent molecular probe, to efficiently differentiate ZnO NPs and Zn(2+), and combined with confocal laser scanning microscopy (CLSM) to in situ study the Zn(2+) release process of ZnO NPs in cancer cell system through detecting the change of Zn(2+) level over time. During the experiments, the authors have designed the test group ZnO-2 in addition to assess the influence of a long-term storage on the characteristics of ZnO NPs in aqueous solution, and the Zn(2+) release process of ZnO NPs in cancer cell system. After three-month storage at room temperature, the release process became earlier and faster, which was consistent with previous results of transmission electron microscope, UV-Vis and PL spectra. It is a good detection method that combination of Zn(2+)-specific fluorescent molecular probe and CLSM, which will be helpful for ZnO NPs using in clinical research.

Detecting plant silica fibres in animal tissue by confocal fluorescence microscopy.

Science.gov (United States)

Hodson, M J; Smith, R J; van Blaaderen, A; Crafton, T; O'Neill, C H

1994-04-01

Silica fibres from the inflorescence bracts of the grass Phalaris canariensis L. cause dermatitis, and have been implicated in the aetiology of oesophageal cancer in northeastern Iran. Here we describe a method for labelling these fibres so that they can be located in mammalian tissue. Fluorescein was covalently linked to isolated, purified fibres with the silane coupling agent 3-aminopropyl triethoxysilane. The labelled hairs were then rubbed into the backs of mice. These were later killed and their skin fixed, stained and sliced at a thickness of 250 microns. A confocal laser scanning microscope gave brilliant images of the fibres at any depth up to 100 microns or more beneath the surface of the slice. Fibres penetrated deeply into the dermis. Several cubic millimetres of tissue could be surveyed in 1 h. The number of fibres present was approximately 2 mm-3 initially, falling to 0.1 mm-3 after 7 days.

Development of HiLo Microscope and its use in In-Vivo Applications

Science.gov (United States)

Patel, Shreyas J.

The functionality of achieving optical sectioning in biomedical research is invaluable as it allows for visualization of a biological sample at different depths while being free of background scattering. Most current microscopy techniques that offer optical sectioning, unfortunately, require complex instrumentation and thus are generally costly. HiLo microscopy, on the other hand, offers the same functionality and advantage at a relatively low cost. Hence, the work described in this thesis involves the design, build, and application of a HiLo microscope. More specifically, a standalone HiLo microscope was built in addition to implementing HiLo microscopy on a standard fluorescence microscope. In HiLo microscopy, optical sectioning is achieved by acquiring two different types of images per focal plane. One image is acquired under uniform illumination and the other is acquired under speckle illumination. These images are processed using an algorithm that extracts in-focus information and removes features and glare that occur as a result of background fluorescence. To show the benefits of the HiLo microscopy, several imaging experiments on various samples were performed under a HiLo microscope and compared against a traditional fluorescence microscope and a confocal microscope, which is considered the gold standard in optical imaging. In-vitro and ex-vivo imaging was performed on a set of pollen grains, and optically cleared mouse brain and heart slices. Each of these experiments showed great reduction in background scattering at different depths under HiLo microscopy. More importantly, HiLo imaging of optically cleared heart slice demonstrated emergence of different vasculature at different depths. Reduction of out-of-focus light increased the spatial resolution and allowed better visualization of capillary vessels. Furthermore, HiLo imaging was tested in an in-vivo model of a rodent dorsal window chamber model. When imaging the same sample under confocal microscope

Confocal Raman microscopy for identification of bacterial species in biofilms

Science.gov (United States)

Beier, Brooke D.; Quivey, Robert G.; Berger, Andrew J.

2011-03-01

Implemented through a confocal microscope, Raman spectroscopy has been used to distinguish between biofilm samples of two common oral bacteria species, Streptococcus sanguinis and mutans, which are associated with healthy and cariogenic plaque, respectively. Biofilms of these species are studied as a model of dental plaque. A prediction model has been calibrated and validated using pure biofilms. This model has been used to identify the species of transferred and dehydrated samples (much like a plaque scraping) as well as hydrated biofilms in situ. Preliminary results of confocal Raman mapping of species in an intact two-species biofilm will be shown.

The learning curve, interobserver, and intraobserver agreement of endoscopic confocal laser endomicroscopy in the assessment of mucosal barrier defects.

Science.gov (United States)

Chang, Jeff; Ip, Matthew; Yang, Michael; Wong, Brendon; Power, Theresa; Lin, Lisa; Xuan, Wei; Phan, Tri Giang; Leong, Rupert W

2016-04-01

Confocal laser endomicroscopy can dynamically assess intestinal mucosal barrier defects and increased intestinal permeability (IP). These are functional features that do not have corresponding appearance on histopathology. As such, previous pathology training may not be beneficial in learning these dynamic features. This study aims to evaluate the diagnostic accuracy, learning curve, inter- and intraobserver agreement for identifying features of increased IP in experienced and inexperienced analysts and pathologists. A total of 180 endoscopic confocal laser endomicroscopy (Pentax EC-3870FK; Pentax, Tokyo, Japan) images of the terminal ileum, subdivided into 6 sets of 30 were evaluated by 6 experienced analysts, 13 inexperienced analysts, and 2 pathologists, after a 30-minute teaching session. Cell-junction enhancement, fluorescein leak, and cell dropout were used to represent increased IP and were either present or absent in each image. For each image, the diagnostic accuracy, confidence, and quality were assessed. Diagnostic accuracy was significantly higher for experienced analysts compared with inexperienced analysts from the first set (96.7% vs 83.1%, P 0.86 for experienced observers. Features representative of increased IP can be rapidly learned with high inter- and intraobserver agreement. Confidence and image quality were significant predictors of accurate interpretation. Previous pathology training did not have an effect on learning. Copyright © 2016 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.

Combined laser and atomic force microscope lithography on aluminum: Mask fabrication for nanoelectromechanical systems

DEFF Research Database (Denmark)

Berini, Abadal Gabriel; Boisen, Anja; Davis, Zachary James

1999-01-01

A direct-write laser system and an atomic force microscope (AFM) are combined to modify thin layers of aluminum on an oxidized silicon substrate, in order to fabricate conducting and robust etch masks with submicron features. These masks are very well suited for the production of nanoelectromecha......A direct-write laser system and an atomic force microscope (AFM) are combined to modify thin layers of aluminum on an oxidized silicon substrate, in order to fabricate conducting and robust etch masks with submicron features. These masks are very well suited for the production...... writing, and to perform submicron modifications by AFM oxidation. The mask fabrication for a nanoscale suspended resonator bridge is used to illustrate the advantages of this combined technique for NEMS. (C) 1999 American Institute of Physics. [S0003-6951(99)00221-1]....

Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics.

Science.gov (United States)

Amos, W B; Reichelt, S; Cattermole, D M; Laufer, J

2003-05-01

In this paper, differential phase imaging (DPC) with transmitted light is implemented by adding a suitable detection system to a standard commercially available scanning confocal microscope. DPC, a long-established method in scanning optical microscopy, depends on detecting the intensity difference between opposite halves or quadrants of a split photodiode detector placed in an aperture plane. Here, DPC is compared with scanned differential interference contrast (DIC) using a variety of biological specimens and objective lenses of high numerical aperture. While DPC and DIC images are generally similar, DPC seems to have a greater depth of field. DPC has several advantages over DIC. These include low cost (no polarizing or strain-free optics are required), absence of a double scanning spot, electronically variable direction of shading and the ability to image specimens in plastic dishes where birefringence prevents the use of DIC. DPC is also here found to need 20 times less laser power at the specimen than DIC.

Upgrading the GSI beamline microscope with a confocal fluorescence lifetime scanner to monitor charged particle induced chromatin decondensation in living cells

Energy Technology Data Exchange (ETDEWEB)

Abdollahi, Elham; Taucher-Scholz, Gisela [Department of Biophysics, GSI Helmholtz Center for Heavy Ion Research, Planckstrasse 1, 64291 Darmstadt (Germany); Durante, Marco [Department of Biophysics, GSI Helmholtz Center for Heavy Ion Research, Planckstrasse 1, 64291 Darmstadt (Germany); Institute for Condensed Matter Physics, Darmstadt University of Technology, 64289 Darmstadt (Germany); Jakob, Burkhard, E-mail: B.Jakob@gsi.de [Department of Biophysics, GSI Helmholtz Center for Heavy Ion Research, Planckstrasse 1, 64291 Darmstadt (Germany)

2015-12-15

We report the upgrade of the GSI beamline microscope coupled to the linear accelerator UNILAC by a confocal FLIM scanner utilizing time correlated single photon counting technique (TCSPC). The system can now be used to address the radiation induced chromatin decondensation in more detail and with higher sensitivity compared to intensity based methods. This decondensation of heterochromatic areas is one of the early DNA damage responses observed after charged particle irradiation and might facilitate the further processing of the induced lesions. We describe here the establishment of different DNA dyes as chromatin compaction probes usable for quantification of the DNA condensation status in living cells utilizing lifetime imaging. In addition, we find an evidence of heterochromatic chromatin decondensation in ion irradiated murine chromocenters detected after subsequent fixation using FLIM measurements.

Characterization of the photocurrents generated by the laser of atomic force microscopes

Energy Technology Data Exchange (ETDEWEB)

Ji, Yanfeng; Hui, Fei; Shi, Yuanyuan; Lanza, Mario, E-mail: mlanza@suda.edu.cn [Institute of Functional Nano and Soft Materials (FUNSOM), Collaborative Innovation Center of Suzhou Nanoscience and Technology, Soochow University, 199 Ren-Ai Road, Suzhou 215123 (China); Iglesias, Vanessa [International Iberian Nanotechnology Laboratory, 4715-330 Braga (Portugal); Lewis, David [Nanonics Imaging, Har Hotzvim, Jerusalem 91487 (Israel); Niu, Jiebin; Long, Shibing; Liu, Ming [Laboratory of Nanofabrication and Novel Device Integration, Institute of Microelectronics, Chinese Academy of Sciences, Beijing 100029 (China); Hofer, Alexander; Frammelsberger, Werner; Benstetter, Guenther [Deggendorf Institute of Technology, Edlmairstr. 6+8, 94469 Deggendorf (Germany); Scheuermann, Andrew; McIntyre, Paul C. [Department of Materials Science and Engineering, Stanford University, Stanford, California 94305 (United States)

2016-08-15

The conductive atomic force microscope (CAFM) has become an essential tool for the nanoscale electronic characterization of many materials and devices. When studying photoactive samples, the laser used by the CAFM to detect the deflection of the cantilever can generate photocurrents that perturb the current signals collected, leading to unreliable characterization. In metal-coated semiconductor samples, this problem is further aggravated, and large currents above the nanometer range can be observed even without the application of any bias. Here we present the first characterization of the photocurrents introduced by the laser of the CAFM, and we quantify the amount of light arriving to the surface of the sample. The mechanisms for current collection when placing the CAFM tip on metal-coated photoactive samples are also analyzed in-depth. Finally, we successfully avoided the laser-induced perturbations using a two pass technique: the first scan collects the topography (laser ON) and the second collects the current (laser OFF). We also demonstrate that CAFMs without a laser (using a tuning fork for detecting the deflection of the tip) do not have this problem.

Confocal comparison of corneal reinnervation after small incision lenticule extraction (SMILE) and femtosecond laser in situ keratomileusis (FS-LASIK).

Science.gov (United States)

Li, Meiyan; Niu, Lingling; Qin, Bing; Zhou, Zimei; Ni, Katherine; Le, Qihua; Xiang, Jun; Wei, Anji; Ma, Weiping; Zhou, Xingtao

2013-01-01

To evaluate corneal reinnervation, and the corresponding corneal sensitivity and keratocyte density after small incision lenticule extraction (SMILE) and femtosecond laser in situ keratomileusis (FS-LASIK). In this prospective, non-randomized observational study, 18 patients (32 eyes) received SMILE surgery, and 22 patients (42 eyes) received FS-LASIK surgery to correct myopia. The corneal subbasal nerve density and microscopic morphological changes in corneal architecture were evaluated by confocal microscopy prior to surgery and at 1 week, 1 month, 3 months, and 6 months after surgery. A correlation analysis was performed between subbasal corneal nerve density and the corresponding keratocyte density and corneal sensitivity. The decrease in subbasal nerve density was less severe in SMILE-treated eyes than in FS-LASIK-treated eyes at 1 week (P = 0.0147), 1 month (P = 0.0243), and 3 months (P = 0.0498), but no difference was detected at the 6-month visit (P = 0.5277). The subbasal nerve density correlated positively with central corneal sensitivity in both groups (r = 0.416, PLASIK group, respectively). The SMILE-treated eyes have a lower risk of developing peripheral empty space with epithelial cells filling in (P = 0.0005). The decrease in subbasal nerve fiber density was less severe in the SMILE group than the FS-LASIK group in the first 3 months following the surgeries. The subbasal nerve density was correlated with central corneal sensitivity.

Activity and three-dimensional distribution of toluene-degrading Pseudomonas putida in a multispecies biofilm assessed by quantitative in situ hybridization and scanning confocal laser microscopy

DEFF Research Database (Denmark)

Møller, Søren; Pedersen, Anne Rathmann; Poulsen, L.K.

1996-01-01

As a representative member of the toluene-degrading population in a biofilter for waste gas treatment, Pseudomonas putida was investigated with a 16S rRNA targeting probe, The three-dimensional distribution of P. putida was visualized in the biofilm matrix by scanning confocal laser microscopy...

Line-scanning confocal microscopy for high-resolution imaging of upconverting rare-earth-based contrast agents

Science.gov (United States)

Higgins, Laura M.; Zevon, Margot; Ganapathy, Vidya; Sheng, Yang; Tan, Mei Chee; Riman, Richard E.; Roth, Charles M.; Moghe, Prabhas V.; Pierce, Mark C.

2015-01-01

Abstract. Rare-earth (RE) doped nanocomposites emit visible luminescence when illuminated with continuous wave near-infrared light, making them appealing candidates for use as contrast agents in biomedical imaging. However, the emission lifetime of these materials is much longer than the pixel dwell times used in scanning intravital microscopy. To overcome this limitation, we have developed a line-scanning confocal microscope for high-resolution, optically sectioned imaging of samples labeled with RE-based nanomaterials. Instrument performance is quantified using calibrated test objects. NaYF4:Er,Yb nanocomposites are imaged in vitro, and in ex vivo tissue specimens, with direct comparison to point-scanning confocal microscopy. We demonstrate that the extended pixel dwell time of line-scanning confocal microscopy enables subcellular-level imaging of these nanomaterials while maintaining optical sectioning. The line-scanning approach thus enables microscopic imaging of this emerging class of contrast agents for preclinical studies, with the potential to be adapted for real-time in vivo imaging in the clinic. PMID:26603495

Ultraviolet Laser SQUID Microscope for GaN Blue Light Emitting Diode Testing

International Nuclear Information System (INIS)

Daibo, M; Kamiwano, D; Kurosawa, T; Yoshizawa, M; Tayama, N

2006-01-01

We carried out non-contacting measurements of photocurrent distributions in GaN blue light emitting diode (LED) chips using our newly developed ultraviolet (UV) laser SQUID microscope. The UV light generates the photocurrent, and then the photocurrent induces small magnetic fields around the chip. An off-axis arranged HTS-SQUID magnetometer is employed to detect a vector magnetic field whose typical amplitude is several hundred femto-tesla. Generally, it is difficult to obtain Ohmic contacts for p-type GaN because of the low hole concentration in the p-type epitaxial layer and the lack of any available metal with a higher work function compared with the p-type GaN. Therefore, a traditional probecontacted electrical test is difficult to conduct for wide band gap semiconductors without an adequately annealed electrode. Using the UV-laser SQUID microscope, the photocurrent can be measured without any electrical contact. We show the photocurrent vector map which was reconstructed from measured magnetic fields data. We also demonstrate how we found the position of a defect of the electrical short circuits in the LED chip

3-D laser confocal microscopy study of the oxidation of NdFeB magnets in atmospheric conditions

International Nuclear Information System (INIS)

Meakin, J.P.; Speight, J.D.; Sheridan, R.S.; Bradshaw, A.; Harris, I.R.; Williams, A.J.; Walton, A.

2016-01-01

Highlights: • Room temperature atmospheric oxidation behaviour of sintered NdFeB. • 3D laser confocal microscopy measurement of oxide phase growth. • Significant height increase of oxide phase only observed at triple points. • Raman spectroscopy identified oxide phase to be Nd 2 O 3 . • Diffusion coefficient determined to be 4 × 10 −13 cm 2 /s. - Abstract: Neodymium iron boron (NdFeB) magnets are used in a number of important applications, such as generators in gearless wind turbines, motors in electric vehicles and electronic goods (e.g.— computer hard disk drives, HDD). Hydrogen can be used as a processing gas to separate and recycle scrap sintered Nd-Fe-B magnets from end-of-life products to form a powder suitable for recycling. However, the magnets are likely to have been exposed to atmospheric conditions prior to processing, and any oxidation could lead to activation problems for the hydrogen decrepitation reaction. Many previous studies on the oxidation of NdFeB magnets have been performed at elevated temperatures; however, few studies have been formed under atmospheric conditions. In this paper a combination of 3-D laser confocal microscopy and Raman spectroscopy have been used to assess the composition, morphology and rate of oxidation/corrosion on scrap sintered NdFeB magnets. Confocal microscopy has been employed to measure the growth of surface reaction products at room temperature, immediately after exposure to air. The results showed that there was a significant height increase at the triple junctions of the Nd-rich grain boundaries. Using Raman spectroscopy, the product was shown to consist of Nd 2 O 3 and formed only on the Nd-rich triple junctions. The diffusion coefficient of the triple junction reaction product growth at 20 °C was determined to be approximately 4 × 10 −13 cm 2 /sec. This value is several orders of magnitude larger than values derived from the diffusion controlled oxide growth observations at elevated

3-D laser confocal microscopy study of the oxidation of NdFeB magnets in atmospheric conditions

Energy Technology Data Exchange (ETDEWEB)

Meakin, J.P., E-mail: jxm764@bham.ac.uk; Speight, J.D.; Sheridan, R.S.; Bradshaw, A.; Harris, I.R.; Williams, A.J.; Walton, A.

2016-08-15

Highlights: • Room temperature atmospheric oxidation behaviour of sintered NdFeB. • 3D laser confocal microscopy measurement of oxide phase growth. • Significant height increase of oxide phase only observed at triple points. • Raman spectroscopy identified oxide phase to be Nd{sub 2}O{sub 3}. • Diffusion coefficient determined to be 4 × 10{sup −13} cm{sup 2}/s. - Abstract: Neodymium iron boron (NdFeB) magnets are used in a number of important applications, such as generators in gearless wind turbines, motors in electric vehicles and electronic goods (e.g.— computer hard disk drives, HDD). Hydrogen can be used as a processing gas to separate and recycle scrap sintered Nd-Fe-B magnets from end-of-life products to form a powder suitable for recycling. However, the magnets are likely to have been exposed to atmospheric conditions prior to processing, and any oxidation could lead to activation problems for the hydrogen decrepitation reaction. Many previous studies on the oxidation of NdFeB magnets have been performed at elevated temperatures; however, few studies have been formed under atmospheric conditions. In this paper a combination of 3-D laser confocal microscopy and Raman spectroscopy have been used to assess the composition, morphology and rate of oxidation/corrosion on scrap sintered NdFeB magnets. Confocal microscopy has been employed to measure the growth of surface reaction products at room temperature, immediately after exposure to air. The results showed that there was a significant height increase at the triple junctions of the Nd-rich grain boundaries. Using Raman spectroscopy, the product was shown to consist of Nd{sub 2}O{sub 3} and formed only on the Nd-rich triple junctions. The diffusion coefficient of the triple junction reaction product growth at 20 °C was determined to be approximately 4 × 10{sup −13} cm{sup 2}/sec. This value is several orders of magnitude larger than values derived from the diffusion controlled oxide growth

Clinical applications of in vivo fluorescence confocal laser scanning microscopy

Science.gov (United States)

Oh, Chilhwan; Park, Sangyong; Kim, Junhyung; Ha, Seunghan; Park, Gyuman; Lee, Gunwoo; Lee, Onseok; Chun, Byungseon; Gweon, Daegab

2008-02-01

Living skin for basic and clinical research can be evaluated by Confocal Laser Scanning Microscope (CLSM) non-invasively. CLSM imaging system can achieve skin image its native state either "in vivo" or "fresh biopsy (ex vivo)" without fixation, sectioning and staining that is necessary for routine histology. This study examines the potential fluorescent CLSM with a various exogenous fluorescent contrast agent, to provide with more resolution images in skin. In addition, in vivo fluorescent CLSM researchers will be extended a range of potential clinical application. The prototype of our CLSM system has been developed by Prof. Gweon's group. The operating parameters are composed of some units, such as illuminated wavelength 488 nm, argon illumination power up to 20mW on the skin, objective lens, 0.9NA oil immersion, axial resolution 1.0μm, field of view 200μm x 100μm (lateral resolution , 0.3μm). In human volunteer, fluorescein sodium was administrated topically and intradermally. Animal studies were done in GFP transgenic mouse, IRC mouse and pig skin. For imaging of animal skin, fluorescein sodium, acridine orange, and curcumine were used for fluorescein contrast agent. We also used the GFP transgenic mouse for fluorescein CLSM imaging. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. Curcumin is a yellow food dye that has similar fluorescent properties to fluorescein sodium. Acridin Orange can be highlight nuclei in viable keratinocyte. In vivo CLSM of transgenic GFP mouse enable on in vivo, high resolution view of GFP expressing skin tissue. GFP signals are brightest in corneocyte, kertinocyte, hair and eccrine gland. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. In

A fluorescence in situ staining method for investigating spores and vegetative cells of Clostridia by confocal laser scanning microscopy and structured illuminated microscopy.

Science.gov (United States)

D'Incecco, P; Ong, L; Gras, S; Pellegrino, L

2018-04-18

Non-pathogenic spore-forming Clostridia are of increasing interest due to their application in biogas production and their capability to spoil different food products. The life cycle for Clostridium includes a spore stage that can assist in survival under environmentally stressful conditions, such as extremes of temperature or pH. Due to their size, spores can be investigated by a range of microscopic techniques, many of which involve sample pre-treatment. We have developed a quick, simple and non-destructive fluorescent staining procedure that allows a clear differentiation between spores and vegetative cells and effectively stains spores, allowing recovery and tracking in subsequent experiments. Hoechst 34580, Propidium iodide and wheat germ agglutinin WGA 488 were used in combination to stain four strains of Clostridia at different life cycle stages. Staining was conducted without drying the sample, preventing changes induced by dehydration and cells observed by confocal laser scanner microscopy or using a super-resolution microscope equipped with a 3D-structured illumination module. Dual staining with Hoechst/Propidium iodide differentiated spores from vegetative cells, provided information on the viability of cells and was successfully applied to follow spore production induced by heating. Super-resolution microscopy of spores probed by Hoechst 34580 also allowed chromatin to be visualised. Direct staining of a cheese specimen using Nile Red and Fast Green allowed in situ observation of spores within the cheese and their position within the cheese matrix. The proposed staining method has broad applicability and can potentially be applied to follow Clostridium spore behaviour in a range of different environments. Copyright © 2018 Elsevier Ltd. All rights reserved.

Integrated Photoacoustic and Fluorescence Confocal Microscopy

OpenAIRE

Wang, Yu; Maslov, Konstantin; Kim, Chulhong; Hu, Song; Wang, Lihong V.

2010-01-01

We have developed a dual-modality imaging system by integrating optical-resolution photoacoustic microscopy and fluorescence confocal microscopy to provide optical absorption and fluorescence contrasts simultaneously. By sharing the same laser source and objective lens, intrinsically registered photoacoustic and fluorescence images are acquired in a single scan. The micrometer resolution allows imaging of both blood and lymphatic vessels down to the capillary level. Simultaneous photoacoustic...

A multi-axis confocal rheoscope for studying shear flow of structured fluids

KAUST Repository

Lin, Neil Y. C.; McCoy, Jonathan H.; Cheng, Xiang; Leahy, Brian; Israelachvili, Jacob N.; Cohen, Itai

2014-01-01

of confinement effects. By using our shear cell in conjunction with a biaxial force measurement device and a high-speed confocal microscope, we are able to measure the real-time biaxial stress while simultaneously imaging the material three-dimensional structure

Confocal microscopy imaging of the biofilm matrix

DEFF Research Database (Denmark)

Schlafer, Sebastian; Meyer, Rikke L

2017-01-01

The extracellular matrix is an integral part of microbial biofilms and an important field of research. Confocal laser scanning microscopy is a valuable tool for the study of biofilms, and in particular of the biofilm matrix, as it allows real-time visualization of fully hydrated, living specimens...... the concentration of solutes and the diffusive properties of the biofilm matrix....

Detection of a fluorescent-labeled avidin-nucleic acid nanoassembly by confocal laser endomicroscopy in the microvasculature of chronically inflamed intestinal mucosa

Directory of Open Access Journals (Sweden)

Buda A

2015-01-01

Full Text Available Andrea Buda,1,* Sonia Facchin,1,* Elisa Dassie,2 Elisabetta Casarin,3 Mark A Jepson,4 Helmut Neumann,5 Giorgia Hatem,1 Stefano Realdon,6 Renata D’Incà,1 Giacomo Carlo Sturniolo,1 Margherita Morpurgo3 1Department of Surgical, Oncological, and Gastroenterological Sciences, University of Padova, 2Department of Molecular Medicine, University of Padova, Padova, Italy; 3Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy; 4School of Biochemistry and Wolfson Bioimaging Facility, University of Bristol, Bristol, UK; 5Ludwig Demlig Endoscopic Center of Excellence, ESGE Endoscopy Training Center, University of Erlangen-Nuremberg, Erlangen, Germany; 6Veneto Institute of Oncology IOV-IRCCS, Padova, Italy *These authors contributed equally to this work Abstract: Inflammatory bowel diseases are chronic gastrointestinal pathologies causing great discomfort in both children and adults. The pathogenesis of inflammatory bowel diseases is not yet fully understood and their diagnosis and treatment are often challenging. Nanoparticle-based strategies have been tested in local drug delivery to the inflamed colon. Here, we have investigated the use of the novel avidin-nucleic acid nanoassembly (ANANAS platform as a potential diagnostic carrier in an experimental model of inflammatory bowel diseases. Fluorescent-labeled ANANAS nanoparticles were administered to mice with chemically induced chronic inflammation of the large intestine. Localization of mucosal nanoparticles was assessed in vivo by dual-band confocal laser endomicroscopy. This technique enables characterization of the mucosal microvasculature and crypt architecture at subcellular resolution. Intravascular nanoparticle distribution was observed in the inflamed mucosa but not in healthy controls, demonstrating the utility of the combination of ANANAS and confocal laser endomicroscopy for highlighting intestinal inflammatory conditions. The specific localization of

Laser-based microstructuring of materials surfaces using low-cost microlens arrays

Science.gov (United States)

Nieto, Daniel; Vara, G.; Diez, J. A.; O`Connor, Gerard M.; Arines, Justo; Gómez-Reino, C.; Flores-Arias, M.

2012-03-01

Since frictional interactions in microscopically small components are becoming increasingly important for the development of new products for all modern technology, we present a laser-based technique for micro-patterning surfaces of materials using low-cost microlens arrays. The microlens used were fabricated on soda-lime glass using a laser direct-write technique, followed by a thermal treatment into an oven. By combining laser direct-write and the thermal treatment it was possible to obtain high quality elements using a low cost infrared laser widely implemented in industry which makes this technique attractive in comparison with other more expensive methods. The main advantage of using microlens arrays for micropatterning surfaces is the possibility of fabricating a large number of identical structures simultaneously, leading to a highly efficient process. In order to study the capabilities of the microlens fabricated for microstructuring materials, identical structures and arrays of holes were fabricated over a variety of materials, such us, stainless steel, polymer and ceramic. The minimum diameter of the individual microstructure generated at surface is 5 μm. Different nanosecond lasers operating at Infrared, Green and UV were used. The topography and morphology of the elements obtained were determined using a confocal microscope SENSOFAR 2300 Plμ.

In vivo detection of basal cell carcinoma: comparison of a reflectance confocal microscope and a multiphoton tomograph

Science.gov (United States)

Ulrich, Martina; Klemp, Marisa; Darvin, Maxim E.; König, Karsten; Lademann, Jürgen; Meinke, Martina C.

2013-06-01

The standard diagnostic procedure for basal cell carcinoma (BCC) is invasive tissue biopsy with time-consuming histological examination. To reduce the number of biopsies, noninvasive optical methods have been developed providing high-resolution skin examination. We present direct comparison of a reflectance confocal microscope (RLSM) and a multiphoton tomograph (MPT) for BCC diagnosis. Both systems are applied to nine patients prior to surgery, and the results are analyzed, including histological results. Both systems prove suitable for detecting typical characteristics of BCC in various stages. The RLSM allows large horizontal overview images to be obtained, enabling the investigator to find the regions of interest quickly, e.g., BCC nests. Elongated cells and palisading structures are easily recognized using both methods. Due to the higher resolution, changes in nucleus diameter or cytoplasm could be visualized with the MPT. Therefore, the nucleus diameter, nucleus/cytoplasm ratio, and cell density are estimated for normal and BCC cells using the MPT. The nucleus of elongated BCC cells is significantly longer than other measured normal skin cells, whereas the cell density and nucleus/cytoplasm ratio of BCC cannot be significantly distinguished from granular cells.

Confocal laser scanning microscopy detection of chlorophylls and carotenoids in chloroplasts and chromoplasts of tomato fruit.

Science.gov (United States)

D'Andrea, Lucio; Amenós, Montse; Rodríguez-Concepción, Manuel

2014-01-01

Plant cells are unique among eukaryotic cells because of the presence of plastids, including chloroplasts and chromoplasts. Chloroplasts are found in green tissues and harbor the photosynthetic machinery (including chlorophyll molecules), while chromoplasts are present in non-photosynthetic tissues and accumulate large amounts of carotenoids. During tomato fruit development, chloroplasts are converted into chromoplasts that accumulate high levels of lycopene, a linear carotenoid responsible for the characteristic red color of ripe fruit. Here, we describe a simple and fast method to detect both types of fully differentiated plastids (chloroplasts and chromoplasts), as well as intermediate stages, in fresh tomato fruits. The method is based on the differential autofluorescence of chlorophylls and carotenoids (lycopene) detected by Confocal Laser Scanning Microscopy.

Assessing the plasmonics of gold nano-triangles with higher order laser modes

Directory of Open Access Journals (Sweden)

Laura E. Hennemann

2012-10-01

Full Text Available Regular arrays of metallic nano-triangles – so called Fischer patterns – are fabricated by nano-sphere lithography. We studied such gold nano-triangle arrays on silicon or glass substrates. A series of different samples was investigated with a parabolic mirror based confocal microscope where the sample is scanned through the laser focus. By employing higher order laser modes (azimuthally and radially polarised laser beams, we can excite the Fischer patterns using either a pure in-plane (x,y electric field or a strongly z-directional (optical axis of the optical microscope electric field. We collected and evaluated the emitted luminescence and thereby investigated the respectively excited plasmonic modes. These varied considerably: firstly with the light polarisation in the focus, secondly with the aspect ratio of the triangles and thirdly with the employed substrate. Moreover, we obtained strongly enhanced Raman spectra of an adenine (sub-monolayer on gold Fischer patterns on glass. We thus showed that gold Fischer patterns are promising surface-enhanced Raman scattering (SERS substrates.

Scanning electron microscopical examination of the impact of laser patterning on microscopic inhomogeneities of Cu(In,Ga)(Se,S)2 absorbers produced by rapid thermal processing

International Nuclear Information System (INIS)

Künecke, U.; Hölzing, A.; Jost, S.; Lechner, R.; Vogt, H.; Heiß, A.; Palm, J.; Hock, R.; Wellmann, P.

2013-01-01

Laser scribing of the Mo back electrode is commonly applied to define the cell structure of Cu(In,Ga)(Se,S) 2 (CIGSSe) thin film solar cells. The patterning process was performed on laboratory samples using ns and ps pulse length laser processes. After structuring, CIGSSe absorbers were processed by rapid thermal processing (RTP) of stacked elemental layer precursors. Microscopic inhomogeneities were investigated on different sample positions. For samples structured with ns pulse, the absorber morphology in the laser line vicinity is different as compared to the morphology in the unstructured cell area. Scanning electron microscopy and energy-dispersive X-ray spectroscopy show significant changes in the absorber grain size and chemical composition. Close to the laser line, the typically observed Ga accumulation on the back contact is less pronounced and more Ga is incorporated closer to the surface leading to a smaller grain size. The observed changes are attributed to partial damaging of a diffusion barrier between glass and Mo induced by the ns laser process, which allows diffusion of sodium from the glass substrate into the absorber during RTP. The enhanced Ga incorporation closer to the surface is an indication for the influence of sodium on the local phase development during RTP. The damages of the diffusion barrier can be effectively prevented by the application of a ps laser scribing process. CIGSSe absorbers processed on samples structured with ps pulse length do not show the described microscopic inhomogeneities around the laser line. - Highlights: ► Scanning electron microscopy on Cu(In,Ga)(Se,S) 2 solar cell absorbers ► Laser patterning with ns laser pulse damages the sodium diffusion barrier. ► Improved laser patterning with ps laser pulse leaves diffusion barrier intact. ► Additional sodium changes phase development during absorber formation. ► Gallium content is increased at surface and decreased at backside of absorber

Application of gold nanoparticles as contrast agents in confocal laser scanning microscopy

Energy Technology Data Exchange (ETDEWEB)

Lemelle, A; Veksler, B; Piletsky, S A; Meglinski, I [Cranfield Health, Cranfield University, Cranfield, MK43 0AL (United Kingdom); Kozhevnikov, I S; Akchurin, G G, E-mail: a.lemelle.s06@cranfield.ac.uk [Physics Faculty, Saratov State University, Saratov 410012 (Russian Federation)

2009-01-15

Confocal laser scanning microscopy (CLSM) is a modern high-resolution optical technique providing detailed image of tissue structure with high (down to microns) spatial resolution. Aiming at a concurrent improvement of imaging depth and image quality the CLSM requires the use of contrast agents. Commonly employed fluorescent contrast agents, such as fluorescent dyes and proteins, suffer from toxicity, photo-bleaching and overlapping with the tissues autofluorescence. Gold nanoparticles are potentially highly attractive to be applied as a contrast agent since they are not subject to photo-bleaching and can target biochemical cells markers associated with the specific diseases. In current report we consider the applicability of gold nano-spheres as a contrast agent to enhance quality of CLSM images of skin tissues in vitro versus the application of optical clearing agent, such as glycerol. The enhancement of CLSM image contrast was observed with an application of gold nano-spheres diffused within the skin tissues. We show that optical clearing agents such as a glycerol provide better CLSM image contrast than gold nano-spheres.

Application of gold nanoparticles as contrast agents in confocal laser scanning microscopy

International Nuclear Information System (INIS)

Lemelle, A; Veksler, B; Piletsky, S A; Meglinski, I; Kozhevnikov, I S; Akchurin, G G

2009-01-01

Confocal laser scanning microscopy (CLSM) is a modern high-resolution optical technique providing detailed image of tissue structure with high (down to microns) spatial resolution. Aiming at a concurrent improvement of imaging depth and image quality the CLSM requires the use of contrast agents. Commonly employed fluorescent contrast agents, such as fluorescent dyes and proteins, suffer from toxicity, photo-bleaching and overlapping with the tissues autofluorescence. Gold nanoparticles are potentially highly attractive to be applied as a contrast agent since they are not subject to photo-bleaching and can target biochemical cells markers associated with the specific diseases. In current report we consider the applicability of gold nano-spheres as a contrast agent to enhance quality of CLSM images of skin tissues in vitro versus the application of optical clearing agent, such as glycerol. The enhancement of CLSM image contrast was observed with an application of gold nano-spheres diffused within the skin tissues. We show that optical clearing agents such as a glycerol provide better CLSM image contrast than gold nano-spheres

Application of gold nanoparticles as contrast agents in confocal laser scanning microscopy

Science.gov (United States)

Lemelle, A.; Veksler, B.; Kozhevnikov, I. S.; Akchurin, G. G.; Piletsky, S. A.; Meglinski, I.

2009-01-01

Confocal laser scanning microscopy (CLSM) is a modern high-resolution optical technique providing detailed image of tissue structure with high (down to microns) spatial resolution. Aiming at a concurrent improvement of imaging depth and image quality the CLSM requires the use of contrast agents. Commonly employed fluorescent contrast agents, such as fluorescent dyes and proteins, suffer from toxicity, photo-bleaching and overlapping with the tissues autofluorescence. Gold nanoparticles are potentially highly attractive to be applied as a contrast agent since they are not subject to photo-bleaching and can target biochemical cells markers associated with the specific diseases. In current report we consider the applicability of gold nano-spheres as a contrast agent to enhance quality of CLSM images of skin tissues in vitro versus the application of optical clearing agent, such as glycerol. The enhancement of CLSM image contrast was observed with an application of gold nano-spheres diffused within the skin tissues. We show that optical clearing agents such as a glycerol provide better CLSM image contrast than gold nano-spheres.

Laser apparatus and method for microscopic and spectroscopic analysis and processing of biological cells

Science.gov (United States)

Gourley, P.L.; Gourley, M.F.

1997-03-04

An apparatus and method are disclosed for microscopic and spectroscopic analysis and processing of biological cells. The apparatus comprises a laser having an analysis region within the laser cavity for containing one or more biological cells to be analyzed. The presence of a cell within the analysis region in superposition with an activated portion of a gain medium of the laser acts to encode information about the cell upon the laser beam, the cell information being recoverable by an analysis means that preferably includes an array photodetector such as a CCD camera and a spectrometer. The apparatus and method may be used to analyze biomedical cells including blood cells and the like, and may include processing means for manipulating, sorting, or eradicating cells after analysis. 20 figs.

Development and Optical Testing of the Camera, Hand Lens, and Microscope Probe with Scannable Laser Spectroscopy (CHAMP-SLS)

Science.gov (United States)

Mungas, Greg S.; Gursel, Yekta; Sepulveda, Cesar A.; Anderson, Mark; La Baw, Clayton; Johnson, Kenneth R.; Deans, Matthew; Beegle, Luther; Boynton, John

2008-01-01

Conducting high resolution field microscopy with coupled laser spectroscopy that can be used to selectively analyze the surface chemistry of individual pixels in a scene is an enabling capability for next generation robotic and manned spaceflight missions, civil, and military applications. In the laboratory, we use a range of imaging and surface preparation tools that provide us with in-focus images, context imaging for identifying features that we want to investigate at high magnification, and surface-optical coupling that allows us to apply optical spectroscopic analysis techniques for analyzing surface chemistry particularly at high magnifications. The camera, hand lens, and microscope probe with scannable laser spectroscopy (CHAMP-SLS) is an imaging/spectroscopy instrument capable of imaging continuously from infinity down to high resolution microscopy (resolution of approx. 1 micron/pixel in a final camera format), the closer CHAMP-SLS is placed to a feature, the higher the resultant magnification. At hand lens to microscopic magnifications, the imaged scene can be selectively interrogated with point spectroscopic techniques such as Raman spectroscopy, microscopic Laser Induced Breakdown Spectroscopy (micro-LIBS), laser ablation mass-spectrometry, Fluorescence spectroscopy, and/or Reflectance spectroscopy. This paper summarizes the optical design, development, and testing of the CHAMP-SLS optics.

Simulation with Python on transverse modes of the symmetric confocal resonator

Science.gov (United States)

Wang, Qing Hua; Qi, Jing; Ji, Yun Jing; Song, Yang; Li, Zhenhua

2017-08-01

Python is a popular open-source programming language that can be used to simulate various optical phenomena. We have developed a suite of programs to help teach the course of laser principle. The complicated transverse modes of the symmetric confocal resonator can be visualized in personal computers, which is significant to help the students understand the pattern distribution of laser resonator.

Super-resolution imaging of ciliary microdomains in isolated olfactory sensory neurons using a custom two-color stimulated emission depletion microscope

Science.gov (United States)

Meyer, Stephanie A.; Ozbay, Baris N.; Potcoava, Mariana; Salcedo, Ernesto; Restrepo, Diego; Gibson, Emily A.

2016-06-01

We performed stimulated emission depletion (STED) imaging of isolated olfactory sensory neurons (OSNs) using a custom-built microscope. The STED microscope uses a single pulsed laser to excite two separate fluorophores, Atto 590 and Atto 647N. A gated timing circuit combined with temporal interleaving of the different color excitation/STED laser pulses filters the two channel detection and greatly minimizes crosstalk. We quantified the instrument resolution to be ˜81 and ˜44 nm, for the Atto 590 and Atto 647N channels. The spatial separation between the two channels was measured to be under 10 nm, well below the resolution limit. The custom-STED microscope is incorporated onto a commercial research microscope allowing brightfield, differential interference contrast, and epifluorescence imaging on the same field of view. We performed immunolabeling of OSNs in mice to image localization of ciliary membrane proteins involved in olfactory transduction. We imaged Ca2+-permeable cyclic nucleotide gated (CNG) channel (Atto 594) and adenylyl cyclase type III (ACIII) (Atto 647N) in distinct cilia. STED imaging resolved well-separated subdiffraction limited clusters for each protein. We quantified the size of each cluster to have a mean value of 88±48 nm and 124±43 nm, for CNG and ACIII, respectively. STED imaging showed separated clusters that were not resolvable in confocal images.

Nano-displacement measurement based on virtual pinhole confocal method

International Nuclear Information System (INIS)

Li, Long; Kuang, Cuifang; Xue, Yi; Liu, Xu

2013-01-01

A virtual pinhole confocal system based on charge-coupled device (CCD) detection and image processing techniques is built to measure axial displacement with 10 nm resolution, preeminent flexibility and excellent robustness when facing spot drifting. Axial displacement of the sample surface is determined by capturing the confocal laser spot using a CCD detector and quantifying the energy collected by programmable virtual pinholes. Experiments indicate an applicable measuring range of 1000 nm (Gaussian fitting r = 0.9902) with a highly linear range of 500 nm (linear fitting r = 0.9993). A concentric subtraction algorithm is introduced to further enhance resolution. Factors affecting measuring precision, sensitivity and signal-to-noise ratio are discussed using theoretical deductions and diffraction simulations. The virtual pinhole technique has promising applications in surface profiling and confocal imaging applications which require easily-customizable pinhole configurations. (paper)

Apertureless near-field scanning optical microscope working with or without laser source.

Science.gov (United States)

Formanek, F; De Wilde, Y; Aigouy, L; Chen, Y

2004-01-01

An apertureless near-field scanning optical microscope (ANSOM), used indifferent configurations, is presented. Our versatile home-made setup, based on a sharp tungsten tip glued onto a quartz tuning fork and working in tapping mode, allows to perform imaging over a broad spectral range. We have recorded optical images in the visible (wavelength, lambda = 655 nm) and in the infrared (lambda = 10.6 microm), proving that the setup routinely achieves an optical resolution of images recorded in the visible (lambda = 655 nm) in an inverted configuration where the tip does not perturb the focused spot of the illumination laser. Approach curves as well as image profiles have revealed that on demodulating the optical signal at higher harmonics, we can obtain an effective probe sharpening which results in an improvement of the resolution. Finally, we have presented optical images recorded in the infrared without any illumination, that is, the usual laser source is replaced by a simple heating of the sample. This has shown that the ANSOM can be used as a near-field thermal optical microscope (NTOM) to probe the near field generated by the thermal emission of the sample.

Confocal comparison of corneal reinnervation after small incision lenticule extraction (SMILE and femtosecond laser in situ keratomileusis (FS-LASIK.

Directory of Open Access Journals (Sweden)

Meiyan Li

Full Text Available PURPOSE: To evaluate corneal reinnervation, and the corresponding corneal sensitivity and keratocyte density after small incision lenticule extraction (SMILE and femtosecond laser in situ keratomileusis (FS-LASIK. METHODS: In this prospective, non-randomized observational study, 18 patients (32 eyes received SMILE surgery, and 22 patients (42 eyes received FS-LASIK surgery to correct myopia. The corneal subbasal nerve density and microscopic morphological changes in corneal architecture were evaluated by confocal microscopy prior to surgery and at 1 week, 1 month, 3 months, and 6 months after surgery. A correlation analysis was performed between subbasal corneal nerve density and the corresponding keratocyte density and corneal sensitivity. RESULTS: The decrease in subbasal nerve density was less severe in SMILE-treated eyes than in FS-LASIK-treated eyes at 1 week (P = 0.0147, 1 month (P = 0.0243, and 3 months (P = 0.0498, but no difference was detected at the 6-month visit (P = 0.5277. The subbasal nerve density correlated positively with central corneal sensitivity in both groups (r = 0.416, P<0.0001, and r = 0.2567, P = 0.0038 for SMILE group and FS-LASIK group, respectively. The SMILE-treated eyes have a lower risk of developing peripheral empty space with epithelial cells filling in (P = 0.0005. CONCLUSIONS: The decrease in subbasal nerve fiber density was less severe in the SMILE group than the FS-LASIK group in the first 3 months following the surgeries. The subbasal nerve density was correlated with central corneal sensitivity.

Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy.

Science.gov (United States)

Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin; Sohn, Dae Kyung

2016-07-01

We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis.

Confocal microscopy patterns in nonmelanoma skin cancer and clinical applications.

Science.gov (United States)

González, S; Sánchez, V; González-Rodríguez, A; Parrado, C; Ullrich, M

2014-06-01

Reflectance confocal microscopy is currently the most promising noninvasive diagnostic tool for studying cutaneous structures between the stratum corneum and the superficial reticular dermis. This tool gives real-time images parallel to the skin surface; the microscopic resolution is similar to that of conventional histology. Numerous studies have identified the main confocal features of various inflammatory skin diseases and tumors, demonstrating the good correlation of these features with certain dermatoscopic patterns and histologic findings. Confocal patterns and diagnostic algorithms have been shown to have high sensitivity and specificity in melanoma and nonmelanoma skin cancer. Possible present and future applications of this noninvasive technology are wide ranging and reach beyond its use in noninvasive diagnosis. This tool can also be used, for example, to evaluate dynamic skin processes that occur after UV exposure or to assess tumor response to noninvasive treatments such as photodynamic therapy. We explain the characteristic confocal features found in the main nonmelanoma skin tumors and discuss possible applications for this novel diagnostic technique in routine dermatology practice. Copyright © 2012 Elsevier España, S.L. and AEDV. All rights reserved.

Laser polishing of additive manufactured Ti alloys

Science.gov (United States)

Ma, C. P.; Guan, Y. C.; Zhou, W.

2017-06-01

Laser-based additive manufacturing has attracted much attention as a promising 3D printing method for metallic components in recent years. However, surface roughness of additive manufactured components has been considered as a challenge to achieve high performance. In this work, we demonstrate the capability of fiber laser in polishing rough surface of additive manufactured Ti-based alloys as Ti-6Al-4V and TC11. Both as-received surface and laser-polished surfaces as well as cross-section subsurfaces were analyzed carefully by White-Light Interference, Confocal Microscope, Focus Ion Beam, Scanning Electron Microscopy, Energy Dispersive Spectrometer, and X-ray Diffraction. Results revealed that as-received Ti-based alloys with surface roughness more than 5 μm could be reduce to less than 1 μm through laser polishing process. Moreover, microstructure, microhardness and wear resistance of laser-polished zone was investigated in order to examine the thermal effect of laser polishing processing on the substrate of additive manufactured Ti alloys. This proof-of-concept process has the potential to effectively improve the surface roughness of additive manufactured metallic alloy by local polishing method without damage to the substrate.

Laser-induced surface deformation microscope for the study of the dynamic viscoelasticity of plasma membrane in a living cell.

Science.gov (United States)

Morisaku, Toshinori; Yui, Hiroharu

2018-05-15

A laser-induced surface deformation (LISD) microscope is developed and applied to measurement of the dynamic relaxation responses of the plasma membrane in a living cell. A laser beam is tightly focused on an optional area of cell surface and the focused light induces microscopic deformation on the surface via radiation pressure. The LISD microscope not only allows non-contact and destruction-free measurement but provides power spectra of the surface responses depending on the frequency of the intensity of the laser beam. An optical system for the LISD is equipped via a microscope, allowing us to measure the relaxation responses in sub-cellular-sized regions of the plasma membrane. In addition, the forced oscillation caused by the radiation pressure for surface deformation extends the upper limit of the frequency range in the obtained power spectra to 106 Hz, which enables us to measure relaxation responses in local regions within the plasma membrane. From differences in power-law exponents at higher frequencies, it is realized that a cancerous cell obeys a weaker single power-law than a normal fibroblast cell. Furthermore, the power spectrum of a keratinocyte cell obeys a power-law with two exponents, indicating that alternative mechanical models to a conventional soft glassy rheology model (where single power-laws explain cells' responses below about 103 Hz) are needed for the understanding over a wider frequency range. The LISD microscope would contribute to investigation of microscopic cell rheology, which is important for clarifying the mechanisms of cell migration and tissue construction.

Microscopic investigation of InGaN/GaN heterostructure laser diode degradation using Kelvin probe force microscopy

International Nuclear Information System (INIS)

Lochthofen, A; Mertin, W; Bacher, G; Furitsch, M; Bruederl, G; Strauss, U; Haerle, V

2008-01-01

We report on Kelvin probe force microscopy (KPFM) measurements on fresh and artificially aged InGaN/GaN laser test structures. In the case of an unbiased laser diode, a comparison of the surface potential between a fresh and a stressed laser diode shows a pronounced modification of the laser facet due to the aging process. Performing KPFM measurements under forward bias, a correlation between the macroscopic I-V characteristics and the microscopic voltage drop across the heterostructure layer sequence is found. This clearly demonstrates the potential of KPFM for investigating InGaN/GaN laser diode degradation

Resolution Enhancement of Scanning Laser Acoustic Microscope Using Transverse Wave

International Nuclear Information System (INIS)

Ko, D. S.; Park, J. S.; Kim, Y. H.

1997-01-01

We studied the resolution enhancement of a novel scanning laser acoustic microscope (SLAM) using transverse waves. Mode conversion of the ultrasonic wave takes place at the liquid-solid interface and some energy of the insonifying longitudinal waves in the water will convert to transverse wave energy within the solid specimen. The resolution of SLAM depends on the size of detecting laser spot and the wavelength of the insonifying ultrasonic waves. Science the wavelength of the transverse wave is shorter than that of the longitudinal wave, we are able to achieve the high resolution by using transverse waves. In order to operate SLAM in the transverse wave mode, we made wedge for changing the incident angle. Our experimental results with model 2140 SLAM and an aluminum specimen showed higher contrast of the SLAM image in the transverse wave mode than that in the longitudinal wave mode

CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR MEASUREMENTS, QUANTITATION AND SPECTROSCOPY

Science.gov (United States)

The confocal laser-scanning microscopy (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of the emitted signal. The accuracy of these measurements demands t...

Characterization of hydrogel microstructure using laser tweezers particle tracking and confocal reflection imaging

International Nuclear Information System (INIS)

Kotlarchyk, M A; Botvinick, E L; Putnam, A J

2010-01-01

Hydrogels are commonly used as extracellular matrix mimetics for applications in tissue engineering and increasingly as cell culture platforms with which to study the influence of biophysical and biochemical cues on cell function in 3D. In recent years, a significant number of studies have focused on linking substrate mechanical properties to cell function using standard methodologies to characterize the bulk mechanical properties of the hydrogel substrates. However, current understanding of the correlations between the microstructural mechanical properties of hydrogels and cell function in 3D is poor, in part because of a lack of appropriate techniques. Here we have utilized a laser tracking system, based on passive optical microrheology instrumentation, to characterize the microstructure of viscoelastic fibrin clots. Trajectories and mean square displacements were observed as bioinert PEGylated (PEG: polyethylene glycol) microspheres (1, 2 or 4.7 μm in diameter) diffused within confined pores created by the protein phase of fibrin hydrogels. Complementary confocal reflection imaging revealed microstructures comprised of a highly heterogeneous fibrin network with a wide range of pore sizes. As the protein concentration of fibrin gels was increased, our quantitative laser tracking measurements showed a corresponding decrease in particle mean square displacements with greater resolution and sensitivity than conventional imaging techniques. This platform-independent method will enable a more complete understanding of how changes in substrate mechanical properties simultaneously influence other microenvironmental parameters in 3D cultures.

Laser-generated plasma plume expansion: Combined continuous-microscopic modeling

Science.gov (United States)

Itina, Tatiana E.; Hermann, Jörg; Delaporte, Philippe; Sentis, Marc

2002-12-01

The physical phenomena involved in the interaction of a laser-generated plasma plume with a background gas are studied numerically. A three-dimensional combined model is developed to describe the plasma plume formation and its expansion in vacuum or into a background gas. The proposed approach takes advantages of both continuous and microscopic descriptions. The simulation technique is suitable for the simulation of high-rate laser ablation for a wide range of background pressure. The model takes into account the mass diffusion and the energy exchange between the ablated and background species, as well as the collective motion of the ablated species and the background-gas particles. The developed approach is used to investigate the influence of the background gas on the expansion dynamics of the plume obtained during the laser ablation of aluminum. At moderate pressures, both plume and gas compressions are weak and the process is mainly governed by the diffusive mixing. At higher pressures, the interaction is determined by the plume-gas pressure interplay, the plume front is strongly compressed, and its center exhibits oscillations. In this case, the snowplough effect takes place, leading to the formation of a compressed gas layer in front of the plume. The background pressure needed for the beginning of the snowplough effect is determined from the plume and gas density profiles obtained at various pressures. Simulation results are compared with experimentally measured density distributions. It is shown that the calculations suggest localized formation of molecules during reactive laser ablation.

Laser-generated plasma plume expansion: Combined continuous-microscopic modeling

International Nuclear Information System (INIS)

Itina, Tatiana E.; Hermann, Joerg; Delaporte, Philippe; Sentis, Marc

2002-01-01

The physical phenomena involved in the interaction of a laser-generated plasma plume with a background gas are studied numerically. A three-dimensional combined model is developed to describe the plasma plume formation and its expansion in vacuum or into a background gas. The proposed approach takes advantages of both continuous and microscopic descriptions. The simulation technique is suitable for the simulation of high-rate laser ablation for a wide range of background pressure. The model takes into account the mass diffusion and the energy exchange between the ablated and background species, as well as the collective motion of the ablated species and the background-gas particles. The developed approach is used to investigate the influence of the background gas on the expansion dynamics of the plume obtained during the laser ablation of aluminum. At moderate pressures, both plume and gas compressions are weak and the process is mainly governed by the diffusive mixing. At higher pressures, the interaction is determined by the plume-gas pressure interplay, the plume front is strongly compressed, and its center exhibits oscillations. In this case, the snowplough effect takes place, leading to the formation of a compressed gas layer in front of the plume. The background pressure needed for the beginning of the snowplough effect is determined from the plume and gas density profiles obtained at various pressures. Simulation results are compared with experimentally measured density distributions. It is shown that the calculations suggest localized formation of molecules during reactive laser ablation

A confocal laser scanning microscopic study on thermoresponsive

Indian Academy of Sciences (India)

Monodisperse poly(N -isopropylacrylamide) (PNIPAM) particles loaded with cadmium telluride (CdTe) quantum dots (QDs) of two different sizes (4.7 nm and 5.6 nm) were synthesized in aqueous medium by bonding the capping agent on the quantum dots to the amide groups of PNIPAM and incubating the samples at 45° ...

Confocal laser-scanning microscopy of capillaries in normal and psoriatic skin

Science.gov (United States)

Archid, Rami; Patzelt, Alexa; Lange-Asschenfeldt, Bernhard; Ahmad, Sufian S.; Ulrich, Martina; Stockfleth, Eggert; Philipp, Sandra; Sterry, Wolfram; Lademann, Juergen

2012-10-01

An important and most likely active role in the pathogenesis of psoriasis has been attributed to changes in cutaneous blood vessels. The purpose of this study was to use confocal laser-scanning microscopy (CLSM) to investigate dermal capillaries in psoriatic and normal skin. The structures of the capillary loops in 5 healthy participants were compared with those in affected skin of 13 psoriasis patients. The diameters of the capillaries and papillae were measured for each group with CLSM. All investigated psoriasis patients showed elongated, widened, and tortuous microvessels in the papillary dermis, whereas all healthy controls showed a single capillary loop in each dermal papilla. The capillaries of the papillary loop and the dermal papilla were significantly enlarged in the psoriatic skin lesions (diameters 24.39±2.34 and 146.46±28.52 μm, respectively) in comparison to healthy skin (diameters 9.53±1.8 and 69.48±17.16 μm, respectively) (P<0.001). CLSM appears to represent a promising noninvasive technique for evaluating dermal capillaries in patients with psoriasis. The diameter of the vessels could be seen as a well-quantifiable indicator for the state of psoriatic skin. CLSM could be useful for therapeutic monitoring to delay possible recurrences.

Analysis of the effects of cerium on calcium ion in the protoplasts of ...

African Journals Online (AJOL)

The laser-scanning confocal microscopy has become a routine technique and indispensable tool for cell biological studies. In this study, the probe Fluo-3 AM was used to research the instantaneous changes of calcium ion (Ca2+) in the protoplasts of Arabidopsis thaliana. The laser-scanning mode of confocal microscope is ...

Reflectance confocal microscopy: non-invasive distinction between actinic keratosis and squamous cell carcinoma

NARCIS (Netherlands)

Peppelman, M.; Nguyen, K.P.; Hoogedoorn, L.; Erp, P.E.J. van; Gerritsen, M.J.P.

2015-01-01

BACKGROUND: Early recognition of squamous cell carcinoma (SCC) is difficult. Non-invasive reflectance confocal microscopic (RCM) imaging of the skin is a promising diagnostic technique. Although several RCM features for SCC and AK have been described, it is not determined whether RCM has the ability

Assessment of nerve ultrastructure by fibre-optic confocal microscopy.

Science.gov (United States)

Cushway, T R; Lanzetta, M; Cox, G; Trickett, R; Owen, E R

1996-01-01

Fibre-optic technology combined with confocality produces a microscope capable of optical thin sectioning. In this original study, tibial nerves have been stained in a rat model with a vital dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide, and analysed by fibre-optic confocal microscopy to produce detailed images of nerve ultrastructure. Schwann cells, nodes of Ranvier and longitudinal myelinated sheaths enclosing axons were clearly visible. Single axons appeared as brightly staining longitudinal structures. This allowed easy tracing of multiple signal axons within the nerve tissue. An accurate measurement of internodal lengths was easily accomplished. This technique is comparable to current histological techniques, but does not require biopsy, thin sectioning or tissue fixing. This study offers a standard for further in vivo microscopy, including the possibility of monitoring the progression of nerve regeneration following microsurgical neurorraphy.

Scanner component and head development for confocal microscopy using moving mirror technology

Science.gov (United States)

Loney, Gregory C.

1993-12-01

One of the challenges in designing a confocal microscope is choosing the scan system configuration. The selection is based largely on the microscope application and involves a few distinct schemes. One scheme, moving mirror using galvanometer and resonant scanners, has been shown to offer an excellent solution exhibited by the large number of commercial systems which utilize them. Perceived shortcomings, such as slow image acquisition, are being dispelled due to the advent of large angle, high frequency resonant scanners. These newer devices offer near video rate performance at good scan efficiency.

A compact narrow-linewidth laser with a low-Q monolithic cavity

International Nuclear Information System (INIS)

Peng, Yu

2013-01-01

We demonstrate an approach to narrowing the linewidth of a diode laser to around 15×10 3 Hz with a compact setup of confocal and parallel monolithic Fabry–Perot cavities (MFCs). Resonances of the confocal and parallel MFCs with low finesse are obtained. Diode lasers with optical feedback from confocal and parallel monolithic MFCs are demonstrated. The frequency could be tuned 80×10 6 Hz by changing the grating position of the external cavity diode laser based on the confocal MFC, and 100×10 6 Hz by tuning the temperature of the plane MFC over 0.02 ° C for the external cavity diode laser based on the parallel MFC. (paper)

Noise analysis of a white-light supercontinuum light source for multiple wavelength confocal laser scanning fluorescence microscopy

Energy Technology Data Exchange (ETDEWEB)

McConnell, Gail [Centre for Biophotonics, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow, G4 0NR (United Kingdom)

2005-08-07

Intensity correlations of a Ti : sapphire, Kr/Ar and a white-light supercontinuum were performed to quantify the typical signal amplitude fluctuations and hence ascertain the comparative output stability of the white-light supercontinuum source for confocal laser scanning microscopy (CLSM). Intensity correlations across a two-pixel sample (n = 1000) of up to 98%, 95% and 94% were measured for the Ti : sapphire, Kr/Ar and white-light supercontinuum source, respectively. The white-light supercontinuum noise level is therefore acceptable for CLSM, with the added advantage of wider wavelength flexibility over traditional CLSM excitation sources. The relatively low-noise white-light supercontinuum was then used to perform multiple wavelength sequential CLSM of guinea pig detrusor to confirm the reliability of the system and to demonstrate system flexibility.

Quantitative and Qualitative Aspects of Gas-Metal-Oxide Mass Transfer in High-Temperature Confocal Scanning Laser Microscopy

Science.gov (United States)

Piva, Stephano P. T.; Pistorius, P. Chris; Webler, Bryan A.

2018-05-01

During high-temperature confocal scanning laser microscopy (HT-CSLM) of liquid steel samples, thermal Marangoni flow and rapid mass transfer between the sample and its surroundings occur due to the relatively small sample size (diameter around 5 mm) and large temperature gradients. The resulting evaporation and steel-slag reactions tend to change the chemical composition in the metal. Such mass transfer effects can change observed nonmetallic inclusions. This work quantifies oxide-metal-gas mass transfer of solutes during HT-CSLM experiments using computational simulations and experimental data for (1) dissolution of MgO inclusions in the presence and absence of slag and (2) Ca, Mg-silicate inclusion changes upon exposure of a Si-Mn-killed steel to an oxidizing gas atmosphere.

Semiautomated confocal imaging of fungal pathogenesis on plants: Microscopic analysis of macroscopic specimens.

Science.gov (United States)

Minker, Katharine R; Biedrzycki, Meredith L; Kolagunda, Abhishek; Rhein, Stephen; Perina, Fabiano J; Jacobs, Samuel S; Moore, Michael; Jamann, Tiffany M; Yang, Qin; Nelson, Rebecca; Balint-Kurti, Peter; Kambhamettu, Chandra; Wisser, Randall J; Caplan, Jeffrey L

2018-02-01

The study of phenotypic variation in plant pathogenesis provides fundamental information about the nature of disease resistance. Cellular mechanisms that alter pathogenesis can be elucidated with confocal microscopy; however, systematic phenotyping platforms-from sample processing to image analysis-to investigate this do not exist. We have developed a platform for 3D phenotyping of cellular features underlying variation in disease development by fluorescence-specific resolution of host and pathogen interactions across time (4D). A confocal microscopy phenotyping platform compatible with different maize-fungal pathosystems (fungi: Setosphaeria turcica, Cochliobolus heterostrophus, and Cercospora zeae-maydis) was developed. Protocols and techniques were standardized for sample fixation, optical clearing, species-specific combinatorial fluorescence staining, multisample imaging, and image processing for investigation at the macroscale. The sample preparation methods presented here overcome challenges to fluorescence imaging such as specimen thickness and topography as well as physiological characteristics of the samples such as tissue autofluorescence and presence of cuticle. The resulting imaging techniques provide interesting qualitative and quantitative information not possible with conventional light or electron 2D imaging. Microsc. Res. Tech., 81:141-152, 2018. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke.

Science.gov (United States)

Talley Watts, Lora; Zheng, Wei; Garling, R Justin; Frohlich, Victoria C; Lechleiter, James Donald

2015-06-23

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Efficiency of the confocal method of laser endomicroscopy in complex diagnoses of diseases of common bile duct

International Nuclear Information System (INIS)

Anaskin, S G; Korniletsky, I D; Panchenkov, D N; Chertyuk, V B; Sazonov, D V; Zabozlayev, F G; Danilevskaya, O V; Mokshina, N V

2017-01-01

One of the more frequent manifestations of diseases of the bile ducts are its’ strictures or stenoses that could be of either malignant or benign nature. Current methods of diagnosing this pathology include computer tomography (CT) scan, magnetic resonance cholangiopancreatography (MRCP), endoscopic ultrasound (EUS) and endoscopic retrograde cholangiopancreatography (ERCP). However, these methods are not always informative, which makes this a current and topical problem. A fundamentally new method that broadens the capabilities of ERCP when diagnosing diseases of the bile duct accompanied by the development of strictures or stenoses is probe-based confocal laser endomicroscopy (pCLE). The method is based on the principle of confocal fluorescence microscopy. The most elaborate complications arise with the presence of the pre-existing pancreatobiliary pathology: pseudotumoral chronic pancreatitis, acute cholangitis, etc. Early stage cholangiocarcinoma diagnosis can be difficult (and not always possible) even with the help of modern research methods. For the timely diagnostic it is advantageous to conduct pCLE and targeted biopsy of the zone with most manifested changes. In all instances, the first use of the pCLE method for diagnostic purposes allowed us to clarify and correctly verify the diagnosis. When concerning the diseases of the bile duct, the modern stage of pCLE development can be of critical importance when other methods are not effective. (paper)

Elastomeric photo-actuators and their investigation by confocal laser scanning microscopy

International Nuclear Information System (INIS)

Czaniková, Klaudia; Ilčíková, Markéta; Mičušík, Matej; Kasák, Peter; Mosnáček, Jaroslav; Omastová, Mária; Krupa, Igor; Pavlova, Ewa; Chorvát Jr, Dušan

2013-01-01

The photo-actuation behavior of nanocomposites based on ethylene–vinylacetate copolymer (EVA) and styrene–isoprene–styrene (SIS) block copolymer filled with well-dispersed and modified multiwalled carbon nanotubes (MWCNTs) is discussed in this paper. The nanocomposites were prepared by casting from solution. To improve the dispersion of the MWCNTs in EVA, the MWCNT surface was modified with a non-covalent surfactant, cholesteryl 1-pyrenecarboxylate (PyChol). To prepare SIS nanocomposites, the MWCNT surface was covalently modified with polystyrene chains. The good dispersion of the filler was confirmed by transmission electron microscopy (TEM). Special, custom-made punch/die molds were used to create a Braille element (BE)-like shape, which under shear forces induces a uniaxial orientation of the MWCNTs within the matrix. The uniaxial orientation of MWCNTs is an essential precondition to ensure the photo-actuating behavior of MWCNTs in polymeric matrices. The orientation of the MWCNTs within the matrices was examined by scanning electron microscopy (SEM). Nanocomposite BEs were illuminated from the bottom by a red light-emitting diode (LED), and the photo-actuation was investigated by confocal laser scanning microscopy (CLSM). When the BEs were exposed to light, a temporary increase in the height of the element was detected. This process was observed to be reversible: after switching off the light, the BEs returned to their original shape and height. (paper)

Fluorescence excitation analysis by two-photon confocal laser scanning microscopy: a new method to identify fluorescent nanoparticles on histological tissue sections

Directory of Open Access Journals (Sweden)

Kahn E

2012-10-01

Full Text Available Edmond Kahn,1 Nicolas Tissot,3 Perrine Frere,3 Aurélien Dauphin,3 Mohamed Boumhras,2,4 Claude-Marie Bachelet,3 Frédérique Frouin,1 Gérard Lizard21Institut National de la Santé et de la Recherche Médicale (INSERM U678/UMR-S UPMC, CHU Pitié-Salpêtrière, Paris, France; 2Equipe Biochimie du Peroxysome, Inflammation et Métabolisme Lipidique EA7270, Faculté des Sciences Gabriel, Université de Bourgogne-INSERM Dijon, France; 3Plateforme d'Imagerie cellulaire, UPMC, Paris, France; 4Laboratory of Biochemistry and Neuroscience, Applied Toxicology Group, Faculty of Science and Technology, Settat, MoroccoAbstract: In the present study, we make use of the ability of two-photon confocal laser scanning microscopes (CLSMs equipped with tunable lasers to produce spectral excitation image sequences. Furthermore, unmixing, which is usually performed on emission image sequences, is performed on these excitation image sequences. We use factor analysis of medical image sequences (FAMIS, which produces factor images, to unmix spectral image sequences of stained structures in tissue sections to provide images of characterized stained cellular structures. This new approach is applied to histological tissue sections of mouse aorta containing labeled iron nanoparticles stained with Texas Red and counterstained with SYTO13, to obtain visual information about the accumulation of these nanoparticles in the arterial wall. The possible presence of Texas Red is determined using a two-photon CLSM associated with FAMIS via the excitation spectra. Texas Red and SYTO13 are thus differentiated, and corresponding factor images specify their possible presence and cellular localization. In conclusion, the designed protocol shows that sequences of images obtained by excitation in a two-photon CLSM enables characterization of Texas Red-stained nanoparticles and other markers. This methodology offers an alternative and complementary solution to the conventional use of emission

Selective treatment of carious dentin using a mid-infrared tunable pulsed laser at 6 μm wavelength range

Science.gov (United States)

Saiki, Masayuki; Ishii, Katsunori; Yoshikawa, Kazushi; Yasuo, Kenzo; Yamamoto, Kazuyo; Awazu, Kunio

2011-03-01

Optical technologies have good potential for caries detection, prevention, excavation, and the realization of minimal intervention dentistry. This study aimed to develop a selective excavation technique of carious tissue using the specific absorption in 6 μm wavelength range. Bovine dentin demineralized with lactic acid solution was used as a carious dentin model. A mid-infrared tunable pulsed laser was obtained by difference-frequency generation technique. The wavelength was tuned to 6.02 and 6.42 μm which correspond to absorption bands called amide I and amide II, respectively. The laser delivers 5 ns pulse width at a repetition rate of 10 Hz. The morphological change after irradiation was observed with a scanning electron microscope, and the measurement of ablation depth was performed with a confocal laser microscope. At λ = 6.02 μm and the average power density of 15 W/cm2, demineralized dentin was removed selectively with less-invasive effect on sound dentin. The wavelength of 6.42 μm also showed the possibility of selective removal. High ablation efficiency and low thermal side effect were observed using the nanosecond pulsed laser with λ = 6.02 μm. In the near future, development of compact laser device will open the minimal invasive laser treatment to the dental clinic.

Confocal Imaging of porous media

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Shah, S.; Crawshaw, D.; Boek, D.

2012-12-01

Carbonate rocks, which hold approximately 50% of the world's oil and gas reserves, have a very complicated and heterogeneous structure in comparison with sandstone reservoir rock. We present advances with different techniques to image, reconstruct, and characterize statistically the micro-geometry of carbonate pores. The main goal here is to develop a technique to obtain two dimensional and three dimensional images using Confocal Laser Scanning Microscopy. CLSM is used in epi-fluorescent imaging mode, allowing for the very high optical resolution of features well below 1μm size. Images of pore structures were captured using CLSM imaging where spaces in the carbonate samples were impregnated with a fluorescent, dyed epoxy-resin, and scanned in the x-y plane by a laser probe. We discuss the sample preparation in detail for Confocal Imaging to obtain sub-micron resolution images of heterogeneous carbonate rocks. We also discuss the technical and practical aspects of this imaging technique, including its advantages and limitation. We present several examples of this application, including studying pore geometry in carbonates, characterizing sub-resolution porosity in two dimensional images. We then describe approaches to extract statistical information about porosity using image processing and spatial correlation function. We have managed to obtain very low depth information in z -axis (~ 50μm) to develop three dimensional images of carbonate rocks with the current capabilities and limitation of CLSM technique. Hence, we have planned a novel technique to obtain higher depth information to obtain high three dimensional images with sub-micron resolution possible in the lateral and axial planes.

Ultrastructure and Light Microscope Analysis of Intact Skin after a Varying Number of Low Level Laser Irradiations in Mice

Directory of Open Access Journals (Sweden)

Mamie Mizusaki Iyomasa

2014-01-01

Full Text Available Low level laser therapy (LLLT has been used to relieve pain, inflammation, and wound healing processes. Thus, the skin is overexposed to laser and this effect is not completely understood. This study analyzed the effects of the number of laser applications (three, six, and 10 on the intact skin of the masseteric region in mice of strain HRS/J. The animals (n=30 were equally divided into control (0 J/cm2 and irradiated (20 J/cm2, and each of these groups was further equally divided according to the number of laser applications (three, six, and 10 and underwent LLLT on alternate days. Samples were analyzed by light microscopy and transmission electron microscope (TEM. The animals receiving applications exhibited open channels more dilated between the keratinocytes and photobiomodulation effect on endothelial cells and fibroblasts by TEM. Under the light microscope after 10 laser applications, the type I collagen decreased (P<0.05 compared to the three and six applications. Under these experimental conditions, all numbers of applications provided photobiomodulatory effect on the epidermis and dermis, without damage. More studies are needed to standardize the energy density and number of applications recommended for laser therapy to have a better cost-benefit ratio associated with treatment.

In vivo cellular imaging with microscopes enabled by MEMS scanners

Science.gov (United States)

Ra, Hyejun

High-resolution optical imaging plays an important role in medical diagnosis and biomedical research. Confocal microscopy is a widely used imaging method for obtaining cellular and sub-cellular images of biological tissue in reflectance and fluorescence modes. Its characteristic optical sectioning capability also enables three-dimensional (3-D) image reconstruction. However, its use has mostly been limited to excised tissues due to the requirement of high numerical aperture (NA) lenses for cellular resolution. Microscope miniaturization can enable in vivo imaging to make possible early cancer diagnosis and biological studies in the innate environment. In this dissertation, microscope miniaturization for in vivo cellular imaging is presented. The dual-axes confocal (DAC) architecture overcomes limitations of the conventional single-axis confocal (SAC) architecture to allow for miniaturization with high resolution. A microelectromechanical systems (MEMS) scanner is the central imaging component that is key in miniaturization of the DAC architecture. The design, fabrication, and characterization of the two-dimensional (2-D) MEMS scanner are presented. The gimbaled MEMS scanner is fabricated on a double silicon-on-insulator (SOI) wafer and is actuated by self-aligned vertical electrostatic combdrives. The imaging performance of the MEMS scanner in a DAC configuration is shown in a breadboard microscope setup, where reflectance and fluorescence imaging is demonstrated. Then, the MEMS scanner is integrated into a miniature DAC microscope. The whole imaging system is integrated into a portable unit for research in small animal models of human biology and disease. In vivo 3-D imaging is demonstrated on mouse skin models showing gene transfer and siRNA silencing. The siRNA silencing process is sequentially imaged in one mouse over time.

Optimising performance of a confocal fluorescence microscope with a differential pinhole

International Nuclear Information System (INIS)

Kakade, Rohan; Walker, John G; Phillips, Andrew J

2016-01-01

The signal-to-noise ratio (SNR)-resolution trade-off is of great importance to bio-imaging applications where the aim is to image the sample using as little light as possible without significantly sacrificing image quality. In this paper the inherent SNR-resolution tradeoff in Confocal Fluorescence Microscopy (CFM) systems is presented by means of an effective tradeoff curve. A CFM system that employs a differential pinhole detection scheme has recently been shown to offer increased resolution, but at the expense of SNR. An optimum profile for the differential pinhole is identified in this paper that offers improved performance over a conventional (circular pinhole) system. The performance enhancement is illustrated through computer simulation. (paper)

A compact scanning soft X-ray microscope

International Nuclear Information System (INIS)

Trail, J.A.

1989-01-01

Soft x-ray microscopes operating at wavelengths between 2.3 nm and 4.4 nm are capable of imaging wet biological cells with a resolution many times that of a visible light microscope. Several such soft x-ray microscopes have been constructed. However, with the exception of contact microscopes, all use synchrotrons as the source of soft x-ray radiation and Fresnel zone plates as the focusing optics. These synchrotron based microscopes are very successful but have the disadvantage of limited access. This dissertation reviews the construction and performance of a compact scanning soft x-ray microscope whose size and accessibility is comparable to that of an electron microscope. The microscope uses a high-brightness laser-produced plasma as the soft x-ray source and normal incidence multilayer-coated mirrors in a Schwarzschild configuration as the focusing optics. The microscope operates at a wavelength of 14 nm, has a spatial resolution of 0.5 μm, and has a soft x-ray photon flux through the focus of 10 4 -10 5 s -1 when operated with only 170 mW of average laser power. The complete system, including the laser, fits on a single 4' x 8' optical table. The significant components of the compact microscope are the laser-produced plasma (LPP) source, the multilayer coatings, and the Schwarzschild objective. These components are reviewed, both with regard to their particular use in the current microscope and with regard to extending the microscope performance to higher resolution, higher speed, and operation at shorter wavelengths. Measurements of soft x-ray emission and debris emission from our present LPP source are presented and considerations given for an optimal LPP source. The LPP source was also used as a broadband soft x-ray source for measurement of normal incidence multilayer mirror reflectance in the 10-25 nm spectral region

Confocal examination of subsurface cracking in ceramic materials.

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Etman, Maged K

2009-10-01

The original ceramic surface finish and its microstructure may have an effect on crack propagation. The purpose of this study was to investigate the relation between crack propagation and ceramic microstructure following cyclic fatigue loading, and to qualitatively evaluate and quantitatively measure the surface and subsurface crack depths of three types of ceramic restorations with different microstructures using a Confocal Laser Scanning Microscope (CLSM) and Scanning Electron Microscope (SEM). Twenty (8 x 4 x 2 mm(3)) blocks of AllCeram (AC), experimental ceramic (EC, IPS e.max Press), and Sensation SL (SSL) were prepared, ten glazed and ten polished of each material. Sixty antagonist enamel specimens were made from the labial surfaces of permanent incisors. The ceramic abraders were attached to a wear machine, so that each enamel specimen presented at 45 degrees to the vertical movement of the abraders, and immersed in artificial saliva. Wear was induced for 80K cycles at 60 cycles/min with a load of 40 N and 2-mm horizontal deflection. The specimens were examined for cracks at baseline, 5K, 10K, 20K, 40K, and 80K cycles. Twenty- to 30-microm deep subsurface cracking appeared in SSL, with 8 to 10 microm in AC, and 7 microm close to the margin of the wear facets in glazed EC after 5K cycles. The EC showed no cracks with increasing wear cycles. Seventy-microm deep subsurface cracks were detected in SSL and 45 microm in AC after 80K cycles. Statistically, there was significant difference among the three materials (p 0.05) in crack depth within the same ceramic material with different surface finishes. The ceramic materials with different microstructures showed different patterns of subsurface cracking.

Superresolution confocal technology for displacement measurements based on total internal reflection

International Nuclear Information System (INIS)

Kuang Cuifang; Hao Xiang; Wang Tingting; Liu Xu; Ali, M. Yakut

2010-01-01

In order to achieve a higher axial resolution for displacement measurement, a novel method is proposed based on total internal reflection filter and confocal microscope principle. A theoretical analysis of the basic measurement principles is presented. The analysis reveals that the proposed confocal detection scheme is effective in enhancing the resolution of nonlinearity of the reflectance curve greatly. In addition, a simple prototype system has been developed based on the theoretical analysis and a series of experiments have been performed under laboratory conditions to verify the system feasibility, accuracy, and stability. The experimental results demonstrate that the axial resolution in displacement measurements is better than 1 nm in a range of 200 nm which is threefold better than that can be achieved using the plane reflector.

Superresolution confocal technology for displacement measurements based on total internal reflection.

Science.gov (United States)

Kuang, Cuifang; Ali, M Yakut; Hao, Xiang; Wang, Tingting; Liu, Xu

2010-10-01

In order to achieve a higher axial resolution for displacement measurement, a novel method is proposed based on total internal reflection filter and confocal microscope principle. A theoretical analysis of the basic measurement principles is presented. The analysis reveals that the proposed confocal detection scheme is effective in enhancing the resolution of nonlinearity of the reflectance curve greatly. In addition, a simple prototype system has been developed based on the theoretical analysis and a series of experiments have been performed under laboratory conditions to verify the system feasibility, accuracy, and stability. The experimental results demonstrate that the axial resolution in displacement measurements is better than 1 nm in a range of 200 nm which is threefold better than that can be achieved using the plane reflector.

Confocal nanoscanning, bead picking (CONA): PickoScreen microscopes for automated and quantitative screening of one-bead one-compound libraries.

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Hintersteiner, Martin; Buehler, Christof; Uhl, Volker; Schmied, Mario; Müller, Jürgen; Kottig, Karsten; Auer, Manfred

2009-01-01

Solid phase combinatorial chemistry provides fast and cost-effective access to large bead based libraries with compound numbers easily exceeding tens of thousands of compounds. Incubating one-bead one-compound library beads with fluorescently labeled target proteins and identifying and isolating the beads which contain a bound target protein, potentially represents one of the most powerful generic primary high throughput screening formats. On-bead screening (OBS) based on this detection principle can be carried out with limited automation. Often hit bead detection, i.e. recognizing beads with a fluorescently labeled protein bound to the compound on the bead, relies on eye-inspection under a wide-field microscope. Using low resolution detection techniques, the identification of hit beads and their ranking is limited by a low fluorescence signal intensity and varying levels of the library beads' autofluorescence. To exploit the full potential of an OBS process, reliable methods for both automated quantitative detection of hit beads and their subsequent isolation are needed. In a joint collaborative effort with Evotec Technologies (now Perkin-Elmer Cellular Technologies Germany GmbH), we have built two confocal bead scanner and picker platforms PS02 and a high-speed variant PS04 dedicated to automated high resolution OBS. The PS0X instruments combine fully automated confocal large area scanning of a bead monolayer at the bottom of standard MTP plates with semiautomated isolation of individual hit beads via hydraulic-driven picker capillaries. The quantification of fluorescence intensities with high spatial resolution in the equatorial plane of each bead allows for a reliable discrimination between entirely bright autofluorescent beads and real hit beads which exhibit an increased fluorescence signal at the outer few micrometers of the bead. The achieved screening speed of up to 200,000 bead assayed in less than 7 h and the picking time of approximately 1 bead

A multi-axis confocal rheoscope for studying shear flow of structured fluids

KAUST Repository

Lin, Neil Y. C.

2014-03-01

We present a new design for a confocal rheoscope that enables uniform uniaxial or biaxial shear. The design consists of two precisely positioned parallel plates with a gap that can be adjusted down to 2 ±0.1 μm, allowing for the exploration of confinement effects. By using our shear cell in conjunction with a biaxial force measurement device and a high-speed confocal microscope, we are able to measure the real-time biaxial stress while simultaneously imaging the material three-dimensional structure. We illustrate the importance of the instrument capabilities by discussing the applications of this instrument in current and future research topics in colloidal suspensions. © 2014 AIP Publishing LLC.

Cell differentiation in cardiac myxomas: confocal microscopy and gene expression analysis after laser capture microdissection.

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Pucci, Angela; Mattioli, Claudia; Matteucci, Marco; Lorenzini, Daniele; Panvini, Francesca; Pacini, Simone; Ippolito, Chiara; Celiento, Michele; De Martino, Andrea; Dolfi, Amelio; Belgio, Beatrice; Bortolotti, Uberto; Basolo, Fulvio; Bartoloni, Giovanni

2018-05-22

Cardiac myxomas are rare tumors with a heterogeneous cell population including properly neoplastic (lepidic), endothelial and smooth muscle cells. The assessment of neoplastic (lepidic) cell differentiation pattern is rather difficult using conventional light microscopy immunohistochemistry and/or whole tissue extracts for mRNA analyses. In a preliminary study, we investigated 20 formalin-fixed and paraffin-embedded cardiac myxomas by means of conventional immunohistochemistry; in 10/20 cases, cell differentiation was also analyzed by real-time RT-PCR after laser capture microdissection of the neoplastic cells, whereas calretinin and endothelial antigen CD31 immunoreactivity was localized in 4/10 cases by double immunofluorescence confocal microscopy. Gene expression analyses of α-smooth muscle actin, endothelial CD31 antigen, alpha-cardiac actin, matrix metalloprotease-2 (MMP2) and tissue inhibitor of matrix metalloprotease-1 (TIMP1) was performed on cDNA obtained from either microdissected neoplastic cells or whole tumor sections. We found very little or absent CD31 and α-Smooth Muscle Actin expression in the microdissected cells as compared to the whole tumors, whereas TIMP1 and MMP2 genes were highly expressed in both ones, greater levels being found in patients with embolic phenomena. α-Cardiac Actin was not detected. Confocal microscopy disclosed two different signals corresponding to calretinin-positive myxoma cells and to endothelial CD31-positive cells, respectively. In conclusion, the neoplastic (lepidic) cells showed a distinct gene expression pattern and no consistent overlapping with endothelial and smooth muscle cells or cardiac myocytes; the expression of TIMP1 and MMP2 might be related to clinical presentation; larger series studies using also systematic transcriptome analysis might be useful to confirm the present results.

Confocal microscopy studies of morphology and apoptosis: ovaries, limbs, embryos and insects

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

CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR QUANTIFYING CYTOMETRIC APPLICATIONS WITH SPECTROSCOPIC INSTRUMENTS

Science.gov (United States)

The confocal laser-scanning microscopy (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of the emitted signal. The accuracy of these measurements demands t...

Confocal Microscopy and Flow Cytometry System Performance: Assessment of QA Parameters that affect data Quanitification

Science.gov (United States)

Flow and image cytometers can provide useful quantitative fluorescence data. We have devised QA tests to be used on both a flow cytometer and a confocal microscope to assure that the data is accurate, reproducible and precise. Flow Cytometry: We have provided two simple perform...

Toward the development of a low-cost laser Doppler module for ophthalmic microscopes

Science.gov (United States)

Cattini, Stefano; Rovati, Luigi

2012-03-01

A laser Doppler module easily integrated into a commercial ophthalmic microscope is proposed. Such setup adds flow measurement capability to standard visual inspection of the fundus. The proposed instrument may provide important clinical information such as the detection of vessel occlusion provided by surgical treatments (i.e. photocoagulation). The measuring system is based on a self-mixing laser diode Doppler flowmeter (SM-DF). Reduced costs, easy implementation and small size represent the main features of SM-DF. Moreover, this technique offers the advantage to have the excitation and measurement beams spatially overlapped, thus both overcoming the alignment difficulty of traditional laser Doppler flowmeter and, well fitting with to limited optical aperture of the pupil. Thanks to an on-board DSP-microcontroller, the optoelectronic module directly estimates the blood flow; USB connection and an ad-hoc developed user-friendly software interface allow displaying the result on a personal computer. Preliminary test demonstrates the applicability of the proposed measuring system.

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

Science.gov (United States)

Lim, Daryl; Ford, Tim N.; Chu, Kengyeh K.; Mertz, Jerome

2011-01-01

We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.

Laser reflection spot as a pattern in a diamond coating – a microscopic study

Directory of Open Access Journals (Sweden)

GORDANA S. RISTIĆ

2009-07-01

Full Text Available Diamond coatings were deposited by the synchronous and coupled action of a hot filament CVD method and a pulsed CO2 laser in spectro-absorbing and spectro-non-absorbing diamond precursor atmospheres. The obtained coatings were structured/patterned, i.e., they were comprised of uncovered, bare locations. An extra effect observed only in the spectro-active diamond precursor atmosphere was the creation of another laser spot in the coating – a reflection spot. In order to establish the practical usability of the latter one, extensive microscopic investigations were performed with consideration of the morphology changes in the spot of the direct laser beam. Normal incidence SEM images of this spot showed a smooth surface, without any pulse radiation damage. AFM imaging revealed the actual surface condition and gave precise data on the surface characteristics.

Emulation and design of terahertz reflection-mode confocal scanning microscopy based on virtual pinhole

Science.gov (United States)

Yang, Yong-fa; Li, Qi

2014-12-01

In the practical application of terahertz reflection-mode confocal scanning microscopy, the size of detector pinhole is an important factor that determines the performance of spatial resolution characteristic of the microscopic system. However, the use of physical pinhole brings some inconvenience to the experiment and the adjustment error has a great influence on the experiment result. Through reasonably selecting the parameter of matrix detector virtual pinhole (VPH), it can efficiently approximate the physical pinhole. By using this approach, the difficulty of experimental calibration is reduced significantly. In this article, an imaging scheme of terahertz reflection-mode confocal scanning microscopy that is based on the matrix detector VPH is put forward. The influence of detector pinhole size on the axial resolution of confocal scanning microscopy is emulated and analyzed. Then, the parameter of VPH is emulated when the best axial imaging performance is reached.

Imaging of Norway spruce early somatic embryos with the ESEM, Cryo-SEM and laser scanning microscope.

Science.gov (United States)

Neděla, Vilém; Hřib, Jiří; Havel, Ladislav; Hudec, Jiří; Runštuk, Jiří

2016-05-01

This article describes the surface structure of Norway spruce early somatic embryos (ESEs) as a typical culture with asynchronous development. The microstructure of extracellular matrix covering ESEs were observed using the environmental scanning electron microscope as a primary tool and using the scanning electron microscope with cryo attachment and laser electron microscope as a complementary tool allowing our results to be proven independently. The fresh samples were observed in conditions of the air environment of the environmental scanning electron microscope (ESEM) with the pressure from 550Pa to 690Pa and the low temperature of the sample from -18°C to -22°C. The samples were studied using two different types of detector to allow studying either the thin surface structure or material composition. The scanning electron microscope with cryo attachment was used for imaging frozen extracellular matrix microstructure with higher resolution. The combination of both electron microscopy methods was suitable for observation of "native" plant samples, allowing correct evaluation of our results, free of error and artifacts. Copyright © 2016 Elsevier Ltd. All rights reserved.

Curved adjustable fibre-optic diode laser in microscopic cholesteatoma surgery: description of use and review of the relevant literature.

Science.gov (United States)

McCaffer, C J; Pabla, L; Watson, C

2018-04-01

The use of lasers in cholesteatoma surgery is common and well accepted. The most commonly used laser fibres are straight and non-adjustable; these have several limitations. This paper describes the use of an alternative laser fibre. This 'How I Do It' paper describes and illustrates the use of an alternative curved adjustable fibre-optic diode laser in microscopic cholesteatoma surgery. The curved, adjustable laser fibre allows accurate and atraumatic disease removal when the use of a straight laser fibre may be less effective or accurate. It reduces potential damage to delicate structures without the need for extra drilling or bone removal. It is suggested that the curved adjustable laser fibre is superior to the traditional straight fibre for cholesteatoma surgery.

Iris ultrastructure in patients with synechiae as revealed by in vivo laser scanning confocal microscopy : In vivo iris ultrastructure in patients with Synechiae by Laser Scanning Confocal Microscopy.

Science.gov (United States)

Li, Ming; Cheng, Hongbo; Guo, Ping; Zhang, Chun; Tang, Song; Wang, Shusheng

2016-04-26

Iris plays important roles in ocular physiology and disease pathogenesis. Currently it is technically challenging to noninvasively examine the human iris ultrastructure in vivo. The purpose of the current study is to reveal human iris ultrastructure in patients with synechiae by using noninvasive in vivo laser scanning confocal microscopy (LSCM). The ultrastructure of iris in thirty one patients, each with synechiae but transparent cornea, was examined by in vivo LSCM. Five characteristic iris ultrastructures was revealed in patients with synechiae by in vivo LSCM, which include: 1. tree trunk-like structure; 2. tree branch/bush-like structure; 3. Fruit-like structure; 4. Epithelioid-like structure; 5. deep structure. Pigment granules can be observed as a loose structure on the top of the arborization structure. In iris-associated diseases with Tyndall's Phenomenon and keratic precipitates, the pigment particles are more likely to fall off from the arborization structure. The ultrastructure of iris in patients with synechiae has been visualized using in vivo LSCM. Five iris ultrastructures can be clearly observed, with some of the structures maybe disease-associated. The fall-off of the pigment particles may cause the Tyndall's Phenomenon positive. In vivo LSCM provides a non-invasive approach to observe the human iris ultrastructure under certain eye disease conditions, which sets up a foundation to visualize certain iris-associated diseases in the future.

Microscopic analysis of the optoelectronic properties of semiconductor gain media for laser applications; Mikroskopische Analyse optoelektronischer Eigenschaften von Halbleiterverstaerkungsmedien fuer Laseranwendungen

Energy Technology Data Exchange (ETDEWEB)

Bueckers, Christina

2010-12-03

A microscopic many-particle theory is applied to model a wide range of semiconductor laser gain materials. The fundamental understanding of the gain medium and the underlying carrier interaction processes allow for the quantitative prediction of the optoelectronic properties governing the laser performance. Detailed theory-experiment-comparisons are shown for a variety of structures demonstrating the application capabilities of the theoretical approach. The microscopically calculated material properties, in particular absorption, optical gain, luminescence and the intrinsic carrier losses due to radiative and Auger-recombination, constitute the critical input to analyse and design laser structures. On this basis, important system features such as laser wavelength or threshold behaviour become predictable. However, the theory is also used in a diagnostic fashion, e.g. to extract otherwise poorly known structural parameter. Thus, novel concepts for the optimisation of laser designs may be developed with regard to the requirements of specific applications. Moreover, the approach allows for the systematic exploration and assessment of completely novel material systems and their application potential. (orig.)

Ex vivo confocal microscopy: an emerging technique in dermatology

Science.gov (United States)

Perrot, Jean Luc; Labeille, Bruno; Cambazard, Frédéric; Rubegni, Pietro

2018-01-01

This review aims to give an overview of the current available applications of ex vivo confocal microscopy (EVCM) in dermatology. EVCM is a relatively new imaging technique that allows microscopic examination of freshly excised unfixed tissue. It enables a rapid examination of the skin sample directly in the surgery room and thus represents an alternative to the intraoperative micrographic control of the surgical margins of cutaneous tumors by standard microscopic examination on cryopreserved sections during Mohs surgery. Although this technique has mainly been developed for the margin’s control of basal cell carcinoma, many other skin tumors have been studied, including melanoma. Use of EVCM is continuing to evolve, and many possible applications are under investigation, such as the study of nails and hair diseases and the diagnosis of skin infections. PMID:29785327

Imaging subsurface damage of grinded fused silica optics by confocal fluorescence microscopy

International Nuclear Information System (INIS)

Neauport, J.; Cormont, P.; Destribats, J.; Legros, P.; Ambard, C.

2009-01-01

We report an experimental investigation of fluorescence confocal microscopy as a tool to measure subsurface damage on grinded fused silica optics. Confocal fluorescence microscopy was performed with an excitation at the wavelength of 405 nm on fixed abrasive diamond grinded fused silica samples. We detail the measured fluorescence spectrums and compare them to those of oil based coolants and grinding slurries. We evidence that oil based coolant used in diamond grinding induces a fluorescence that marks the subsurface damages and eases its observation. Such residual traces might also be involved in the laser damage process. (authors)

Waveguides fabricated by femtosecond laser exploiting both depressed cladding and stress-induced guiding core.

Science.gov (United States)

Dong, Ming-Ming; Wang, Cheng-Wei; Wu, Zheng-Xiang; Zhang, Yang; Pan, Huai-Hai; Zhao, Quan-Zhong

2013-07-01

We report on the fabrication of stress-induced optical channel waveguides and waveguide splitters with laser-depressed cladding by femtosecond laser. The laser beam was focused into neodymium doped phosphate glass by an objective producing a destructive filament. By moving the sample along an enclosed routine in the horizontal plane followed by a minor descent less than the filament length in the vertical direction, a cylinder with rarified periphery and densified center region was fabricated. Lining up the segments in partially overlapping sequence enabled waveguiding therein. The refractive-index contrast, near- and far-field mode distribution and confocal microscope fluorescence image of the waveguide were obtained. 1-to-2, 1-to-3 and 1-to-4 splitters were also machined with adjustable splitting ratio. Compared with traditional femtosecond laser writing methods, waveguides prepared by this approach showed controllable mode conduction, strong field confinement, large numerical aperture, low propagation loss and intact core region.

Comparison of in vivo and ex vivo laser scanning microscopy and multiphoton tomography application for human and porcine skin imaging

Energy Technology Data Exchange (ETDEWEB)

Darvin, M E; Richter, H; Zhu, Y J; Meinke, M C; Knorr, F; Lademann, J [Center of Experimental and Applied Cutaneous Physiology, Department of Dermatology, Venerology and Allergology, Charité - Universitätsmedizin Berlin (Germany); Gonchukov, S A [National Research Nuclear University ' ' MEPhI' ' (Russian Federation); Koenig, K [JenLab GmbH, Schillerstr. 1, 07745 Jena (Germany)

2014-07-31

Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed by using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted. (laser biophotonics)

Comparison of in vivo and ex vivo laser scanning microscopy and multiphoton tomography application for human and porcine skin imaging

International Nuclear Information System (INIS)

Darvin, M E; Richter, H; Zhu, Y J; Meinke, M C; Knorr, F; Lademann, J; Gonchukov, S A; Koenig, K

2014-01-01

Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed by using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted. (laser biophotonics)

The con focal laser scanning microscope: a powerful tool for the investigation of micro devices and nano structures

International Nuclear Information System (INIS)

Montereali, R.M.; Baldacchini, G.; Bonfigli, F.; Vincenti, M.A.; Almaviva, S.

2008-01-01

In the last years the Con focal Laser Scanning Microscope (CLSM), a versatile and powerful optical instrument, gained a strong increase of interest in the scientific community, not only for biological applications, but also for the characterization of materials, microstructures and devices. The conditions that favoured its wide diffusion are surely the large availability of laser sources and powerful computer-imaging and data-processing systems at relatively low cost; however, the main reason that contributed to its popularity is the ability to obtain tri dimensional reconstruction of a great variety of biological and non-biological samples with sub micrometric resolution. In this report we show the main properties and characteristics of the Con focal Microscope Nikon Eclipse 80-i C1, which has operated sinc more than two years in the Solid State Laser and Spectroscopy Laboratory of the ENEA Research Center in Frascati. Some of the results obtained in the characterization of luminescent micro and nano structures based on lithium fluoride color centers will be presented [it

Confocal microscopy of thick tissue sections: 3D visualizaiton of rat kidney glomeruli

Science.gov (United States)

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

Confocal Microscopy of thick tissue sections: 3D Visualization of rat kidney glomeruli

Science.gov (United States)

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

Multi-spectral confocal microendoscope for in-vivo imaging

Science.gov (United States)

Rouse, Andrew Robert

The concept of in-vivo multi-spectral confocal microscopy is introduced. A slit-scanning multi-spectral confocal microendoscope (MCME) was built to demonstrate the technique. The MCME employs a flexible fiber-optic catheter coupled to a custom built slit-scan confocal microscope fitted with a custom built imaging spectrometer. The catheter consists of a fiber-optic imaging bundle linked to a miniature objective and focus assembly. The design and performance of the miniature objective and focus assembly are discussed. The 3mm diameter catheter may be used on its own or routed though the instrument channel of a commercial endoscope. The confocal nature of the system provides optical sectioning with 3mum lateral resolution and 30mum axial resolution. The prism based multi-spectral detection assembly is typically configured to collect 30 spectral samples over the visible chromatic range. The spectral sampling rate varies from 4nm/pixel at 490nm to 8nm/pixel at 660nm and the minimum resolvable wavelength difference varies from 7nm to 18nm over the same spectral range. Each of these characteristics are primarily dictated by the dispersive power of the prism. The MCME is designed to examine cellular structures during optical biopsy and to exploit the diagnostic information contained within the spectral domain. The primary applications for the system include diagnosis of disease in the gastro-intestinal tract and female reproductive system. Recent data from the grayscale imaging mode are presented. Preliminary multi-spectral results from phantoms, cell cultures, and excised human tissue are presented to demonstrate the potential of in-vivo multi-spectral imaging.

A cryogenic scanning laser microscope for investigation of dynamical states in long Josephson junctions

DEFF Research Database (Denmark)

Holm, Jesper; Mygind, Jesper

1995-01-01

on measurements on different oscillator samples, performed with a novel Cryogenic Scanning Laser Microscope (CSLM) having a spatial resolution of less than ±2.5 μm over a 500 μm×50 μm wide scanning area in the temperature range 2 K-300 K. Even though the dynamical states are extremely sensitive to external noise...... tunnel current is one of the most important internal junction parameters which together with the boundary conditions determine the dynamics, it is of vital importance to experimentally determine the current density throughout the entire junction with high spatial resolution. Here we report...... this microscope enables us to make stable in-situ measurements on operating Josephson junctions. Recent results are presented and discussed....

Superresolution size determination in fluorescence microscopy: A comparison between spatially modulated illumination and confocal laser scanning microscopy

International Nuclear Information System (INIS)

Spoeri, Udo; Failla, Antonio Virgilio; Cremer, Christoph

2004-01-01

Recently developed far field light optical methods are a powerful tool to analyze biological nanostructures and their dynamics, in particular including the interior of three-dimensionally conserved cells. In this article, the recently described method of spatially modulated illumination (SMI) microscopy has been further extended to the online determination of the extension of small, subwavelength sized, fluorescent objects (nanosizing). Using fluorescence excitation with 488 nm, the determination of fluorescent labeled object diameters down to 40 nm corresponding to about 1/12th of the wavelength used for one-photon excitation could be shown. The results of the SMI nanosizing procedure for a detailed, systematic variation of the object diameter are presented together with a fast algorithm for online size evaluation. In addition, we show a direct comparison of the diameter of 'colocalization volumes' between SMI nanosizing and conventional confocal laser scanning microscopy

Laser-Based Surface Modification of Microstructure for Carbon Fiber-Reinforced Plastics

Science.gov (United States)

Yang, Wenfeng; Sun, Ting; Cao, Yu; Li, Shaolong; Liu, Chang; Tang, Qingru

2018-05-01

Bonding repair is a powerful feature of carbon fiber-reinforced plastics (CFRP). Based on the theory of interface bonding, the interface adhesion strength and reliability of the CFRP structure will be directly affected by the microscopic features of the CFRP surface, including the microstructure, physical, and chemical characteristics. In this paper, laser-based surface modification was compared to Peel-ply, grinding, and polishing to comparatively evaluate the surface microstructure of CFRP. The surface microstructure, morphology, fiber damage, height and space parameters were investigated by scanning electron microscopy (SEM) and laser confocal microscopy (LCM). Relative to the conventional grinding process, laser modification of the CFRP surface can result in more uniform resin removal and better processing control and repeatability. This decreases the adverse impact of surface fiber fractures and secondary damage. The surface properties were significantly optimized, which has been reflected such things as the obvious improvement of surface roughness, microstructure uniformity, and actual area. The improved surface microstructure based on laser modification is more conducive to interface bonding of CFRP structure repair. This can enhance the interfacial adhesion strength and reliability of repair.

Probing thermal evanescent waves with a scattering-type near-field microscope

International Nuclear Information System (INIS)

Kajihara, Y; Kosaka, K; Komiyama, S

2011-01-01

Long wavelength infrared (LWIR) waves contain many important spectra of matters like molecular motions. Thus, probing spontaneous LWIR radiation without external illumination would reveal detailed mesoscopic phenomena that cannot be probed by any other measurement methods. Here we developed a scattering-type scanning near-field optical microscope (s-SNOM) and demonstrated passive near-field microscopy at 14.5 µm wavelength. Our s-SNOM consists of an atomic force microscope and a confocal microscope equipped with a highly sensitive LWIR detector, called a charge-sensitive infrared phototransistor (CSIP). In our s-SNOM, photons scattered by a tungsten probe are collected by an objective of the confocal LWIR microscope and are finally detected by the CSIP. To suppress the far-field background, we vertically modulated the probe and demodulated the signal with a lock-in amplifier. With the s-SNOM, a clear passive image of 3 µm pitch Au/SiC gratings was successfully obtained and the spatial resolution was estimated to be 60 nm (λ/240). The radiation from Au and GaAs was suggested to be due to thermally excited charge/current fluctuations and surface phonons, respectively. This s-SNOM has the potential to observe mesoscopic phenomena such as molecular motions, biomolecular protein interactions and semiconductor conditions in the future

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. © 2015 Institute of Food Technologists®

Fluorescence Confocal Microscopy for Ex Vivo Diagnosis of Conjunctival Tumors: A Pilot Study.

Science.gov (United States)

Iovieno, Alfonso; Longo, Caterina; De Luca, Mariacarla; Piana, Simonetta; Fontana, Luigi; Ragazzi, Moira

2016-08-01

To evaluate the potential use of fluorescence confocal microscopy (FCM) for ex vivo diagnosis and excision margin assessment of conjunctival neoplasms. Validity study. setting: Single institution. Consecutive patients with clinically suspicious conjunctival lesions. Conjunctival lesions were excised in toto using a standard "no-touch technique" by a single surgeon (A.I.). Collected specimens were examined with a commercially available laser scanning fluorescence confocal microscope after immersion in a 0.6 mM solution of acridine orange dye for 10-20 seconds. Specimens were subsequently processed with standard histologic analysis. FCM diagnosis of the nature and extension of conjunctival lesions. Sixteen consecutive patients were included in the study (11 male, 5 female; mean age 58.1 ± 26.1 years, range 10-90 years). The median time needed to process and analyze a sample with FCM was 15 minutes. Eleven of 16 lesions were identified by FCM as squamous (2 benign papillomas, 2 grade 2 conjunctival intraepithelial neoplasias, 7 in situ squamous carcinomas) and 5 as nonsquamous (1 pingueculum, 1 dermolipoma, 2 melanocytic nevi, 1 melanoma). In all cases FCM was able to detect horizontal and vertical extension of the lesion. All FCM findings were confirmed by corresponding subsequent histologic examination. FCM provides a fast ex vivo preliminary diagnosis of suspicious conjunctival lesions with good histologic details and margin assessment, and may represent a novel tool for intraoperative and postsurgical management of conjunctival tumors. This is the first study to investigate ex vivo FCM application in ophthalmology. Copyright © 2016 Elsevier Inc. All rights reserved.

3D image restoration for confocal microscopy: toward a wavelet deconvolution for the study of complex biological structures

Science.gov (United States)

Boutet de Monvel, Jacques; Le Calvez, Sophie; Ulfendahl, Mats

2000-05-01

Image restoration algorithms provide efficient tools for recovering part of the information lost in the imaging process of a microscope. We describe recent progress in the application of deconvolution to confocal microscopy. The point spread function of a Biorad-MRC1024 confocal microscope was measured under various imaging conditions, and used to process 3D-confocal images acquired in an intact preparation of the inner ear developed at Karolinska Institutet. Using these experiments we investigate the application of denoising methods based on wavelet analysis as a natural regularization of the deconvolution process. Within the Bayesian approach to image restoration, we compare wavelet denoising with the use of a maximum entropy constraint as another natural regularization method. Numerical experiments performed with test images show a clear advantage of the wavelet denoising approach, allowing to `cool down' the image with respect to the signal, while suppressing much of the fine-scale artifacts appearing during deconvolution due to the presence of noise, incomplete knowledge of the point spread function, or undersampling problems. We further describe a natural development of this approach, which consists of performing the Bayesian inference directly in the wavelet domain.

Effect of the Femtosecond Laser on an Intracorneal Inlay for Surgical Compensation of Presbyopia during Cataract Surgery: Scanning Electron Microscope Imaging.

Science.gov (United States)

Ibarz, Marta; Rodríguez-Prats, Jose Luis; Hernández-Verdejo, Jose Luis; Tañá, Pedro

2017-02-01

To investigate the effect of the femtosecond laser-assisted cataract surgery (FLACS) on porcine eyes implanted with a Kamra corneal inlay and to describe how the inlay may change the effect of the femtosecond laser on the lens. FLACS was performed on six porcine eyes and a Kamra corneal inlay had been implanted, exploring the lens under the surgical microscope. Another Kamra corneal inlay was attached to the upper part of the transparent hemisphere used for calibration of the femtosecond laser. Capsulorhexis, arcuate incisions, and phacofragmentation were carried out. The Kamra corneal inlay was compared with a nontreated one using a scanning electron microscope (SEM), and the hemisphere was analyzed with a surgical microscope. Capsulorhexis and phacofragmentation were completed in all the porcine eyes, although accuracy to determine the exact effect on the lens was not possible to achieve. The effect of the femtosecond laser on the PMMA hemisphere through the Kamra corneal inlay showed the capsulorhexis was placed outside the outer margin of the inlay and a sharply sculpted fragmentation pattern with a three-dimensional (donut-shaped) annulus untreated beneath it. SEM images of the nontreated and the treated inlays were comparable. No ultrastructural changes were found in the treated Kamra corneal inlay. FLACS can be performed with a Kamra corneal inlay for surgical compensation of presbyopia without the risk of damaging the inlay. The Kamra corneal inlay acts as a screen that avoids the laser to reach the areas beneath its shadow, but not the exposed areas of the lens.

A statistical pixel intensity model for segmentation of confocal laser scanning microscopy images.

Science.gov (United States)

Calapez, Alexandre; Rosa, Agostinho

2010-09-01

Confocal laser scanning microscopy (CLSM) has been widely used in the life sciences for the characterization of cell processes because it allows the recording of the distribution of fluorescence-tagged macromolecules on a section of the living cell. It is in fact the cornerstone of many molecular transport and interaction quantification techniques where the identification of regions of interest through image segmentation is usually a required step. In many situations, because of the complexity of the recorded cellular structures or because of the amounts of data involved, image segmentation either is too difficult or inefficient to be done by hand and automated segmentation procedures have to be considered. Given the nature of CLSM images, statistical segmentation methodologies appear as natural candidates. In this work we propose a model to be used for statistical unsupervised CLSM image segmentation. The model is derived from the CLSM image formation mechanics and its performance is compared to the existing alternatives. Results show that it provides a much better description of the data on classes characterized by their mean intensity, making it suitable not only for segmentation methodologies with known number of classes but also for use with schemes aiming at the estimation of the number of classes through the application of cluster selection criteria.

Adaptive optics plug-and-play setup for high-resolution microscopes with multi-actuator adaptive lens

Science.gov (United States)

Quintavalla, M.; Pozzi, P.; Verhaegen, Michelle; Bijlsma, Hielke; Verstraete, Hans; Bonora, S.

2018-02-01

Adaptive Optics (AO) has revealed as a very promising technique for high-resolution microscopy, where the presence of optical aberrations can easily compromise the image quality. Typical AO systems however, are almost impossible to implement on commercial microscopes. We propose a simple approach by using a Multi-actuator Adaptive Lens (MAL) that can be inserted right after the objective and works in conjunction with an image optimization software allowing for a wavefront sensorless correction. We presented the results obtained on several commercial microscopes among which a confocal microscope, a fluorescence microscope, a light sheet microscope and a multiphoton microscope.

Using confocal laser scanning microscopy to probe the milk fat globule membrane and associated proteins.

Science.gov (United States)

Gallier, Sophie; Gragson, Derek; Jiménez-Flores, Rafael; Everett, David

2010-04-14

The bovine milk fat globule membrane (MFGM) is an important, biologically relevant membrane due to its functional and health properties. Its composition has been thoroughly studied, but its structure, especially the lateral organization of its components, still remains unclear. We have used confocal laser scanning microscopy (CLSM) to investigate the surface structure of the MFGM in globules with different degrees of processing using two types of fluorescently labeled phospholipid probes and a protein dye. Using this technique, we have observed heterogeneities in the distribution of MFGM lipids and proteins relating to the processing and size of the globules. The effect of pretreating the milk (centrifugation, pasteurization-homogenization and churning) was studied by double-staining the surface of the milk fat globules, followed by observation using CLSM, and by determining the phospholipid profile of raw milk, raw cream, processed milk and buttermilk powder. Our findings agree with other techniques by showing that the composition of the MFGM changes with processing through the loss of phospholipids and the adsorption of caseins and whey proteins onto the surface.

Identification of nodal tissue in the living heart using rapid scanning fiber-optics confocal microscopy and extracellular fluorophores.

Science.gov (United States)

Huang, Chao; Kaza, Aditya K; Hitchcock, Robert W; Sachse, Frank B

2013-09-01

Risks associated with pediatric reconstructive heart surgery include injury of the sinoatrial node (SAN) and atrioventricular node (AVN), requiring cardiac rhythm management using implantable pacemakers. These injuries are the result of difficulties in identifying nodal tissues intraoperatively. Here we describe an approach based on confocal microscopy and extracellular fluorophores to quantify tissue microstructure and identify nodal tissue. Using conventional 3-dimensional confocal microscopy we investigated the microstructural arrangement of SAN, AVN, and atrial working myocardium (AWM) in fixed rat heart. AWM exhibited a regular striated arrangement of the extracellular space. In contrast, SAN and AVN had an irregular, reticulated arrangement. AWM, SAN, and AVN tissues were beneath a thin surface layer of tissue that did not obstruct confocal microscopic imaging. Subsequently, we imaged tissues in living rat hearts with real-time fiber-optics confocal microscopy. Fiber-optics confocal microscopy images resembled images acquired with conventional confocal microscopy. We investigated spatial regularity of tissue microstructure from Fourier analysis and second-order image moments. Fourier analysis of fiber-optics confocal microscopy images showed that the spatial regularity of AWM was greater than that of nodal tissues (37.5 ± 5.0% versus 24.3 ± 3.9% for SAN and 23.8 ± 3.7% for AVN; Pfiber-optics confocal microscopy. Application of the approach in pediatric reconstructive heart surgery may reduce risks of injuring nodal tissues.

Thermal maturity of Tasmanites microfossils from confocal laser scanning fluorescence microscopy

Science.gov (United States)

Hackley, Paul C.; Kus, Jolanta

2015-01-01

We report here, for the first time, spectral properties of Tasmanites microfossils determined by confocal laser scanning fluorescence microscopy (CLSM, using Ar 458 nm excitation). The Tasmanites occur in a well-characterized natural maturation sequence (Ro 0.48–0.74%) of Devonian shale (n = 3 samples) from the Appalachian Basin. Spectral property λmax shows excellent agreement (r2 = 0.99) with extant spectra from interlaboratory studies which used conventional fluorescence microscopy techniques. This result suggests spectral measurements from CLSM can be used to infer thermal maturity of fluorescent organic materials in geologic samples. Spectra of regions with high fluorescence intensity at fold apices and flanks in individual Tasmanites are blue-shifted relative to less-deformed areas in the same body that have lower fluorescence intensity. This is interpreted to result from decreased quenching moiety concentration at these locations, and indicates caution is needed in the selection of measurement regions in conventional fluorescence microscopy, where it is common practice to select high intensity regions for improved signal intensity and better signal to noise ratios. This study also documents application of CLSM to microstructural characterization of Tasmanites microfossils. Finally, based on an extant empirical relation between conventional λmax values and bitumen reflectance, λmax values from CLSM of Tasmanites microfossils can be used to calculate a bitumen reflectance equivalent value. The results presented herein can be used as a basis to broaden the future application of CLSM in the geological sciences into hydrocarbon prospecting and basin analysis.

Dimensional metrology of lab-on-a-chip internal structures: a comparison of optical coherence tomography with confocal fluorescence microscopy.

Science.gov (United States)

Reyes, D R; Halter, M; Hwang, J

2015-07-01

The characterization of internal structures in a polymeric microfluidic device, especially of a final product, will require a different set of optical metrology tools than those traditionally used for microelectronic devices. We demonstrate that optical coherence tomography (OCT) imaging is a promising technique to characterize the internal structures of poly(methyl methacrylate) devices where the subsurface structures often cannot be imaged by conventional wide field optical microscopy. The structural details of channels in the devices were imaged with OCT and analyzed with an in-house written ImageJ macro in an effort to identify the structural details of the channel. The dimensional values obtained with OCT were compared with laser-scanning confocal microscopy images of channels filled with a fluorophore solution. Attempts were also made using confocal reflectance and interferometry microscopy to measure the channel dimensions, but artefacts present in the images precluded quantitative analysis. OCT provided the most accurate estimates for the channel height based on an analysis of optical micrographs obtained after destructively slicing the channel with a microtome. OCT may be a promising technique for the future of three-dimensional metrology of critical internal structures in lab-on-a-chip devices because scans can be performed rapidly and noninvasively prior to their use. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Development of X-ray photoelectron microscope with a compact X-ray source generated by line-focused laser irradiation

International Nuclear Information System (INIS)

Yamaguchi, N.; Takahashi, Z.; Nishimura, Y.; Watanabe, K.; Okamoto, Y.; Sakata, A.; Azuma, H.; Hara, T.

2005-01-01

A laboratory-sized X-ray photoelectron microscope was constructed using a compact X-ray source produced by line-focused laser irradiation. The system is a scanning type photoelectron microscope where X-ray beam is micro-focused via Schwarzschild optics. A compact laser-plasma X-ray source has been developed with a YAG laser, a line-focus lens assembly, an Al tape-target driver and a debris prevention system. The 13.1 nm X-ray was delivered along line plasma whose length was 0.6 or 11 mm with higher intensity than that from a point-focused source. The Schwarzschild optics having the designed demagnification of 224, which was coated with Mo/Si multilayers for 13.1 nm X-ray, was set on the beamline 1 m distant from the source. The electron energy analyser was a spherical capacitor analyser with the photoelectron image detection system that was suited for detection of vast photoelectrons excited by an X-ray pulse of ns-order duration. The spatial resolution less than 5 μm has been confirmed from the variation of As 3d electron intensity along the position of the GaAs sample coated with a photo-resist test pattern

Estudio comparativo entre microscopía confocal y microscopía especular en la valoración del endotelio en córneas con distrofia de Fuchs

OpenAIRE

Charafeddin, Wissam

2011-01-01

La distròfia endotelial de Fuchs es caracteritza per la formació de guttas endotelials i en estadis avançats pot induir edema corneal i pèrdua d'agudesa visual. A diferència del microscopi especular, el microscopi confocal té un disseny que permet evitar la llum aberrant causada per l'edema corneal o opacitats estromals. Comparant ambdues probes en la valoració de l'endoteli corneal en pacients amb distròfia de Fuchs, s'observa millor qualitat d'imatge per microscòpia confocal tot i que no es...

Influence of Ultrasonic Surface Rolling on Microstructure and Wear Behavior of Selective Laser Melted Ti-6Al-4V Alloy

Directory of Open Access Journals (Sweden)

Zhen Wang

2017-10-01

Full Text Available The present article studied the effect of ultrasonic surface rolling process (USRP on the microstructure and wear behavior of a selective laser melted Ti-6Al-4V alloy. Surface characteristics were investigated using optical microscope, nano-indentation, scanning electron microscope, transmission electron microscope and laser scanning confocal microscope. Results indicated that the thickness of pore-free surfaces increased to 100~200 μm with the increasing ultrasonic surface rolling numbers. Severe work hardening occurred in the densified layer, resulting in the formation of refined grains, dislocation walls and deformation twins. After 1000 N 6 passes, about 15.5% and 14.1% increment in surficial Nano-hardness and Vickers-hardness was obtained, respectively. The hardness decreased gradually from the top surface to the substrate. Wear tests revealed that the friction coefficient declined from 0.74 (polished surface to 0.64 (USRP treated surface and the wear volume reduced from 0.205 mm−3 to 0.195 mm−3. The difference in wear volume between USRP treated and polished samples increased with sliding time. The enhanced wear resistance was concluded to be associated with the improvement of hardness and shear resistance and also the inhibition of delamination initiation.

Confocal Raman spectrocopy for the analysis of nail polish evidence.

Science.gov (United States)

López-López, Maria; Vaz, Joana; García-Ruiz, Carmen

2015-06-01

Nail polishes are cosmetic paints that may be susceptible of forensic analysis offering useful information to assist in a crime reconstruction. Although the nail polish appearance could allow a quick visual identification of the sample, this analysis is subjected to the perception and subjective interpretation of the forensic examiner. The chemical analysis of the nail polishes offers great deal of information not subjected to analyst interpretation. Confocal Raman spectroscopy is a well-suited technique for the analysis of paints due to its non-invasive and non-destructive nature and its ability to supply information about the organic and inorganic components of the sample. In this work, 77 regular and gel nail polishes were analyzed with confocal Raman spectroscopy using two laser wavelengths (532 and 780 nm). The sample behavior under the two laser wavelengths and the differences in the spectra taken at different points of the sample were studied for each nail polish. Additionally, the spectra obtained for all the nail polishes were visually compared. The results concluded that the longer laser wavelength prevents sample burning and fluorescence effects; the similarity among the spectra collected within the sample is not directly related with the presence of glitter particles; and 64% of the samples analyzed showed a characteristic spectrum. Additionally, the use of confocal Raman spectroscopy for the forensic analysis of nail polishes evidence in the form of flakes or smudges on different surfaces were studied. The results showed that both types of evidence can be analyzed by the technique. Also, two non-invasive sampling methods for the collection of the evidence from the nails of the suspect or the victim were proposed: (i) to use acetone-soaked cotton swabs to remove the nail varnishes and (ii) to scrape the nail polish from the nail with a blade. Both approaches, each exhibiting advantages and drawbacks in terms of transport and handling were appropriate

Resolution enhancement of pump-probe microscope with an inverse-annular filter

Science.gov (United States)

Kobayashi, Takayoshi; Kawasumi, Koshi; Miyazaki, Jun; Nakata, Kazuaki

2018-04-01

Optical pump-probe microscopy can provide images by detecting changes in probe light intensity induced by stimulated emission, photoinduced absorbance change, or photothermal-induced refractive index change in either transmission or reflection mode. Photothermal microscopy, which is one type of optical pump-probe microscopy, has intrinsically super resolution capability due to the bilinear dependence of signal intensity of pump and probe. We introduce new techniques for further resolution enhancement and fast imaging in photothermal microscope. First, we introduce a new pupil filter, an inverse-annular pupil filter in a pump-probe photothermal microscope, which provides resolution enhancement in three dimensions. The resolutions are proved to be improved in lateral and axial directions by imaging experiment using 20-nm gold nanoparticles. The improvement in X (perpendicular to the common pump and probe polarization direction), Y (parallel to the polarization direction), and Z (axial direction) are by 15 ± 6, 8 ± 8, and 21 ± 2% from the resolution without a pupil filter. The resolution enhancement is even better than the calculation using vector field, which predicts the corresponding enhancement of 11, 8, and 6%. The discussion is made to explain the unexpected results. We also demonstrate the photothermal imaging of thick biological samples (cells from rabbit intestine and kidney) stained with hematoxylin and eosin dye with the inverse-annular filter. Second, a fast, high-sensitivity photothermal microscope is developed by implementing a spatially segmented balanced detection scheme into a laser scanning microscope using a Galvano mirror. We confirm a 4.9 times improvement in signal-to-noise ratio in the spatially segmented balanced detection compared with that of conventional detection. The system demonstrates simultaneous bi-modal photothermal and confocal fluorescence imaging of transgenic mouse brain tissue with a pixel dwell time of 20 µs. The

Design and operation of an inexpensive far-field laser scanning microscope suitable for use in an undergraduate laboratory course

Science.gov (United States)

Pallone, Arthur; Hawk, Eric

2013-03-01

Scanning microscope applications span the science disciplines yet their costs limit their use at educational institutions. The basic concepts of scanning microscopy are simple. The microscope probe - whether it produces a photon, electron or ion beam - moves relative to the surface of the sample object. The beam interacts with the sample to produce a detected signal that depends on the desired property to be measured at the probe location on the sample. The microscope transforms the signal for output in a form desired by the user. Undergraduate students can easily construct a far-field laser scanning microscope that illustrates each of these principles from parts available at local electronics and hardware stores and use the microscope to explore properties of devices such as light dependent resistors and biological samples such as leaves. Students can record, analyze and interpret results using a computer and free software.

Confocal multispot microscope for fast and deep imaging in semicleared tissues

Science.gov (United States)

Adam, Marie-Pierre; Müllenbroich, Marie Caroline; Di Giovanna, Antonino Paolo; Alfieri, Domenico; Silvestri, Ludovico; Sacconi, Leonardo; Pavone, Francesco Saverio

2018-02-01

Although perfectly transparent specimens are imaged faster with light-sheet microscopy, less transparent samples are often imaged with two-photon microscopy leveraging its robustness to scattering; however, at the price of increased acquisition times. Clearing methods that are capable of rendering strongly scattering samples such as brain tissue perfectly transparent specimens are often complex, costly, and time intensive, even though for many applications a slightly lower level of tissue transparency is sufficient and easily achieved with simpler and faster methods. Here, we present a microscope type that has been geared toward the imaging of semicleared tissue by combining multispot two-photon excitation with rolling shutter wide-field detection to image deep and fast inside semicleared mouse brain. We present a theoretical and experimental evaluation of the point spread function and contrast as a function of shutter size. Finally, we demonstrate microscope performance in fixed brain slices by imaging dendritic spines up to 400-μm deep.

Ultrasonically synthesized organic liquid-filled chitosan microcapsules: part 2: characterization using AFM (atomic force microscopy) and combined AFM-confocal laser scanning fluorescence microscopy.

Science.gov (United States)

Mettu, Srinivas; Ye, Qianyu; Zhou, Meifang; Dagastine, Raymond; Ashokkumar, Muthupandian

2018-04-25

Atomic Force Microscopy (AFM) is used to measure the stiffness and Young's modulus of individual microcapsules that have a chitosan cross-linked shell encapsulating tetradecane. The oil filled microcapsules were prepared using a one pot synthesis via ultrasonic emulsification of tetradecane and crosslinking of the chitosan shell in aqueous solutions of acetic acid. The concentration of acetic acid in aqueous solutions of chitosan was varied from 0.2% to 25% v/v. The effect of acetic acid concentration and size of the individual microcapsules on the strength was probed. The deformations and forces required to rupture the microcapsules were also measured. Three dimensional deformations of microcapsules under large applied loads were obtained by the combination of Laser Scanning Confocal Microscopy (LSCM) with Atomic Force Microscopy (AFM). The stiffness, and hence the modulus, of the microcapsules was found to decrease with an increase in size with the average stiffness ranging from 82 to 111 mN m-1 and average Young's modulus ranging from 0.4 to 6.5 MPa. The forces required to rupture the microcapsules varied from 150 to 250 nN with deformations of the microcapsules up to 62 to 110% relative to their radius, respectively. Three dimensional images obtained using laser scanning confocal microscopy showed that the microcapsules retained their structure and shape after being subjected to large deformations and subsequent removal of the loads. Based on the above observations, the oil filled chitosan crosslinked microcapsules are an ideal choice for use in the food and pharmaceutical industries as they would be able to withstand the process conditions encountered.

Nondestructive 3D confocal laser imaging with deconvolution of seven whole stardust tracks with complementary XRF and quantitative analysis

International Nuclear Information System (INIS)

Greenberg, M.; Ebel, D.S.

2009-01-01

We present a nondestructive 3D system for analysis of whole Stardust tracks, using a combination of Laser Confocal Scanning Microscopy and synchrotron XRF. 3D deconvolution is used for optical corrections, and results of quantitative analyses of several tracks are presented. The Stardust mission to comet Wild 2 trapped many cometary and ISM particles in aerogel, leaving behind 'tracks' of melted silica aerogel on both sides of the collector. Collected particles and their tracks range in size from submicron to millimeter scale. Interstellar dust collected on the obverse of the aerogel collector is thought to have an average track length of ∼15 (micro)m. It has been our goal to perform a total non-destructive 3D textural and XRF chemical analysis on both types of tracks. To that end, we use a combination of Laser Confocal Scanning Microscopy (LCSM) and X Ray Florescence (XRF) spectrometry. Utilized properly, the combination of 3D optical data and chemical data provides total nondestructive characterization of full tracks, prior to flattening or other destructive analysis methods. Our LCSM techniques allow imaging at 0.075 (micro)m/pixel, without the use of oil-based lenses. A full textural analysis on track No.82 is presented here as well as analysis of 6 additional tracks contained within 3 keystones (No.128, No.129 and No.140). We present a method of removing the axial distortion inherent in LCSM images, by means of a computational 3D Deconvolution algorithm, and present some preliminary experiments with computed point spread functions. The combination of 3D LCSM data and XRF data provides invaluable information, while preserving the integrity of the samples for further analysis. It is imperative that these samples, the first extraterrestrial solids returned since the Apollo era, be fully mapped nondestructively in 3D, to preserve the maximum amount of information prior to other, destructive analysis.

Diving under a microscope--a new simple and versatile in vitro diving device for fluorescence and confocal microscopy allowing the controls of hydrostatic pressure, gas pressures, and kinetics of gas saturation.

Science.gov (United States)

Wang, Qiong; Belhomme, Marc; Guerrero, François; Mazur, Aleksandra; Lambrechts, Kate; Theron, Michaël

2013-06-01

How underwater diving effects the function of the arterial wall and the activities of endothelial cells is the focus of recent studies on decompression sickness. Here we describe an in vitro diving system constructed to achieve real-time monitoring of cell activity during simulated dives under fluorescent microscopy and confocal microscopy. A 1-mL chamber with sapphire windows on both sides and located on the stage of an inverted microscope was built to allow in vitro diving simulation of isolated cells or arteries in which activities during diving are monitored in real-time via fluorescent microscopy and confocal microscopy. Speed of compression and decompression can range from 20 to 2000 kPa/min, allowing systemic pressure to range up to 6500 kPa. Diving temperature is controlled at 37°C. During air dive simulation oxygen partial pressure is optically monitored. Perfusion speed can range from 0.05 to 10 mL/min. The system can support physiological viability of in vitro samples for real-time monitoring of cellular activity during diving. It allows regulations of pressure, speeds of compression and decompression, temperature, gas saturation, and perfusion speed. It will be a valuable tool for hyperbaric research.

Observation of microorganism colonies using a scanning-laser-beam pH-sensing microscope

International Nuclear Information System (INIS)

Nakao, M.; Inoue, S.; Oishi, R.; Yoshinobu, T.; Iwasaki, H.

1995-01-01

The extracellular pH-distribution of colonies of Saccharomyces cerevisiae (yeast) and Escherichia coli (E. coli) were observed using a newly-developed scanning-laser-beam pH-sensing microscope. Colonies were incubated either on top of agarose plates or between the pH-sensing surface and the agar. In the latter case, colony growth was observed in-situ. The colonies could be observed within a period as short as 8 h for E. coli. The pH-distribution profiles by the colonies were found to be very sharp, in agreement with simulation results. (author)

Cheese Matrix Microstructure Studied by Advanced Microscopic Techniques

Czech Academy of Sciences Publication Activity Database

Burdiková, Z.; Hickey, C.; Auty, M. A. E.; Pala, J.; Švindrych, Z.; Steinmetz, I.; Krzyžánek, Vladislav; Hrubanová, Kamila; Sheehan, J. J.

2014-01-01

Roč. 20, S3 (2014), s. 1336-1337 ISSN 1431-9276 R&D Projects: GA MŠk(CZ) LO1212 Institutional support: RVO:68081731 Keywords : cheese matrix * cryo-SEM * confocal laser scanning microscopy Subject RIV: EA - Cell Biology Impact factor: 1.877, year: 2014

Mechanisms of biliary stent clogging: confocal laser scanning and scanning electron microscopy.

Science.gov (United States)

van Berkel, A M; van Marle, J; Groen, A K; Bruno, M J

2005-08-01

Endoscopic insertion of plastic biliary endoprostheses is a well-established treatment for obstructive jaundice. The major limitation of this technique is late stent occlusion. In order to compare events involved in biliary stent clogging and identify the distribution of bacteria in unblocked stents, confocal laser scanning (CLS) and scanning electron microscopy (SEM) were carried out on two different stent materials - polyethylene (PE) and hydrophilic polymer-coated polyurethane (HCPC). Ten consecutive patients with postoperative benign biliary strictures were included in the study. Two 10-Fr stents 9 cm in length, one made of PE and the other of HCPC, were inserted. The stents were electively exchanged after 3 months and examined using CLS and SEM. No differences were seen between the two types of stent. The inner stent surface was covered with a uniform amorphous layer. On top of this layer, a biofilm of living and dead bacteria was found, which in most cases was unstructured. The lumen was filled with free-floating colonies of bacteria and crystals, surrounded by mobile laminar structures of mucus. An open network of large dietary fibers was seen in all of the stents. The same clogging events occurred in both PE and HCPC stents. The most remarkable observation was the identification of networks of large dietary fibers, resulting from duodenal reflux, acting as a filter. The build-up of this intraluminal framework of dietary fibers appears to be a major factor contributing to the multifactorial process of stent clogging.

Detection of fluorescent organic nanoparticles by confocal laser endomicroscopy in a rat model of Barrett’s esophageal adenocarcinoma

Directory of Open Access Journals (Sweden)

Dassie E

2015-10-01

. Non-operated and operated rats in which gastroesophageal reflux was surgically induced received both types of NPs (NP-DCM and NP-DCM-ASYNYDA by intravenous route. Localization of mucosal NPs was assessed in vivo by confocal laser endomicroscopy, a technique which enables a “real time” and in situ visualization of the tissue at a cellular level. After injection of NP-DCM and NP-DCM-ASYNYDA, fluorescence was observed in rats affected by esophageal cancer, whereas no signal was observed in control non-operated rats, or in rats with simple esophagitis or Barrett’s esophagus mucosa. Fluorescence was observable in vivo 30 minutes after the administration of NPs. Interestingly, NP-DCM-ASYNYDA induced strong fluorescence intensity 24 hours after administration. These observations suggested that NPs could reach the tumor cells, likely by enhanced permeability and retention effect, and the peptide ASYNYDA gave them high specificity for esophageal cancer cells. Thus, the combination of NP platform and confocal laser endomicroscopy could play an important role for highlighting esophageal cancer conditions. This result supports the potential of this strategy as a targeted carrier for photoactive and bioactive molecules in esophageal cancer diagnosis and treatment. Keywords: confocal laser endomicroscopy, Barrett’s esophagus, diagnostics, esophageal adenocarcinoma, fluorescent nanoparticles, heptapeptide

Evaluation of Wear on Macro-Surface Textures Generated by ns Fiber Laser

Science.gov (United States)

Harish, V.; Soundarapandian, S.; Vijayaraghavan, L.; Bharatish, A.

2018-03-01

The demand for improved performance and long term reliability of mechanical systems dictate the use of advanced materials and surface engineering techniques. A small change in the surface topography can lead to substantial improvements in the tribological behaviour of the contact surfaces. One way of altering the surface topography is by surface texturing by introducing dimples or channels on the surfaces. Surface texturing is already a successful technique which finds a wide area of applications ranging from heavy industries to small scale devices. This paper reports the effect of macro texture shapes generated using a nanosecond fiber laser on wear of high carbon chromium steel used in large size bearings having rolling contacts. Circular and square shaped dimples were generated on the surface to assess the effect of sliding velocities on friction coefficient. Graphite was used as solid lubricant to minimise the effect of wear on textured surfaces. The laser parameters such as power, scan speed and passes were optimised to obtain macro circular and square dimples which was characterised using a laser confocal microscope. The friction coefficients of the circular and square dimples were observed to lie in the same range due to minimum wear on the surface. On the contrary, at medium and higher sliding velocities, square dimples exhibited lower friction coefficient values compared to circular dimples. The morphology of textured specimen was characterised using Scanning Electron Microscope.

Comparison between laser terahertz emission microscope and conventional methods for analysis of polycrystalline silicon solar cell

Directory of Open Access Journals (Sweden)

Hidetoshi Nakanishi

2015-11-01

Full Text Available A laser terahertz emission microscope (LTEM can be used for noncontact inspection to detect the waveforms of photoinduced terahertz emissions from material devices. In this study, we experimentally compared the performance of LTEM with conventional analysis methods, e.g., electroluminescence (EL, photoluminescence (PL, and laser beam induced current (LBIC, as an inspection method for solar cells. The results showed that LTEM was more sensitive to the characteristics of the depletion layer of the polycrystalline solar cell compared with EL, PL, and LBIC and that it could be used as a complementary tool to the conventional analysis methods for a solar cell.

[Revealing the chemical changes of tea cell wall induced by anthracnose with confocal Raman microscopy].

Science.gov (United States)

Li, Xiao-li; Luo, Liu-bin; Hu, Xiao-qian; Lou, Bing-gan; He, Yong

2014-06-01

Healthy tea and tea infected by anthracnose were first studied by confocal Raman microscopy to illustrate chemical changes of cell wall in the present paper. Firstly, Raman spectra of both healthy and infected sample tissues were collected with spatial resolution at micron-level, and ultrastructure of healthy and infected tea cells was got from scanning electron microscope. These results showed that there were significant changes in Raman shift and Raman intensity between healthy and infected cell walls, indicating that great differences occurred in chemical compositions of cell walls between healthy and infected samples. In details, intensities at many Raman bands which were closely associated with cellulose, pectin, esters were reduced after infection, revealing that the content of chemical compounds such as cellulose, pectin, esters was decreased after infection. Subsequently, chemical imaging of both healthy and infected tea cell walls were realized based on Raman fingerprint spectra of cellulose and microscopic spatial structure. It was found that not only the content of cellulose was reduced greatly after infection, but also the ordered structure of cellulose was destroyed by anthracnose infection. Thus, confocal Raman microscopy was shown to be a powerful tool to detect the chemical changes in cell wall of tea caused by anthracnose without any chemical treatment or staining. This research firstly applied confocal Raman microscopy in phytopathology for the study of interactive relationship between host and pathogen, and it will also open a new way for intensive study of host-pathogen at cellular level.

Fluorescence confocal endomicroscopy in biological imaging

Science.gov (United States)

Delaney, Peter; Thomas, Steven; Allen, John; McLaren, Wendy; Murr, Elise; Harris, Martin

2007-02-01

In vivo fluorescence microscopic imaging of biological systems in human disease states and animal models is possible with high optical resolution and mega pixel point-scanning performance using optimised off-the-shelf turn-key devices. There are however various trade-offs between tissue access and instrument performance when miniaturising in vivo microscopy systems. A miniature confocal scanning technology that was developed for clinical human endoscopy has been configured into a portable device for direct hand-held interrogation of living tissue in whole animal models (Optiscan FIVE-1 system). Scanning probes of 6.3mm diameter with a distal tip diameter of 5.0mm were constructed either in a 150mm length for accessible tissue, or a 300mm probe for laparoscopic interrogation of internal tissues in larger animal models. Both devices collect fluorescence confocal images (excitation 488 nm; emission >505 or >550 nm) comprised of 1024 x 1204 sampling points/image frame, with lateral resolution 0.7um; axial resolution 7um; FOV 475 x 475um. The operator can dynamically control imaging depth from the tissue surface to approx 250um in 4um steps via an internally integrated zaxis actuator. Further miniaturisation is achieved using an imaging contact probe based on scanning the proximal end of a high-density optical fibre bundle (~30,000 fibres) of small animal organs, albeit at lower resolution (30,000 sampling points/image). In rodent models, imaging was performed using various fluorescent staining protocols including fluorescently labelled receptor ligands, labelled antibodies, FITC-dextrans, vital dyes and labelled cells administered topically or intravenously. Abdominal organs of large animals were accessed laparoscopically and contrasted using i.v. fluorescein-sodium. Articular cartilage of sheep and pigs was fluorescently stained with calcein-AM or fluorescein. Surface and sub-surface cellular and sub-cellular details could be readily visualised in vivo at high

Automated Image Analysis Corrosion Working Group Update: February 1, 2018

Energy Technology Data Exchange (ETDEWEB)

Wendelberger, James G. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

2018-02-01

These are slides for the automated image analysis corrosion working group update. The overall goals were: automate the detection and quantification of features in images (faster, more accurate), how to do this (obtain data, analyze data), focus on Laser Scanning Confocal Microscope (LCM) data (laser intensity, laser height/depth, optical RGB, optical plus laser RGB).

The development of laser surgery and medicine in China

Science.gov (United States)

Chen, Mingzhe

2005-07-01

The first Chinese ruby laser was created in 1961 and it was adopted for the retina coagulation experiment in 1965. Since 1970's, lasers had been widely applied clinically including the diseases suitable to physical therapy or acupuncture. The Chinese HpD was first produced in 1981 and first case of PDT was treated using Chinese HpD and Chinese lasers in the same year. Its success brought attention establishing a research group supported by the government in 1982. A nationwide systemic research project on PDT was then carried out. The step taken for PDT also accelerated the development of various fields of laser medicine and surgery. Laser treatments had been commonly adopted in the clinics and hospitals for the diseases of the superficial lesions and the lesions can be reached by the endoscopes non-invasively in 1980's. Since 1990's, the interventional laser therapies adopted mainly were percutaneous laser angioplasty, laser treatments through laparoscope, thoracoscope, arthroscope, neuro-endoscope etc. Ultrasound guided percutaneous laser heat coagulation for small hepatic cancer revealed good results and ultrasound guided percutaneous PDT for advanced large liver cancer revealed unexpected results after five years follow-up. At present: There are more long-term follow-up patients in the clinical trial; more advanced commercial available lasers and new techniques are adopted. Since the popularization of scanning electron microscope, laser scanning confocal microscope, laser induced auto-fluorescence system, high sensitivity fluorescence microscopic imaging system etc. in the laboratories, the basic studies can be more advanced and some times, the sub-cellular level can be reached; ultra-structure histo-morphology and gene studies are involved. In dermatology, Q-switched Alexandrite laser and other Q-switched lasers are used mainly for the treatment of skin pigmentation and vascular diseases; pulsed dye laser, ultra-pulsed CO2 laser are used in resurfacing, facial

Portable oral cancer detection using a miniature confocal imaging probe with a large field of view

Science.gov (United States)

Wang, Youmin; Raj, Milan; McGuff, H. Stan; Bhave, Gauri; Yang, Bin; Shen, Ting; Zhang, Xiaojing

2012-06-01

We demonstrate a MEMS micromirror enabled handheld confocal imaging probe for portable oral cancer detection, where a comparatively large field of view (FOV) was generated through the programmable Lissajous scanning pattern of the MEMS micromirror. Miniaturized handheld MEMS confocal imaging probe was developed, and further compared with the desktop confocal prototype under clinical setting. For the handheld confocal imaging system, optical design simulations using CODE VR® shows the lateral and axial resolution to be 0.98 µm and 4.2 µm, where experimental values were determined to be 3 µm and 5.8 µm, respectively, with a FOV of 280 µm×300 µm. Fast Lissajous imaging speed up to 2 fps was realized with improved Labview and Java based real-time imaging software. Properties such as 3D imaging through autofocusing and mosaic imaging for extended lateral view (6 mm × 8 mm) were examined for carcinoma real-time pathology. Neoplastic lesion tissues of giant cell fibroma and peripheral ossifying fibroma, the fibroma inside the paraffin box and ex vivo gross tissues were imaged by the bench-top and handheld imaging modalities, and further compared with commercial microscope imaging results. The MEMS scanner-based handheld confocal imaging probe shows great promise as a potential clinical tool for oral cancer diagnosis and treatment.

Portable oral cancer detection using a miniature confocal imaging probe with a large field of view

International Nuclear Information System (INIS)

Wang, Youmin; Raj, Milan; Bhave, Gauri; Yang, Bin; Zhang, Xiaojing; McGuff, H. Stan; Shen, Ting

2012-01-01

We demonstrate a MEMS micromirror enabled handheld confocal imaging probe for portable oral cancer detection, where a comparatively large field of view (FOV) was generated through the programmable Lissajous scanning pattern of the MEMS micromirror. Miniaturized handheld MEMS confocal imaging probe was developed, and further compared with the desktop confocal prototype under clinical setting. For the handheld confocal imaging system, optical design simulations using CODE V R® shows the lateral and axial resolution to be 0.98 µm and 4.2 µm, where experimental values were determined to be 3 µm and 5.8 µm, respectively, with a FOV of 280 µm×300 µm. Fast Lissajous imaging speed up to 2 fps was realized with improved Labview and Java based real-time imaging software. Properties such as 3D imaging through autofocusing and mosaic imaging for extended lateral view (6 mm × 8 mm) were examined for carcinoma real-time pathology. Neoplastic lesion tissues of giant cell fibroma and peripheral ossifying fibroma, the fibroma inside the paraffin box and ex vivo gross tissues were imaged by the bench-top and handheld imaging modalities, and further compared with commercial microscope imaging results. The MEMS scanner-based handheld confocal imaging probe shows great promise as a potential clinical tool for oral cancer diagnosis and treatment. (paper)

First images from the Stanford tabletop scanning soft x-ray microscope

International Nuclear Information System (INIS)

Trail, J.A.; Byer, R.L.

1988-01-01

The authors have constructed a scanning soft x-ray microscope which uses a laser-produced plasma as the soft x-ray source and normal incidence multilayer coated mirrors in a Schwarzschild configuration as the focusing optics. The microscope operates at a wavelength of 140 angstrom, has a spatial resolution of 0.5 μm, and has a soft x-ray photon flux through the focus of 10 4 s -1 when operated with only 170 mW of average laser power. The microscope is compact; the complete system, including the laser, fits on a single optical table. In this paper they describe the microscope and present images of metallic microstructures

Real-Time Demonstration of Split Skin Graft Inosculation and Integra Dermal Matrix Neovascularization Using Confocal Laser Scanning Microscopy

Science.gov (United States)

Greenwood, John; Amjadi, Mahyar; Dearman, Bronwyn; Mackie, Ian

2009-01-01

Objectives: During the first 48 hours after placement, an autograft “drinks” nutrients and dissolved oxygen from fluid exuding from the underlying recipient bed (“plasmatic imbibition”). The theory of inosculation (that skin grafts subsequently obtain nourishment via blood vessel “anastomosis” between new vessels invading from the wound bed and existing graft vessels) was hotly debated from the late 19th to mid-20th century. This study aimed to noninvasively observe blood flow in split skin grafts and Integra™ dermal regeneration matrix to provide further proof of inosculation and to contrast the structure of vascularization in both materials, reflecting mechanism. Methods: Observations were made both clinically and using confocal microscopy on normal skin, split skin graft, and Integra™. The VivaScope™ allows noninvasive, real-time, in vivo images of tissue to be obtained. Results: Observations of blood flow and tissue architecture in autologous skin graft and Integra™ suggest that 2 very different processes are occurring in the establishment of circulation in each case. Inosculation provides rapid circulatory return to skin grafts whereas slower neovascularization creates an unusual initial Integra™ circulation. Conclusions: The advent of confocal laser microscopy like the VivaScope 1500™, together with “virtual” journals such as ePlasty, enables us to provide exciting images and distribute them widely to a “reading” audience. The development of the early Integra™ vasculature by neovascularization results in a large-vessel, high-volume, rapid flow circulation contrasting markedly from the inosculatory process in skin grafts and the capillary circulation in normal skin and merits further (planned) investigation. PMID:19787028

Topotactic changes on η-Mo4O11 caused by biased atomic force microscope tip and cw-laser

Science.gov (United States)

Borovšak, Miloš; Šutar, Petra; Goreshnik, Evgeny; Mihailovic, Dragan

2015-11-01

We present topotactic changes on Mo4O11 crystals induced by a biased atomic force microscope tip and continuous laser. The transformation does not change the topography of the samples, while the surface potential shows remarkable changes on areas where the biased AFM tip was applied. No structural changes were observed by Raman spectroscopy, but AFM scans revealed changes to surface potential due to laser illumination. The observed phenomenon could be potentially useful for memristive memory devices considering the fact that properties of other molybdenum oxides vary from metallic to insulators.

Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles.

Science.gov (United States)

Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

2016-01-01

Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs.

Poly(diacetylene) Monolayers Studied with a Fluorescence Scanning Near-Field Optical Microscope

NARCIS (Netherlands)

Moers, Marco H.P.; Moers, M.H.P.; Gaub, Hermann E.; van Hulst, N.F.

1994-01-01

A novel and powerful method to study the optical properties of thin lipid films which a resolution superior to confocal microscopy is presented. With a scanning near-field optical microscope, fluorescence images of a Langmuir-Blodgett film of diethylene glycol diamine pentacosadiynoic amide are

Confocal microscopy and spectroscopy of nanocrystals on a high-Q microsphere resonator

International Nuclear Information System (INIS)

Goetzinger, S; Menezes, L de S; Benson, O; Talapin, D V; Gaponik, N; Weller, H; Rogach, A L; Sandoghdar, V

2004-01-01

We report on experiments where we used a home-made confocal microscope to excite single nanocrystals on a high-Q microsphere resonator. In that way spectra of an individual quantum emitter could be recorded. The Q factor of the microspheres coated with nanocrystals was still up to 10 9 . We also demonstrate the use of a prism coupler as a well-defined output port to collect the fluorescence of an ensemble of nanocrystals coupled to whispering-gallery modes

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning.

Science.gov (United States)

Perner, Birgit; Schnerwitzki, Danny; Graf, Michael; Englert, Christoph

2016-04-07

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development. The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed. In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance. The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

Ex vivo confocal microscopy: a new diagnostic technique for mucormycosis.

Science.gov (United States)

Leclercq, A; Cinotti, E; Labeille, B; Perrot, J L; Cambazard, F

2016-05-01

Skin-dedicated ex vivo confocal microscopy (EVCM) has so far mainly been employed to identify cutaneous tumours on freshly excised samples. We present two cases where EVCM has been used to diagnose cutaneous mucormycosis. The skin biopsies were evaluated by the skin-dedicated ex vivo confocal microscope VivaScope 2500(®) (MAVIG GmbH, Munich Germany) under both reflectance and fluorescence mode. Conventional direct optical examination on skin scraping and histological examination were later performed. Mucormycetes observed by EVCM presented as hyper-reflective elongated 20 μm in diameter structures with perpendicular ramifications. Fungi were found both under reflectance and fluorescence mode and were better visible with acridine orange under fluorescence EVCM. Conventional direct optical examination on skin scraping and histological examination found the same elongated and branching structures confirming the presence of Mucormycetes. Ex vivo confocal microscopy has both the advantages of being fast as the direct optical examination, and to be able to show the localisation of the fungi in the tissue like the histological examination. In our cases, EVCM allowed to rapidly confirm the clinical diagnosis of mucormycosis, which is essential for the treatment of this fungal infection. Further studies are needed to compare the performance of EVCM with the findings of conventional histological and mycological examinations. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Intracellular photoinduced oxidative stress by zinc phthalocyanine photosensitization: a study of the early events in real time using confocal microscopy

Science.gov (United States)

Alexandratou, Eleni; Yova, Dido; Handris, Panagiotis; Kletsas, Dimitris; Loukas, Spyros

2003-10-01

Oxidative stress has been implicated in several biological and pathological aspects. Reactive oxygen species (ROS) have been proposed to act as signal transduction molecules activating reactions leading to cell rescue or to cell apoptosis/necrosis. In the present study, oxidative stress was induced by photosensitization of zinc phthalocyanine (ZnPc) in human fibroblasts using a photodynamic dose that did not lead to apoptosis or necrosis. The induction of oxidative stress was performed at the microscope stage in preassigned time. The cascade of phenomena evoked was studied in real time and at the single cell level using confocal laser scanning microscopy. Using specific vital fluorescent probes, alterations induced by oxidative stress in mitochondria membrane potential, in intracellular pH and in calcium concentration were recorded. Image processing and analysis techniques were used to quantify the observed changes. Subcellular localization of the photosensitizer was studied in order to determine the primary and immediate ROS target. It was found that ZnPc is mainly localized in the mitochondria region.

Proper alignment of the microscope.

Science.gov (United States)

Rottenfusser, Rudi

2013-01-01

The light microscope is merely the first element of an imaging system in a research facility. Such a system may include high-speed and/or high-resolution image acquisition capabilities, confocal technologies, and super-resolution methods of various types. Yet more than ever, the proverb "garbage in-garbage out" remains a fact. Image manipulations may be used to conceal a suboptimal microscope setup, but an artifact-free image can only be obtained when the microscope is optimally aligned, both mechanically and optically. Something else is often overlooked in the quest to get the best image out of the microscope: Proper sample preparation! The microscope optics can only do its job when its design criteria are matched to the specimen or vice versa. The specimen itself, the mounting medium, the cover slip, and the type of immersion medium (if applicable) are all part of the total optical makeup. To get the best results out of a microscope, understanding the functions of all of its variable components is important. Only then one knows how to optimize these components for the intended application. Different approaches might be chosen to discuss all of the microscope's components. We decided to follow the light path which starts with the light source and ends at the camera or the eyepieces. To add more transparency to this sequence, the section up to the microscope stage was called the "Illuminating Section", to be followed by the "Imaging Section" which starts with the microscope objective. After understanding the various components, we can start "working with the microscope." To get the best resolution and contrast from the microscope, the practice of "Koehler Illumination" should be understood and followed by every serious microscopist. Step-by-step instructions as well as illustrations of the beam path in an upright and inverted microscope are included in this chapter. A few practical considerations are listed in Section 3. Copyright © 2013 Elsevier Inc. All rights

Determination of the parameters of a microscopic object from a complex response of a differential microscope

International Nuclear Information System (INIS)

Baranov, D V; Egorov, Alexander A; Zolotov, Evgenii M; Svidzinsky, K K

1998-01-01

An analysis of the amplitude and phase of a complex response of a heterodyne differential microscope was used to demonstrate experimentally the feasibility of determination of the parameters of a composite microscopic object representing a combination of a step with a groove. (laser applications and other topics in quantum electronics)

3D Volumetric Analysis of Fluid Inclusions Using Confocal Microscopy

Science.gov (United States)

Proussevitch, A.; Mulukutla, G.; Sahagian, D.; Bodnar, B.

2009-05-01

Fluid inclusions preserve valuable information regarding hydrothermal, metamorphic, and magmatic processes. The molar quantities of liquid and gaseous components in the inclusions can be estimated from their volumetric measurements at room temperatures combined with knowledge of the PVTX properties of the fluid and homogenization temperatures. Thus, accurate measurements of inclusion volumes and their two phase components are critical. One of the greatest advantages of the Laser Scanning Confocal Microscopy (LSCM) in application to fluid inclsion analsyis is that it is affordable for large numbers of samples, given the appropriate software analysis tools and methodology. Our present work is directed toward developing those tools and methods. For the last decade LSCM has been considered as a potential method for inclusion volume measurements. Nevertheless, the adequate and accurate measurement by LSCM has not yet been successful for fluid inclusions containing non-fluorescing fluids due to many technical challenges in image analysis despite the fact that the cost of collecting raw LSCM imagery has dramatically decreased in recent years. These problems mostly relate to image analysis methodology and software tools that are needed for pre-processing and image segmentation, which enable solid, liquid and gaseous components to be delineated. Other challenges involve image quality and contrast, which is controlled by fluorescence of the material (most aqueous fluid inclusions do not fluoresce at the appropriate laser wavelengths), material optical properties, and application of transmitted and/or reflected confocal illumination. In this work we have identified the key problems of image analysis and propose some potential solutions. For instance, we found that better contrast of pseudo-confocal transmitted light images could be overlayed with poor-contrast true-confocal reflected light images within the same stack of z-ordered slices. This approach allows one to narrow

Simple concept for a wide-field lensless digital holographic microscope using a laser diode

Directory of Open Access Journals (Sweden)

Adinda-Ougba A.

2015-09-01

Full Text Available Wide-field, lensless digital holographic microscopy is a new microscopic imaging technique for telemedicine and for resource limited setting [1]. In this contribution we propose a very simple wide-field lensless digital holographic microscope using a laser diode. It is based on in-line digital holography which is capable to provide amplitude and phase images of a sample resulting from numerical reconstruction. The numerical reconstruction consists of the angular spectrum propagation method together with a phase retrieval algorithm. Amplitude and phase images of the sample with a resolution of ∽2 µm and with ∽24 mm2 field of view are obtained. We evaluate our setup by imaging first the 1951 USAF resolution test chart to verify the resolution. Second, we record holograms of blood smear and diatoms. The individual specimen can be easily identified after the numerical reconstruction. Our system is a very simple, compact and low-cost possibility of realizing a microscope capable of imaging biological samples. The availability of the phase provide topographic information of the sample extending the application of this system to be not only for biological sample but also for transparent microstructure. It is suitable for fault detection, shape and roughness measurements of these structures.

In vivo confocal microscopy in dermatology: from research to clinical application

Science.gov (United States)

Ulrich, Martina; Lange-Asschenfeldt, Susanne

2013-06-01

Confocal laser scanning microscopy (CLSM) represents an emerging technique for the noninvasive histomorphological analysis of skin in vivo and has shown its applicability for dermatological research as well as its value as an adjunct tool in the clinical management of skin cancer patients. Herein, we aim to give an overview on the current clinical indications for CLSM in dermatology and also highlight the diverse applications of CLSM in dermatological research.

Visualization of femtosecond laser pulse-induced microincisions inside crystalline lens tissue.

Science.gov (United States)

Stachs, Oliver; Schumacher, Silvia; Hovakimyan, Marine; Fromm, Michael; Heisterkamp, Alexander; Lubatschowski, Holger; Guthoff, Rudolf

2009-11-01

To evaluate a new method for visualizing femtosecond laser pulse-induced microincisions inside crystalline lens tissue. Laser Zentrum Hannover e.V., Hannover, Germany. Lenses removed from porcine eyes were modified ex vivo by femtosecond laser pulses (wavelength 1040 nm, pulse duration 306 femtoseconds, pulse energy 1.0 to 2.5 microJ, repetition rate 100 kHz) to create defined planes at which lens fibers separate. The femtosecond laser pulses were delivered by a 3-dimension (3-D) scanning unit and transmitted by focusing optics (numerical aperture 0.18) into the lens tissue. Lens fiber orientation and femtosecond laser-induced microincisions were examined using a confocal laser scanning microscope (CLSM) based on a Rostock Cornea Module attached to a Heidelberg Retina Tomograph II. Optical sections were analyzed in 3-D using Amira software (version 4.1.1). Normal lens fibers showed a parallel pattern with diameters between 3 microm and 9 microm, depending on scanning location. Microincision visualization showed different cutting effects depending on pulse energy of the femtosecond laser. The effects ranged from altered tissue-scattering properties with all fibers intact to definite fiber separation by a wide gap. Pulse energies that were too high or overlapped too tightly produced an incomplete cutting plane due to extensive microbubble generation. The 3-D CLSM method permitted visualization and analysis of femtosecond laser pulse-induced microincisions inside crystalline lens tissue. Thus, 3-D CLSM may help optimize femtosecond laser-based procedures in the treatment of presbyopia.

3-D laser confocal microscopy study of the oxidation of NdFeB magnets in atmospheric conditions

Science.gov (United States)

Meakin, J. P.; Speight, J. D.; Sheridan, R. S.; Bradshaw, A.; Harris, I. R.; Williams, A. J.; Walton, A.

2016-08-01

Neodymium iron boron (NdFeB) magnets are used in a number of important applications, such as generators in gearless wind turbines, motors in electric vehicles and electronic goods (e.g.- computer hard disk drives, HDD). Hydrogen can be used as a processing gas to separate and recycle scrap sintered Nd-Fe-B magnets from end-of-life products to form a powder suitable for recycling. However, the magnets are likely to have been exposed to atmospheric conditions prior to processing, and any oxidation could lead to activation problems for the hydrogen decrepitation reaction. Many previous studies on the oxidation of NdFeB magnets have been performed at elevated temperatures; however, few studies have been formed under atmospheric conditions. In this paper a combination of 3-D laser confocal microscopy and Raman spectroscopy have been used to assess the composition, morphology and rate of oxidation/corrosion on scrap sintered NdFeB magnets. Confocal microscopy has been employed to measure the growth of surface reaction products at room temperature, immediately after exposure to air. The results showed that there was a significant height increase at the triple junctions of the Nd-rich grain boundaries. Using Raman spectroscopy, the product was shown to consist of Nd2O3 and formed only on the Nd-rich triple junctions. The diffusion coefficient of the triple junction reaction product growth at 20 °C was determined to be approximately 4 × 10-13 cm2/sec. This value is several orders of magnitude larger than values derived from the diffusion controlled oxide growth observations at elevated temperatures in the literature. This indicates that the growth of the room temperature oxidation products are likely defect enhanced processes at the NdFeB triple junctions.

Europium Uptake and Partitioning in Oat (Avena sativa) Roots as studied By Laser-Induced Fluorescence Spectroscopy and Confocal Microscopy Profiling Technique

International Nuclear Information System (INIS)

Fellows, Robert J.; Wang, Zheming; Ainsworth, Calvin C.

2003-01-01

The uptake of Eu3+ by elongating oat plant roots was studied by fluorescence spectroscopy, fluorescence lifetime measurement, as well as laser excitation time-resolved confocal fluorescence profiling technique. The results of this work indicated that the initial uptake of Eu(III) by oat root was most evident within the apical meristem of the root just proximal to the root cap. Distribution of assimilated Eu(III) within the roots differentiation and elongation zone was non-uniform. Higher concentrations were observed within the vascular cylinder, specifically in the phloem and developing xylem parenchyma. Elevated levels of the metal were also observed in the root hairs of the mature root. The concentration of assimilated Eu3+ dropped sharply from the apical meristem to the differentiation and elongation zone and then gradually decreased as the distance from the root cap increased. Fluorescence spectroscopic characteristics of the assimilated Eu3+ suggested that the Eu3+ exists a s inner-sphere mononuclear complexes inside the root. This work has also demonstrated the effectiveness of a time-resolved Eu3+ fluorescence spectroscopy and confocal fluorescence profiling techniques for the in vivo, real-time study of metal[Eu3+] accumulation by a functioning intact plant root. This approach can prove valuable for basic and applied studies in plant nutrition and environmental uptake of actinide radionuclides

Chromatic confocal microscopy for multi-depth imaging of epithelial tissue

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Olsovsky, Cory; Shelton, Ryan; Carrasco-Zevallos, Oscar; Applegate, Brian E.; Maitland, Kristen C.

2013-01-01

We present a novel chromatic confocal microscope capable of volumetric reflectance imaging of microstructure in non-transparent tissue. Our design takes advantage of the chromatic aberration of aspheric lenses that are otherwise well corrected. Strong chromatic aberration, generated by multiple aspheres, longitudinally disperses supercontinuum light onto the sample. The backscattered light detected with a spectrometer is therefore wavelength encoded and each spectrum corresponds to a line image. This approach obviates the need for traditional axial mechanical scanning techniques that are difficult to implement for endoscopy and susceptible to motion artifact. A wavelength range of 590-775 nm yielded a >150 µm imaging depth with ~3 µm axial resolution. The system was further demonstrated by capturing volumetric images of buccal mucosa. We believe these represent the first microstructural images in non-transparent biological tissue using chromatic confocal microscopy that exhibit long imaging depth while maintaining acceptable resolution for resolving cell morphology. Miniaturization of this optical system could bring enhanced speed and accuracy to endomicroscopic in vivo volumetric imaging of epithelial tissue. PMID:23667789

Application of advanced light microscopic techniques to gain deeper insights into cheese matrix physico-chemistry

Czech Academy of Sciences Publication Activity Database

Burdikova, Z.; Svindrych, Z.; Hickey, C.; Wilkinson, M. G.; Auty, M. A. E.; Samek, Ota; Bernatová, Silvie; Krzyžánek, Vladislav; Periasamy, A.; Sheehan, J. J.

2015-01-01

Roč. 95, č. 5 (2015), s. 687-700 ISSN 1958-5586 R&D Projects: GA MŠk(CZ) LO1212; GA MŠk ED0017/01/01 Institutional support: RVO:68081731 Keywords : confocal microscopy * cheese matrix * fluorescence lifetime * second harmonic generation * two-photon excitation * confocal Raman microscopy Subject RIV: BH - Optics, Masers, Lasers Impact factor: 1.435, year: 2015

Comparison of in vivo and ex vivo laser scanning microscopy and multiphoton tomography application for human and porcine skin imaging

Science.gov (United States)

Darvin, M. E.; Richter, H.; Zhu, Y. J.; Meinke, M. C.; Knorr, F.; Gonchukov, S. A.; Koenig, K.; Lademann, J.

2014-07-01

Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed by using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted.

UV-laser-based microscopic dissection of tree rings - a novel sampling tool for δ(13) C and δ(18) O studies.

Science.gov (United States)

Schollaen, Karina; Heinrich, Ingo; Helle, Gerhard

2014-02-01

UV-laser-based microscopic systems were utilized to dissect and sample organic tissue for stable isotope measurements from thin wood cross-sections. We tested UV-laser-based microscopic tissue dissection in practice for high-resolution isotopic analyses (δ(13) C/δ(18) O) on thin cross-sections from different tree species. The method allows serial isolation of tissue of any shape and from millimetre down to micrometre scales. On-screen pre-defined areas of interest were automatically dissected and collected for mass spectrometric analysis. Three examples of high-resolution isotopic analyses revealed that: in comparison to δ(13) C of xylem cells, woody ray parenchyma of deciduous trees have the same year-to-year variability, but reveal offsets that are opposite in sign depending on whether wholewood or cellulose is considered; high-resolution tree-ring δ(18) O profiles of Indonesian teak reflect monsoonal rainfall patterns and are sensitive to rainfall extremes caused by ENSO; and seasonal moisture signals in intra-tree-ring δ(18) O of white pine are weighted by nonlinear intra-annual growth dynamics. The applications demonstrate that the use of UV-laser-based microscopic dissection allows for sampling plant tissue at ultrahigh resolution and unprecedented precision. This new technique facilitates sampling for stable isotope analysis of anatomical plant traits like combined tree eco-physiological, wood anatomical and dendroclimatological studies. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Quantification of whey in fluid milk using confocal Raman microscopy and artificial neural network.

Science.gov (United States)

Alves da Rocha, Roney; Paiva, Igor Moura; Anjos, Virgílio; Furtado, Marco Antônio Moreira; Bell, Maria José Valenzuela

2015-06-01

In this work, we assessed the use of confocal Raman microscopy and artificial neural network as a practical method to assess and quantify adulteration of fluid milk by addition of whey. Milk samples with added whey (from 0 to 100%) were prepared, simulating different levels of fraudulent adulteration. All analyses were carried out by direct inspection at the light microscope after depositing drops from each sample on a microscope slide and drying them at room temperature. No pre- or posttreatment (e.g., sample preparation or spectral correction) was required in the analyses. Quantitative determination of adulteration was performed through a feed-forward artificial neural network (ANN). Different ANN configurations were evaluated based on their coefficient of determination (R2) and root mean square error values, which were criteria for selecting the best predictor model. In the selected model, we observed that data from both training and validation subsets presented R2>99.99%, indicating that the combination of confocal Raman microscopy and ANN is a rapid, simple, and efficient method to quantify milk adulteration by whey. Because sample preparation and postprocessing of spectra were not required, the method has potential applications in health surveillance and food quality monitoring. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A novel method for enhancing the lateral resolution and image SNR in confocal microscopy

Science.gov (United States)

Chen, Youhua; Zhu, Dazhao; Fang, Yue; Kuang, Cuifang; Liu, Xu

2017-12-01

There is always a tradeoff between the resolution and the signal-to-noise ratio (SNR) in confocal microscopy. In particular, the pinhole size is very important for maintaining a balance between them. In this paper, we propose a method for improving the lateral resolution and image SNR in confocal microscopy without making any changes to the hardware. By using the fluorescence emission difference (FED) approach, we divide the images acquired by different pinhole sizes into one image acquired by the central pinhole and several images acquired by ring-shaped pinholes. Then, they are added together with the deconvolution method. Simulation and experimental results for fluorescent particles and cells show that our method can achieve a far better resolution than a large pinhole and a higher SNR than a small pinhole. Moreover, our method can improve the performance of classic confocal laser scanning microscopy (CLSM) to a certain extent, especially CLSM with a continuously variable pinhole.

Analysis of cell-tissue grafts under weightless conditions using confocal fluorescence microscopy

Science.gov (United States)

Volova, L. T.; Milyakova, M. N.; Rossinskaya, V. V.; Boltovskaya, V. V.; Kulagina, L. N.; Kurganskaya, L. V.; Timchenko, P. E.; Timchenko, E. V.; Zherdeva Taskina, Larisa A.

2015-03-01

The research results of monitoring of viable cells in a cellular-tissue graft using confocal laser fluorescence microscopy at 488 nm and 561 nm with the use of fluorophore propidium iodide (propidium iodide, PI Sigma Aldrich USA) are presented. The processing of the received images was carried out using the software ANDOR. It is experimentally shown that the method of confocal fluorescence microscopy is one of the informational methods for detecting cells populated in a 3-D bio-carrier with a resolution of at least 400 nm. Analysis of the received micrographs suggests that the cells that were in a bio-carrier for 30 days in a synchronous ground-based experiment retained their viability compared to a similar space-based experiment in which the cells were hardly detected in a bio-carrier.

Combining total internal reflection sum frequency spectroscopy spectral imaging and confocal fluorescence microscopy.

Science.gov (United States)

Allgeyer, Edward S; Sterling, Sarah M; Gunewardene, Mudalige S; Hess, Samuel T; Neivandt, David J; Mason, Michael D

2015-01-27

Understanding surface and interfacial lateral organization in material and biological systems is critical in nearly every field of science. The continued development of tools and techniques viable for elucidation of interfacial and surface information is therefore necessary to address new questions and further current investigations. Sum frequency spectroscopy (SFS) is a label-free, nonlinear optical technique with inherent surface specificity that can yield critical organizational information on interfacial species. Unfortunately, SFS provides no spatial information on a surface; small scale heterogeneities that may exist are averaged over the large areas typically probed. Over the past decade, this has begun to be addressed with the advent of SFS microscopy. Here we detail the construction and function of a total internal reflection (TIR) SFS spectral and confocal fluorescence imaging microscope directly amenable to surface investigations. This instrument combines, for the first time, sample scanning TIR-SFS imaging with confocal fluorescence microscopy.

ADVANCED 3D LASER MICROSCOPY FOR MEASUREMENTS AND ANALYSIS OF VITRIFIED BONDED ABRASIVE TOOLS

Directory of Open Access Journals (Sweden)

WOJCIECH KAPLONEK

2012-12-01

Full Text Available In many applications, when a precise non-contact assessment of an abrasive tools’ surface is required, alternative measurement methods are often used. Their use offers numerous advantages (referential method as they introduce new qualities into routinely realized measurements. Over the past few years there has been a dynamic increase in the interest for using new types of classical confocal microscopy. These new types are often defined as 3D laser microscopy. This paper presents select aspects of one such method’s application – confocal laser scanning microscopy – for diagnostic analysis of abrasive tools. In addition this paper also looks at the basis for operation, the origins and the development of this measurement technique.The experimental part of this paper presents the select results of tests carried out on grinding wheel active surfaces with sintered microcrystalline corundum grains SG™ bound with glass-crystalline bond. The 3D laser measuring microscopes LEXT OLS3100 and LEXT OLS4000 by Olympus were used in the experiments. Analysis of the obtained measurement data was carried out in dedicated OLS 5.0.9 and OLS4100 2.1 programs, supported by specialist TalyMap Platinum 5.0 software. The realized experiments confirmed the possibility of using the offered measurement method. This concerns both the assessment of grinding wheel active surfaces and their defects, as well as the internal structures of the tools (grain-bond connections. The method presented is an interesting alternative to the typical methods used in the diagnostics of abrasive tools.

Fluorescence lifetime measurement with confocal endomicroscopy for direct analysis of tissue biochemistry in vivo

Directory of Open Access Journals (Sweden)

Youngjae Won

2016-08-01

Full Text Available Confocal endomicroscopy is a powerful tool for in vivo real-time imaging at cellular resolution inside a living body without tissue resection. Microscopic fluorescence lifetime measurement can provide information about localized biochemical conditions such as pH and the concentrations of oxygen and calcium. We hypothesized that combining these techniques could assist accurate cancer discrimination by providing both biochemical and morphological information. We designed a dual-mode experimental setup for confocal endomicroscopic imaging and fluorescence lifetime measurement and applied it to a mouse xenograft model of activated human pancreatic cancer generated by subcutaneous injection of AsPC-1 tumor cells. Using this method with pH-sensitive sodium fluorescein injection, we demonstrated discrimination between normal and cancerous tissues in a living mouse. With further development, this method may be useful for clinical cancer detection.

Experimental verification of subthreshold laser therapy using conventional pattern scan laser.

Directory of Open Access Journals (Sweden)

Tomoyasu Shiraya

Full Text Available Leading-edge therapeutic laser technologies are not available at every medical facility; therefore, alternative approaches incorporating novel advances in digital and laser technology into more readily available conventional methods have generated significant research interest. Using a rabbit model, this study investigated whether the algorithm used in the Endpoint Management (EM software system of the latest devices could enable subthreshold laser treatment in conventional retinal tissue laser therapy systems.Two types of devices were used, the PASCAL Streamline 577 and the MC 500-Vixi™, and the laser method was classified into three categories: EM; single-shot using PASCAL with arbitrary energy settings (PSS-SDM; and MC500-VixiTM (VX-SDM, which were performed in eight eyes from four Dutch-Belted rabbits. In EM, 100 mW (100% was set as a landmark, and the laser energy parameters were gradually decreased to 80%, 60%, 50%, 40%, 30%, 20%, and 10%, using a 2 × 3 square pattern. In PSS-SDM and VX-SDM, as control, the laser energy was gradually decreased to 100, 80, 60, 50, 40, 30, 20, and 10 mW. The laser settings were fixed at 200 μm, 20 ms, and a wavelength of 577 μm. To identify and compare the extent of tissue damage at each spot size, optical coherence tomography (OCT and histological findings were used to construct a three-dimensional histopathology image using a confocal laser scanning fluorescence microscope.The spot size at 50% setting on EM was 7183 μm2; PSS-SDM required 50 mW (5503 μm2 to 60 mW (10279 μm2 and VX-SDM required 50 mW (7423 μm2 to create the approximate spot size. Furthermore, at 50 mW of PSS-SDM and VX-SDM, the extent of tissue damage in all three methods was generally in accord with the outer nuclear layer by OCT and inner nuclear layer by histopathological imaging.These findings suggest that it may be possible to perform subthreshold laser therapy using approximations from the EM algorithm.

Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy.

Science.gov (United States)

Schulz, Olaf; Pieper, Christoph; Clever, Michaela; Pfaff, Janine; Ruhlandt, Aike; Kehlenbach, Ralph H; Wouters, Fred S; Großhans, Jörg; Bunt, Gertrude; Enderlein, Jörg

2013-12-24

We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions.

Injection moulding of plastic parts with laser textured surfaces with optical applications

Science.gov (United States)

Pina-Estany, J.; García-Granada, A. A.; Corull-Massana, E.

2018-05-01

The purpose of this work is to manufacture micro and nanotextured surfaces on plastic injection moulds with the aim of replicating them and obtaining plastic parts with optical applications. Different patterns are manufactured with nanosecond and femtosecond lasers in order to obtain three different optical applications: (i) homogeneous light diffusion (ii) 1D light directionality and (iii) 2D light directionality. Induction heating is used in the injections in order to improve the textures degree of replication. The steel mould and the plastic parts are analyzed with a confocal/focus variation microscope and with a surface roughness tester. A mock-up and a luminance camera are used to evaluate the homogeneity and luminance of the homogeneous light diffusion application in comparison with the current industrial solutions.

All-optical microscope autofocus based on an electrically tunable lens and a totally internally reflected IR laser.

Science.gov (United States)

Bathe-Peters, M; Annibale, P; Lohse, M J

2018-02-05

Microscopic imaging at high spatial-temporal resolution over long time scales (minutes to hours) requires rapid and precise stabilization of the microscope focus. Conventional and commercial autofocus systems are largely based on piezoelectric stages or mechanical objective actuators. Objective to sample distance is either measured by image analysis approaches or by hardware modules measuring the intensity of reflected infrared light. We propose here a truly all-optical microscope autofocus taking advantage of an electrically tunable lens and a totally internally reflected infrared probe beam. We implement a feedback-loop based on the lateral position of a totally internally reflected infrared laser on a quadrant photodetector, as an indicator of the relative defocus. We show here how to treat the combined contributions due to mechanical defocus and deformation of the tunable lens. As a result, the sample can be kept in focus without any mechanical movement, at rates up to hundreds of Hertz. The device requires only reflective optics and can be implemented at a fraction of the cost required for a comparable piezo-based actuator.

Confocal Raman microscopy for in depth analysis in the field of cultural heritage

Science.gov (United States)

Lorenzetti, G.; Striova, J.; Zoppi, A.; Castellucci, E. M.

2011-05-01

In the field of cultural heritage, the main concern when a sample is analyzed is its safeguard, and this means that non-destructive techniques are required. In this work, we show how confocal Raman microscopy (CRM) may be successfully applied in the study of works of art as a valuable alternative to other well established techniques. CRM with a metallurgical objective was tested for the in depth study of thin samples that are of interest in the field of cultural heritage. The sensitivity of the instrumentation was first evaluated by analyzing single layers of pure polyethylene terephthalate (PET) films having a thickness of 12, 25, and 50 μm, respectively, and a multilayer sample of polypropylene (PP) and polyethylene (PE). Subsequently, the technique was applied to the analysis of historical dyed cotton yarns in order to check whether it was possible to achieve a better discrimination of the fibres' signals for an easier identification. A substantial improvement of the signal to noise ratio was found in the confocal arrangement with respect to the non-confocal one, suggesting the use of this technique for this kind of analysis in the field of cultural heritage. Furthermore, Raman spectroscopy in confocal configuration was exploited in the evaluation of cleaning performed on the mural painting specimens, treated with acrylic resin (Paraloid B72). Confocal Raman experiments were performed before and after laser cleaning (at different conditions) in order to monitor the presence and to approximate the polymer thickness: the method proved to be a valid comparative tool in assessment of cleaning efficiencies.

Laser stimulation can activate autophagy in HeLa cells

International Nuclear Information System (INIS)

Wang, Yisen; Hu, Minglie; Wang, Chingyue; Lan, Bei; Cao, Youjia; He, Hao

2014-01-01

For decades, lasers have been a daily tool in most biological research for fluorescent excitation by confocal or multiphoton microscopy. More than 20 years ago, cell photodamage caused by intense laser stimulation was noticed by generating reactive oxygen species, which was then thought as the main damage effect by photons. In this study, we show that laser stimulation can induce autophagy, an important cell lysosomal pathway responding to immune stimulation and starvation, without any biochemical treatment. Two different types of laser stimulations are found to be capable of activating autophagy: continuous scanning by continuous-wave visible lasers and a short-time flash of femtosecond laser irradiation. The autophagy generation is independent from wavelength, power, and scanning duration of the visible lasers. In contrast, the power of femtosecond laser is very critical to autophagy because the multiphoton excited Ca 2+ dominates autophagy signaling. In general, we show here the different mechanisms of autophagy generation by such laser stimulation, which correspond to confocal microscopy and cell surgery, respectively. Those results can help further understanding of photodamage and autophagy signaling.

Laser stimulation can activate autophagy in HeLa cells

Energy Technology Data Exchange (ETDEWEB)

Wang, Yisen; Hu, Minglie; Wang, Chingyue [Ultrafast Laser Laboratory, Key Laboratory of Optoelectronic Information Technology (Ministry of Education), College of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin (China); Lan, Bei; Cao, Youjia [Key Laboratory of Microbial Functional Genomics of Ministry of Education, College of Life Sciences, Nankai University, Tianjin (China); He, Hao, E-mail: haohe@tju.edu.cn [Ultrafast Laser Laboratory, Key Laboratory of Optoelectronic Information Technology (Ministry of Education), College of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin (China); Med-X Research Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai (China)

2014-10-27

For decades, lasers have been a daily tool in most biological research for fluorescent excitation by confocal or multiphoton microscopy. More than 20 years ago, cell photodamage caused by intense laser stimulation was noticed by generating reactive oxygen species, which was then thought as the main damage effect by photons. In this study, we show that laser stimulation can induce autophagy, an important cell lysosomal pathway responding to immune stimulation and starvation, without any biochemical treatment. Two different types of laser stimulations are found to be capable of activating autophagy: continuous scanning by continuous-wave visible lasers and a short-time flash of femtosecond laser irradiation. The autophagy generation is independent from wavelength, power, and scanning duration of the visible lasers. In contrast, the power of femtosecond laser is very critical to autophagy because the multiphoton excited Ca{sup 2+} dominates autophagy signaling. In general, we show here the different mechanisms of autophagy generation by such laser stimulation, which correspond to confocal microscopy and cell surgery, respectively. Those results can help further understanding of photodamage and autophagy signaling.

Confocal Laser Endomicroscopy for the Diagnosis of Urothelial Carcinoma in the Bladder and the Upper Urinary Tract: Protocols for Two Prospective Explorative Studies.

Science.gov (United States)

Liem, Esmee Iml; Freund, Jan Erik; Baard, Joyce; de Bruin, D Martijn; Laguna Pes, M Pilar; Savci-Heijink, C Dilara; van Leeuwen, Ton G; de Reijke, Theo M; de la Rosette, Jean Jmch

2018-02-07

Visual confirmation of a suspicious lesion in the urinary tract is a major corner stone in diagnosing urothelial carcinoma. However, during cystoscopy (for bladder tumors) and ureterorenoscopy (for tumors of the upper urinary tract) no real-time histopathologic information can be obtained. Confocal laser endomicroscopy (CLE) is an optical imaging technique that allows for in vivo high-resolution imaging and may allow real-time tumor grading of urothelial lesions. The primary objective of both studies is to develop descriptive criteria for in vivo CLE images of urothelial carcinoma (low-grade, high-grade, carcinoma in situ) and normal urothelium by comparing CLE images with corresponding histopathology. In these two prospective clinical trials, CLE imaging will be performed of suspicious lesions and normal tissue in the urinary tract during surgery, prior to resection or biopsy. In the bladder study, CLE will be performed in 60 patients using the Cystoflex UHD-R probe. In the upper urinary tract study, CLE will be performed in 25 patients during ureterorenoscopy, who will undergo radical treatment (nephroureterectomy or segmental ureter resection) thereafter. All CLE images will be analyzed frame by frame by three independent, blinded observers. Histopathology and CLE-based diagnosis of the lesions will be evaluated. Both studies comply with the IDEAL stage 2b recommendations. Presently, recruitment of patients is ongoing in both studies. Results and outcomes are expected in 2018. For development of CLE-based diagnosis of urothelial carcinoma in the bladder and the upper urinary tract, a structured conduct of research is required. This study will provide more insight in tissue-specific CLE criteria for real-time tumor grading of urothelial carcinoma. Confocal Laser Endomicroscopy: ClinicalTrials.gov NCT03013894; https://clinicaltrials.gov /ct2/show/NCT03013894?term=NCT03013894&rank=1 (Archived by WebCite at http://www.webcitation.org/6wiPZ378I); and Dutch Central

[Influence of implants prepared by selective laser melting on early bone healing].

Science.gov (United States)

Liu, J Y; Chen, F; Ge, Y J; Wei, L; Pan, S X; Feng, H L

2018-02-18

To evaluate the influence of the rough surface of dental implants prepared by selective laser melting (SLM) on early bone healing around titanium implants. A total of sixteen titanium implants were involved in our research, of which eight implants were prepared by SLM (TIXOS Cylindrical, Leader-Novaxa, Milan, Italy; 3.3 mm×10 mm, internal hex) and the other eight were sandblasted, large-grit and acid-etched (SLA) implants (IMPLUS Cylindrical, Leader-Novaxa, Milan, Italy; 3.3 mm×10 mm, internal hex). All of the dental implants were inserted into the healed extraction sockets of the mandible of two adult male Beagle dogs. Half of the dental implants were designed to be healed beneath the mucosa and the other half were intended to be healed transgingivally and were immediately loaded by acrylic resin bridge restoration. Three types of tetracycline fluorescent labels, namely calcein blue, alizarin complexone and calcein, were administered into the veins of the Beagle dogs 2, 4, and 8 weeks after implant placement respectively for fluorescent evaluation of newly formed bone peri-implant. Both Beagle dogs were euthanized 12 weeks after implant insertion and the mandible block specimens containing the titanium implants and surrounding bone and soft tissue of each dog were carefully sectioned and dissected. A total of 16 hard tissue slices were obtained and stained with toluidine blue for microscopic examination and histomorphometric measurements. Histological observation was made for each slice under light microscope and laser scanning confocal microscope (LSCM). Comparison on new bone formation around titanium implants of each group was made and mineral apposition rate (MAR) was calculated for each group. Dental implants prepared by selective laser melting had achieved satisfying osseointegration to surrounding bone tissue after the healing period of 12 weeks. Newly formed bone tissue was observed creeping on the highly porous surface of the SLM implant and growing

Towards modeling of cardiac micro-structure with catheter-based confocal microscopy: a novel approach for dye delivery and tissue characterization.

Science.gov (United States)

Lasher, Richard A; Hitchcock, Robert W; Sachse, Frank B

2009-08-01

This work presents a methodology for modeling of cardiac tissue micro-structure. The approach is based on catheter-based confocal imaging systems, which are emerging as tools for diagnosis in various clinical disciplines. A limitation of these systems is that a fluorescent marker must be available in sufficient concentration in the imaged region. We introduce a novel method for the local delivery of fluorescent markers to cardiac tissue based on a hydro-gel carrier brought into contact with the tissue surface. The method was tested with living rabbit cardiac tissue and applied to acquire three-dimensional image stacks with a standard inverted confocal microscope and two-dimensional images with a catheter-based confocal microscope. We processed these image stacks to obtain spatial models and quantitative data on tissue microstructure. Volumes of atrial and ventricular myocytes were 4901 +/- 1713 and 10 299 +/-3598 mum (3) (mean+/-sd), respectively. Atrial and ventricular myocyte volume fractions were 72.4 +/-4.7% and 79.7 +/- 2.9% (mean +/-sd), respectively. Atrial and ventricular myocyte density was 165 571 +/- 55 836 and 86 957 +/- 32 280 cells/mm (3) (mean+/-sd), respectively. These statistical data and spatial descriptions of tissue microstructure provide important input for modeling studies of cardiac tissue function. We propose that the described methodology can also be used to characterize diseased tissue and allows for personalized modeling of cardiac tissue.

Mycelial pellet intrastructure visualization and viability prediction in a culture of Streptomyces fradiae using confocal scanning laser microscopy

International Nuclear Information System (INIS)

Park, Y.; Tamura, S.; Koike, Y.; Toriya, M.; Okabe, M.

1997-01-01

The intrastructure of mycelial pellets of Streptomyces fradiae, which produces tylosin, was visualized following labeling with fluorescein isothiocyanate (FITC) and propidium iodide (PI) using confocal scanning laser microscopy. A PI-labeled inactive core was present inside the mycelial pellet in both fermentor and air-lift reactor cultures. The thickness of the active mycelial layer on the pellet surface was calculated from a density sliced image to be 60 and 68 μm respectively, in the fermentor and in air-lift reactor cultures. Using image analysis, the active mycelial concentration of pellets in the fermentor culture was predicted to be 2.1 times higher than that in the air-lift reactor culture. The tylosin production rate in the fermentor reached 0.78 g/l/d, which was 2.5-fold that in the air-lift reactor culture. These results indicate that the higher tylosin production rate in the fermentor culture was due to the higher active mycelial concentration in the fermentor compared to that in the air-lift reactor. (author)

Confocal laser-induced fluorescence detector for narrow capillary system with yoctomole limit of detection.

Science.gov (United States)

Weaver, Mitchell T; Lynch, Kyle B; Zhu, Zaifang; Chen, Huang; Lu, Joann J; Pu, Qiaosheng; Liu, Shaorong

2017-04-01

Laser-induced fluorescence (LIF) detectors for low-micrometer and sub-micrometer capillary on-column detection are not commercially available. In this paper, we describe in details how to construct a confocal LIF detector to address this issue. We characterize the detector by determining its limit of detection (LOD), linear dynamic range (LDR) and background signal drift; a very low LOD (~70 fluorescein molecules or 12 yoctomole fluorescein), a wide LDR (greater than 3 orders of magnitude) and a small background signal drift (~1.2-fold of the root mean square noise) are obtained. For detecting analytes inside a low-micrometer and sub-micrometer capillary, proper alignment is essential. We present a simple protocol to align the capillary with the optical system and use the position-lock capability of a translation stage to fix the capillary in position during the experiment. To demonstrate the feasibility of using this detector for narrow capillary systems, we build a 2-μm-i.d. capillary flow injection analysis (FIA) system using the newly developed LIF prototype as a detector and obtain an FIA LOD of 14 zeptomole fluorescein. We also separate a DNA ladder sample by bare narrow capillary - hydrodynamic chromatography and use the LIF prototype to monitor the resolved DNA fragments. We obtain not only well-resolved peaks but also the quantitative information of all DNA fragments. Copyright © 2016 Elsevier B.V. All rights reserved.

Minimizing inter-microscope variability in dental microwear texture analysis

Science.gov (United States)

Arman, Samuel D.; Ungar, Peter S.; Brown, Christopher A.; DeSantis, Larisa R. G.; Schmidt, Christopher; Prideaux, Gavin J.

2016-06-01

A common approach to dental microwear texture analysis (DMTA) uses confocal profilometry in concert with scale-sensitive fractal analysis to help understand the diets of extinct mammals. One of the main benefits of DMTA over other methods is the repeatable, objective manner of data collection. This repeatability, however, is threatened by variation in results of DMTA of the same dental surfaces yielded by different microscopes. Here we compare DMTA data of five species of kangaroos measured on seven profilers of varying specifications. Comparison between microscopes confirms that inter-microscope differences are present, but we show that deployment of a number of automated treatments to remove measurement noise can help minimize inter-microscope differences. Applying these same treatments to a published hominin DMTA dataset shows that they alter some significant differences between dietary groups. Minimising microscope variability while maintaining interspecific dietary differences requires then that these factors are balanced in determining appropriate treatments. The process outlined here offers a solution for allowing comparison of data between microscopes, which is essential for ongoing DMTA research. In addition, the process undertaken, including considerations of other elements of DMTA protocols also promises to streamline methodology, remove measurement noise and in doing so, optimize recovery of a reliable dietary signature.

Quantitative detection of caffeine in human skin by confocal Raman spectroscopy--A systematic in vitro validation study.

Science.gov (United States)

Franzen, Lutz; Anderski, Juliane; Windbergs, Maike

2015-09-01

For rational development and evaluation of dermal drug delivery, the knowledge of rate and extent of substance penetration into the human skin is essential. However, current analytical procedures are destructive, labor intense and lack a defined spatial resolution. In this context, confocal Raman microscopy bares the potential to overcome current limitations in drug depth profiling. Confocal Raman microscopy already proved its suitability for the acquisition of qualitative penetration profiles, but a comprehensive investigation regarding its suitability for quantitative measurements inside the human skin is still missing. In this work, we present a systematic validation study to deploy confocal Raman microscopy for quantitative drug depth profiling in human skin. After we validated our Raman microscopic setup, we successfully established an experimental procedure that allows correlating the Raman signal of a model drug with its controlled concentration in human skin. To overcome current drawbacks in drug depth profiling, we evaluated different modes of peak correlation for quantitative Raman measurements and offer a suitable operating procedure for quantitative drug depth profiling in human skin. In conclusion, we successfully demonstrate the potential of confocal Raman microscopy for quantitative drug depth profiling in human skin as valuable alternative to destructive state-of-the-art techniques. Copyright © 2015 Elsevier B.V. All rights reserved.

Imaging the Microscopic Structure of Shear Thinning and Thickening Colloidal Suspensions

KAUST Repository

Cheng, X.

2011-09-01

The viscosity of colloidal suspensions varies with shear rate, an important effect encountered in many natural and industrial processes. Although this non-Newtonian behavior is believed to arise from the arrangement of suspended particles and their mutual interactions, microscopic particle dynamics are difficult to measure. By combining fast confocal microscopy with simultaneous force measurements, we systematically investigate a suspension\\'s structure as it transitions through regimes of different flow signatures. Our measurements of the microscopic single-particle dynamics show that shear thinning results from the decreased relative contribution of entropic forces and that shear thickening arises from particle clustering induced by hydrodynamic lubrication forces. This combination of techniques illustrates an approach that complements current methods for determining the microscopic origins of non-Newtonian flow behavior in complex fluids.

Complex composition film condensation in the sluice device of an electron microscope

International Nuclear Information System (INIS)

Kukuev, V.I.; Lesovoj, M.V.; Vlasov, D.A.; Malygin, M.V.; Domashevskaya, Eh.P.; Tomashpol'skij, Yu.Ya.

1994-01-01

Based on the sluice device of an electron microscope a system is developed for material laser evaporation and vapor condensation on a substrate, situated in the microscope specimen holder. Substrate heating by laser radiation to 100 deg C is used. The system is applied for investigating growth of high-temperature superconductor films

In-situ observation of recrystallization in an AlMgScZr alloy using confocal laser scanning microscopy

International Nuclear Information System (INIS)

Taendl, J.; Nambu, S.; Orthacker, A.; Kothleitner, G.; Inoue, J.; Koseki, T.; Poletti, C.

2015-01-01

In this work we present a novel in-situ approach to study the recrystallization behavior of age hardening alloys. We use confocal laser scanning microscopy (CLSM) at 400 °C to investigate the static recrystallization of an AlMg4Sc0.4Zr0.12 alloy in-situ. The results are combined with electron backscatter diffraction (EBSD) and transmission electron microscopy (TEM) analyses. It was found that CLSM is a powerful tool to visualize both the local initiation and temporal sequence of recrystallization. After fast nucleation and initial growth, the grain growth rate decreases and the grain boundary migration stops after some minutes due to Zener pinning from Al 3 (Sc,Zr) precipitates produced during the heat treatment. EBSD and TEM analyses confirm both the boundary movements and the particle-boundary interactions. - Highlights: • First time that CLSM is used to study recrystallization in-situ. • The start and end of recrystallization can be directly observed. • The procedure is easy to apply and requires only simple data interpretation. • In-situ observations on the surface correlate to modifications inside the bulk. • In-situ observations correlate to EBSD and EFTEM analyses.

In-situ observation of recrystallization in an AlMgScZr alloy using confocal laser scanning microscopy

Energy Technology Data Exchange (ETDEWEB)

Taendl, J., E-mail: johannes.taendl@tugraz.atl [Institute of Materials Science and Welding, Graz University of Technology, Graz (Austria); Nambu, S. [Department of Materials Engineering, The University of Tokyo, Tokyo 113-8656 (Japan); Orthacker, A.; Kothleitner, G. [Institute of Electron Microscopy and Nanoanalysis, Graz University of Technology, Graz (Austria); Graz Center for Electron Microscopy, Graz (Austria); Inoue, J.; Koseki, T. [Department of Materials Engineering, The University of Tokyo, Tokyo 113-8656 (Japan); Poletti, C. [Institute of Materials Science and Welding, Graz University of Technology, Graz (Austria)

2015-10-15

In this work we present a novel in-situ approach to study the recrystallization behavior of age hardening alloys. We use confocal laser scanning microscopy (CLSM) at 400 °C to investigate the static recrystallization of an AlMg4Sc0.4Zr0.12 alloy in-situ. The results are combined with electron backscatter diffraction (EBSD) and transmission electron microscopy (TEM) analyses. It was found that CLSM is a powerful tool to visualize both the local initiation and temporal sequence of recrystallization. After fast nucleation and initial growth, the grain growth rate decreases and the grain boundary migration stops after some minutes due to Zener pinning from Al{sub 3}(Sc,Zr) precipitates produced during the heat treatment. EBSD and TEM analyses confirm both the boundary movements and the particle-boundary interactions. - Highlights: • First time that CLSM is used to study recrystallization in-situ. • The start and end of recrystallization can be directly observed. • The procedure is easy to apply and requires only simple data interpretation. • In-situ observations on the surface correlate to modifications inside the bulk. • In-situ observations correlate to EBSD and EFTEM analyses.

In vivo assessment of the structure of skin microcirculation by reflectance confocal-laser-scanning microscopy

Science.gov (United States)

Sugata, Keiichi; Osanai, Osamu; Kawada, Hiromitsu

2012-02-01

One of the major roles of the skin microcirculation is to supply oxygen and nutrition to the surrounding tissue. Regardless of the close relationship between the microcirculation and the surrounding tissue, there are few non-invasive methods that can evaluate both the microcirculation and its surrounding tissue at the same site. We visualized microcapillary plexus structures in human skin using in vivo reflectance confocal-laser-scanning microscopy (CLSM), Vivascope 3000® (Lucid Inc., USA) and Image J software (National Institutes of Health, USA) for video image processing. CLSM is a non-invasive technique that can visualize the internal structure of the skin at the cellular level. In addition to internal morphological information such as the extracellular matrix, our method reveals capillary structures up to the depth of the subpapillary plexus at the same site without the need for additional optical systems. Video images at specific depths of the inner forearm skin were recorded. By creating frame-to-frame difference images from the video images using off-line video image processing, we obtained images that emphasize the brightness depending on changes of intensity coming from the movement of blood cells. Merging images from different depths of the skin elucidates the 3-dimensional fine line-structure of the microcirculation. Overall our results show the feasibility of a non-invasive, high-resolution imaging technique to characterize the skin microcirculation and the surrounding tissue.

Combining confocal laser scanning microscopy with serial section reconstruction in the study of adult neurogenesis.

Directory of Open Access Journals (Sweden)

Federico eLuzzati

2011-05-01

Full Text Available Current advances in imaging techniques have extended the possibility of visualizing small structures within large volumes of both fixed and live specimens without sectioning. These techniques have contributed valuable information to study neuronal plasticity in the adult brain. However, technical limits still hamper the use of these approaches to investigate neurogenic regions located far from the ventricular surface such as parenchymal neurogenic niches, or the scattered neuroblasts induced by brain lesions. Here, we present a method to combine confocal laser scanning microscopy (CLSM and serial section reconstruction in order to reconstruct large volumes of brain tissue at cellular resolution. In this method a series of thick sections are imaged with CLSM and the resulting stacks of images are registered and 3D reconstructed. This approach is based on existing freeware software and can be performed on ordinary laboratory personal computers (PC. By using this technique we have investigated the morphology and spatial organization of a group of doublecortin (DCX+ neuroblasts located in the lateral striatum of the late post-natal guinea pig. The 3D study unravelled a complex network of long and poorly ramified cell processes, often fascicled and mostly oriented along the internal capsule fibre bundles. These data support CLSM serial section reconstruction as a reliable alternative to the whole mount approaches to analyze cyto-architectural features of adult germinative niches.

Clinical impact of confocal laser endomicroscopy in the management of gastrointestinal lesions with an uncertain diagnosis.

Science.gov (United States)

Robles-Medranda, Carlos; Vargas, Maria; Ospina, Jesenia; Puga-Tejada, Miguel; Valero, Manuel; Soria, Miguel; Bravo, Gladys; Robles-Jara, Carlos; Lukashok, Hannah Pitanga

2017-08-16

To evaluate the clinical impact of confocal laser endomicroscopy (CLE) in the diagnosis and management of patients with an uncertain diagnosis. A retrospective chart review was performed. Patients who underwent CLE between November 2013 and October 2015 and exhibited a poor correlation between endoscopic and histological findings were included. Baseline characteristics, indications, previous diagnostic studies, findings at the time of CLE, clinical management and histological results were analyzed. Interventions based on CLE findings were also analyzed. We compared the diagnostic accuracy of CLE and target biopsies of surgical specimens. A total of 144 patients were included. Of these, 51% (74/144) were female. The mean age was 51 years old. In all, 41/144 (28.4%) lesions were neoplastic (13 bile duct, 10 gastric, 8 esophageal, 6 colonic, 1 duodenal, 1 rectal, 1 ampulloma and 1 pancreatic). The sensitivity, specificity, positive predictive value, negative predictive value, and observed agreement when CLE was used to detect N-lesions were 85.37%, 87.38%, 72.92%, 93.75% and 86.81%, respectively. Cohen's Kappa was 69.20%, thus indicating good agreement. Changes in management were observed in 54% of the cases. CLE is a new diagnostic tool that has a significant clinical impact on the diagnosis and treatment of patients with uncertain diagnosis.

Exploring the diversity of Listeria monocytogenes biofilm architecture by high-throughput confocal laser scanning microscopy and the predominance of the honeycomb-like morphotype.

Science.gov (United States)

Guilbaud, Morgan; Piveteau, Pascal; Desvaux, Mickaël; Brisse, Sylvain; Briandet, Romain

2015-03-01

Listeria monocytogenes is involved in food-borne illness with a high mortality rate. The persistence of the pathogen along the food chain can be associated with its ability to form biofilms on inert surfaces. While most of the phenotypes associated with biofilms are related to their spatial organization, most published data comparing biofilm formation by L. monocytogenes isolates are based on the quantitative crystal violet assay, which does not give access to structural information. Using a high-throughput confocal-imaging approach, the aim of this work was to decipher the structural diversity of biofilms formed by 96 L. monocytogenes strains isolated from various environments. Prior to large-scale analysis, an experimental design was created to improve L. monocytogenes biofilm formation in microscopic-grade microplates, with special emphasis on the growth medium composition. Microscopic analysis of biofilms formed under the selected conditions by the 96 isolates revealed only weak correlation between the genetic lineages of the isolates and the structural properties of the biofilms. However, a gradient in their geometric descriptors (biovolume, mean thickness, and roughness), ranging from flat multilayers to complex honeycomb-like structures, was shown. The dominant honeycomb-like morphotype was characterized by hollow voids hosting free-swimming cells and localized pockets containing mixtures of dead cells and extracellular DNA (eDNA). Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Confocal fluorescence microscopy investigation of visible emitting defects induced by electron beam lithography in LIF films

Energy Technology Data Exchange (ETDEWEB)

Montereali, R.M.; Bigotta, S.; Pace, A.; Piccinini, M. [ENEA, Divisione Fisica Applicata, Centro Ricerche Frascati, Frascati, RM (Italy); Burattini, E.; Grilli, A.; Raco, A. [Istituto Nazionale di Fisica Nucleare, Laboratori Nazionali di Fisica, Frascati, Rome (Italy); Giammatteo, M. [Unita' Istituto Nazionale di Fisica Nucleare, Frascati, RM (Italy)]|[L' Aquila Univ., L' Aquila (Italy). Centro di Microscopia Elettronica; Picozzi, P.; Santucci, S. [Unita' Istituto Nazionale di Fisica Nucleare, Frascati, RM (Italy)]|[L' Aquila Univ., L' Aquila (Italy). Dipt. di Fisica

2000-07-01

Low energy electron irradiation of lithium fluoride (LiF), in the form of bulk crystals and films, gives rise to the stable formation of primary F defects and aggregated color centers in a thin layer located at the surface of the investigated material. For the first time a confocal light scanning microscope (CLSM) in fluorescence mode was used to reconstruct the depth distribution of efficiently emitting laser active color centers in a stripe-like region induced by 12 and 16 keV electrons on LiF films thermally evaporated on glass. The formation of the F{sub 3}{sup +} and F{sub 2} aggregated defects appears restricted to the electron penetration and proportional to their energy depth profile, as obtained from Monte Carlo simulations. [Italian] L'irraggiamento con elettroni di bassa energia del fluoruro di litio (LiF), in forma di cristalli e film, induce la formazione di difetti primari F e centri di colore aggregati stabili in un sottile strato localizzato alla superficie del materiale investigato. Per la prima volta un microscopio confocale a scansione (CLSM) in modalita' fluorescenza e' stato usato per ricostruire la distribuzione di centri di colore laser attivi ad alta efficienza di emissione nel visibile, in strisce colorate ottenute con elettroni da 12 e 16 keV su film di LiF evaporati termicamente su vetro. La formazione dei difetti aggregati F2 e F3+ risulta ristretta spazialmente nella regione di penetrazione degli elettroni e proporzionale al profilo della distribuzione dell'energia da essi depositata, ricavata tramite simulazioni Monte Carlo.

Fluorescence microscopy.

Science.gov (United States)

Sanderson, Michael J; Smith, Ian; Parker, Ian; Bootman, Martin D

2014-10-01

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. © 2014 Cold Spring Harbor Laboratory Press.

Evaluation of baseline structural factors for predicting glaucomatous visual-field progression using optical coherence tomography, scanning laser polarimetry and confocal scanning laser ophthalmoscopy.

Science.gov (United States)

Sehi, M; Bhardwaj, N; Chung, Y S; Greenfield, D S

2012-12-01

The objective of this study is to assess whether baseline optic nerve head (ONH) topography and retinal nerve fiber layer thickness (RNFLT) are predictive of glaucomatous visual-field progression in glaucoma suspect (GS) and glaucomatous eyes, and to calculate the level of risk associated with each of these parameters. Participants with ≥28 months of follow-up were recruited from the longitudinal Advanced Imaging for Glaucoma Study. All eyes underwent standard automated perimetry (SAP), confocal scanning laser ophthalmoscopy (CSLO), time-domain optical coherence tomography (TDOCT), and scanning laser polarimetry using enhanced corneal compensation (SLPECC) every 6 months. Visual-field progression was assessed using pointwise linear-regression analysis of SAP sensitivity values (progressor) and defined as significant sensitivity loss of >1 dB/year at ≥2 adjacent test locations in the same hemifield at P<0.01. Cox proportional hazard ratios (HR) were calculated to determine the predictive ability of baseline ONH and RNFL parameters for SAP progression using univariate and multivariate models. Seventy-three eyes of 73 patients (43 GS and 30 glaucoma, mean age 63.2±9.5 years) were enrolled (mean follow-up 51.5±11.3 months). Four of 43 GS (9.3%) and 6 of 30 (20%) glaucomatous eyes demonstrated progression. Mean time to progression was 50.8±11.4 months. Using multivariate models, abnormal CSLO temporal-inferior Moorfields classification (HR=3.76, 95% confidence interval (CI): 1.02-6.80, P=0.04), SLPECC inferior RNFLT (per -1 μm, HR=1.38, 95% CI: 1.02-2.2, P=0.02), and TDOCT inferior RNFLT (per -1 μm, HR=1.11, 95% CI: 1.04-1.2, P=0.001) had significant HRs for SAP progression. Abnormal baseline ONH topography and reduced inferior RNFL are predictive of SAP progression in GS and glaucomatous eyes.

Topotactic changes on η-Mo4O11 caused by biased atomic force microscope tip and cw-laser

International Nuclear Information System (INIS)

Borovšak, Miloš; Šutar, Petra; Goreshnik, Evgeny; Mihailovic, Dragan

2015-01-01

Highlights: • We report influencing electronic properties of η-Mo 4 O 11 . • With the biased AFM tip we induce the surface potential changes on η-Mo 4 O 11 . • We used cw-laser to induced similar effect on surface potential on η-Mo 4 O 11 . • We do not influence the surface and topography of the samples. • No change in topography of samples indicates the topotactic transformation. - Abstract: We present topotactic changes on Mo 4 O 11 crystals induced by a biased atomic force microscope tip and continuous laser. The transformation does not change the topography of the samples, while the surface potential shows remarkable changes on areas where the biased AFM tip was applied. No structural changes were observed by Raman spectroscopy, but AFM scans revealed changes to surface potential due to laser illumination. The observed phenomenon could be potentially useful for memristive memory devices considering the fact that properties of other molybdenum oxides vary from metallic to insulators.

Research on microstructure properties of the TiC/Ni-Fe-Al coating prepared by laser cladding technology

Science.gov (United States)

Jiao, Junke; Xu, Zifa; Zan, Shaoping; Zhang, Wenwu; Sheng, Liyuan

2017-10-01

In this paper, the laser cladding method was used to preparation the TiC reinforced Ni-Fe-Al coating on the Ni base superalloy. The Ti/Ni-Fe-Al powder was preset on the Ni base superalloy and the powder layer thickness is 0.5mm. A fiber laser was used the melting Ti/Ni-Fe-Al powder in an inert gas environment. The shape of the cladding layer was tested using laser scanning confocal microscope (LSCM) under different cladding parameters such as the laser power, the melting velocity and the defocused amount. The microstructure, the micro-hardness was tested by LSCM, SEM, Vickers hardness tester. The test result showed that the TiC particles was distributed uniformly in the cladding layer and hardness of the cladding layer was improved from 180HV to 320HV compared with the Ni-Fe-Al cladding layer without TiC powder reinforced, and a metallurgical bonding was produced between the cladding layer and the base metal. The TiC powder could make the Ni-Fe-Al cladding layer grain refining, and the more TiC powder added in the Ni-Fe-Al powder, the smaller grain size was in the cladding layer.

Visualizing 3-D microscopic specimens

Science.gov (United States)

Forsgren, Per-Ola; Majlof, Lars L.

1992-06-01

The confocal microscope can be used in a vast number of fields and applications to gather more information than is possible with a regular light microscope, in particular about depth. Compared to other three-dimensional imaging devices such as CAT, NMR, and PET, the variations of the objects studied are larger and not known from macroscopic dissections. It is therefore important to have several complementary ways of displaying the gathered information. We present a system where the user can choose display techniques such as extended focus, depth coding, solid surface modeling, maximum intensity and other techniques, some of which may be combined. A graphical user interface provides easy and direct control of all input parameters. Motion and stereo are available options. Many three- dimensional imaging devices give recordings where one dimension has different resolution and sampling than the other two which requires interpolation to obtain correct geometry. We have evaluated algorithms with interpolation in object space and in projection space. There are many ways to simplify the geometrical transformations to gain performance. We present results of some ways to simplify the calculations.

Confocal Raman spectroscopy to trace lipstick with their smudges on different surfaces.

Science.gov (United States)

López-López, Maria; Özbek, Nil; García-Ruiz, Carmen

2014-06-01

Lipsticks are very popular cosmetic products that can be transferred by contact to different surfaces, being important forensic evidence with an intricate analysis if they are found in a crime scene. This study evaluates the use of confocal Raman microscopy at 780 nm excitation wavelength for the nondestructive identification of 49 lipsticks of different brands and colors, overcoming the lipstick fluorescence problem reported by previous works using other laser wavelengths. Although the lipsticks samples showed some fluorescence, this effect was not so intense to completely overwhelm the Raman spectra. Lipsticks smudges on twelve different surfaces commonly stained with these samples were also analyzed. In the case of the surfaces, some of them provided several bands to the smudge spectra compromising the identification of the lipstick. For these samples spectral subtraction of the interfering bands from the surface was performed. Finally, five different red lipsticks with very similar color were measured on different surfaces to evaluate the lipstick traceability with their smudges even on interfering surfaces. Although previous spectral subtraction was needed in some cases, all the smudged were linked to their corresponding lipsticks even when they are smeared on the interfering surfaces. As a consequence, confocal Raman microscopy using the 780 nm excitation laser is presented as a nondestructive powerful tool for the identification of these tricky samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Purinergic receptors have different effects in rat exocrine pancreas. Calcium signals monitored by fura-2 using confocal microscopy

DEFF Research Database (Denmark)

Novak, Ivana; Nitschke, Roland; Amstrup, Jan

2002-01-01

Pancreatic ducts have several types of purinergic P2 receptors, however, nothing is known about P2 receptors in acini. The aim was to establish whether acini express functional P2 receptors coupled to intracellular Ca2+ signals and to measure the signals ratiometrically in a confocal laser scanning...

Confocal Raman microscopy

CERN Document Server

Dieing, Thomas; Hollricher, Olaf

2018-01-01