Fast Confocal Raman Imaging Using a 2-D Multifocal Array for Parallel Hyperspectral Detection.

PubMed

Kong, Lingbo; Navas-Moreno, Maria; Chan, James W

2016-01-19

We present the development of a novel confocal hyperspectral Raman microscope capable of imaging at speeds up to 100 times faster than conventional point-scan Raman microscopy under high noise conditions. The microscope utilizes scanning galvomirrors to generate a two-dimensional (2-D) multifocal array at the sample plane, generating Raman signals simultaneously at each focus of the array pattern. The signals are combined into a single beam and delivered through a confocal pinhole before being focused through the slit of a spectrometer. To separate the signals from each row of the array, a synchronized scan mirror placed in front of the spectrometer slit positions the Raman signals onto different pixel rows of the detector. We devised an approach to deconvolve the superimposed signals and retrieve the individual spectra at each focal position within a given row. The galvomirrors were programmed to scan different focal arrays following Hadamard encoding patterns. A key feature of the Hadamard detection is the reconstruction of individual spectra with improved signal-to-noise ratio. Using polystyrene beads as test samples, we demonstrated not only that our system images faster than a conventional point-scan method but that it is especially advantageous under noisy conditions, such as when the CCD detector operates at fast read-out rates and high temperatures. This is the first demonstration of multifocal confocal Raman imaging in which parallel spectral detection is implemented along both axes of the CCD detector chip. We envision this novel 2-D multifocal spectral detection technique can be used to develop faster imaging spontaneous Raman microscopes with lower cost detectors.

Two-photon microscopy and spectroscopy based on a compact confocal scanning head

NASA Astrophysics Data System (ADS)

Diaspro, Alberto; Chirico, Giberto; Federici, Federico; Cannone, Fabio; Beretta, Sabrina; Robello, Mauro; Olivini, Francesca; Ramoino, Paola

2001-07-01

We have combined a confocal laser scanning head modified for TPE (two-photon excitation) microscopy with some spectroscopic modules to study single molecules and molecular aggregates. The behavior of the TPE microscope unit has been characterized by means of point spread function measurements and of the demonstration of its micropatterning abilities. One-photon and two-photon mode can be simply accomplished by switching from a mono-mode optical fiber (one-photon) coupled to conventional laser sources to an optical module that allows IR laser beam (two- photon/TPE) delivery to the confocal laser scanning head. We have then described the characterization of the two-photon microscope for spectroscopic applications: fluorescence correlation, lifetime and fluorescence polarization anisotropy measurements. We describe the measurement of the response of the two-photon microscope to the light polarization and discuss fluorescence polarization anisotropy measurements on Rhodamine 6G as a function of the viscosity and on a globular protein, the Beta-lactoglobulin B labeled with Alexa 532 at very high dilutions. The average rotational and translational diffusion coefficients measured with fluorescence polarization anisotropy and fluorescence correlation methods are in good agreement with the protein size, therefore validating the use of the microscope for two-photon spectroscopy on biomolecules.

Confocal laser endomicroscopy in the "in vivo" histological diagnosis of the gastrointestinal tract.

PubMed

De Palma, Giovanni D

2009-12-14

Recent technological advances in miniaturization have allowed for a confocal scanning microscope to be integrated into a conventional flexible endoscope, or into trans-endoscopic probes, a technique now known as confocal endomicroscopy or confocal laser endomicroscopy. This newly-developed technology has enabled endoscopists to collect real-time in vivo histological images or "virtual biopsies" of the gastrointestinal mucosa during endoscopy, and has stimulated significant interest in the application of this technique in clinical gastroenterology. This review aims to evaluate the current data on the technical aspects and the utility of this new technology in clinical gastroenterology and its potential impact in the future, particularly in the screening or surveillance of gastrointestinal neoplasia.

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

NASA Astrophysics Data System (ADS)

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.

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

PubMed

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.

Fibre optic confocal imaging (FOCI) for subsurface microscopy of the colon in vivo.

PubMed Central

Delaney, P M; King, R G; Lambert, J R; Harris, M R

1994-01-01

Fibre optic confocal imaging (FOCI) is a new type of microscopy which has been recently developed (Delaney et al. 1993). In contrast to conventional light microscopy, FOCI and other confocal techniques allow clear imaging of subsurface structures within translucent objects. However, unlike conventional confocal microscopes which are bulky (because of a need for accurate alignment of large components) FOCI allows the imaging end to be miniaturised and relatively mobile. FOCI is thus particularly suited for clear subsurface imaging of structures within living animals or subjects. The aim of the present study was to assess the suitability of using FOCI for imaging of subsurface structures within the colon, both in vitro (human and rat biopsies) and in vivo (in rats). Images were obtained in fluorescence mode (excitation 488 nm, detection above 515 nm) following topical application of fluorescein. By this technique the glandular structure of the colon was imaged. FOCI is thus suitable for subsurface imaging of the colon in vivo. Images Fig. 2 Fig. 3 PMID:8157487

Examining the contents of isolated Xenopus germinal vesicles.

PubMed

Gall, Joseph G; Wu, Zheng'an

2010-05-01

One can manually isolate the giant oocyte nucleus or germinal vesicle (GV) of Xenopus from a living oocyte with nothing more complicated than jewelers' forceps and a dissecting microscope. Similarly, one can remove the nuclear envelope by hand and allow the lampbrush chromosomes and other nuclear organelles to spread on a microscope slide. After centrifugation, the nuclear contents adhere tightly to the slide, where they can be subjected to immunostaining or fluorescent in situ hybridization for visualization by conventional or confocal microscopy. Preparations of isolated GV contents reveal details of nuclear structure that are almost impossible to attain by more conventional techniques.

Multimodal optical workstation for simultaneous linear, nonlinear microscopy and nanomanipulation: upgrading a commercial confocal inverted microscope.

PubMed

Mathew, Manoj; Santos, Susana I C O; Zalvidea, Dobryna; Loza-Alvarez, Pablo

2009-07-01

In this work we propose and build a multimodal optical workstation that extends a commercially available confocal microscope (Nikon Confocal C1-Si) to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools such as nanosurgery. The setup allows both subsystems (confocal and nonlinear) to work independently and simultaneously. The workstation enables, for instance, nanosurgery along with simultaneous confocal and brightfield imaging. The nonlinear microscopy capabilities are added around the commercial confocal microscope by exploiting all the flexibility offered by this microscope and without need for any mechanical or electronic modification of the confocal microscope systems. As an example, the standard differential interference contrast condenser and diascopic detector in the confocal microscope are readily used as a forward detection mount for second harmonic generation imaging. The various capabilities of this workstation, as applied directly to biology, are demonstrated using the model organism Caenorhabditis elegans.

Any Way You Slice It—A Comparison of Confocal Microscopy Techniques

PubMed Central

Jonkman, James

2015-01-01

The confocal fluorescence microscope has become a popular tool for life sciences researchers, primarily because of its ability to remove blur from outside of the focal plane of the image. Several different kinds of confocal microscopes have been developed, each with advantages and disadvantages. This article will cover the grid confocal, classic confocal laser-scanning microscope (CLSM), the resonant scanning-CLSM, and the spinning-disk confocal microscope. The way each microscope technique works, the best applications the technique is suited for, the limitations of the technique, and new developments for each technology will be presented. Researchers who have access to a range of different confocal microscopes (e.g., through a local core facility) should find this paper helpful for choosing the best confocal technology for specific imaging applications. Others with funding to purchase an instrument should find the article helpful in deciding which technology is ideal for their area of research. PMID:25802490

Easy performance of 6-color confocal immunofluorescence with 4-laser line microscopes.

PubMed

Eissing, Nathalie; Heger, Lukas; Baranska, Anna; Cesnjevar, Robert; Büttner-Herold, Maike; Söder, Stephan; Hartmann, Arndt; Heidkamp, Gordon F; Dudziak, Diana

2014-09-01

Confocal laser scanning microscopy is an advanced technique for imaging tissue samples in vitro and in vivo at high optical resolution. The development of new fluorochrome variants do not only make it possible to perform multicolor flow cytometry of single cells, but in combination with high resolution laser scanning systems also to investigate the distribution of cells in lymphoid tissues by confocal immunofluorescence analyses, thus allowing the distinction of various cell populations directly in the tissue. Here, we provide a protocol for the visualization of at least six differently fluorochrome-labeled antibodies at the same time using a conventional confocal laser scanning microscope with four laser lines (405 nm, 488 nm, 555 nm, and 639 nm laser wavelength) in both murine and human tissue samples. We further demonstrate that compensation correction algorithms are not necessary to reduce spillover of fluorochromes into other channels when the used fluorochromes are combined according to their specific emission bands and the varying Stokes shift for co-excited fluorochromes with the same laser line. Copyright © 2014 Elsevier B.V. All rights reserved.

Use of a white light supercontinuum laser for confocal interference-reflection microscopy

PubMed Central

Chiu, L-D; Su, L; Reichelt, S; Amos, WB

2012-01-01

Shortly after its development, the white light supercontinuum laser was applied to confocal scanning microscopy as a more versatile substitute for the multiple monochromatic lasers normally used for the excitation of fluorescence. This light source is now available coupled to commercial confocal fluorescence microscopes. We have evaluated a supercontinuum laser as a source for a different purpose: confocal interferometric imaging of living cells and artificial models by interference reflection. We used light in the range 460–700 nm where this source provides a reasonably flat spectrum, and obtained images free from fringe artefacts caused by the longer coherence length of conventional lasers. We have also obtained images of cytoskeletal detail that is difficult to see with a monochromatic laser. PMID:22432542

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

PubMed Central

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.

PubMed

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.

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

PubMed Central

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

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

PubMed

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.

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

PubMed Central

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

2018-01-01

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. PMID:29675328

Differential polarization laser scanning microscopy: biological applications

NASA Astrophysics Data System (ADS)

Steinbach, G.; Besson, F.; Pomozi, I.; Garab, G.

2005-09-01

With the aid of a differential polarization (DP) apparatus, developed in our laboratory and attached to our laser scanning confocal microscope, we can measure the magnitude and spatial distribution of 8 different DP quantities: linear and circular dichroism (LD&CD), linear and circular anisotropy of the emission (R and CPL, confocal), fluorescence detected dichroisms (FDLD&FDCD, confocal), linear birefringence (LB), and the degree of polarization of fluorescence emission (P, confocal). The attachment uses high frequency modulation and subsequent demodulation, via lock-in amplifier, of the detected intensity values, and records and displays pixel-by-pixel the measured DP quantity. These microscopic DP data carry important physical information on the molecular architecture of anisotropically organized samples. Microscopic DP measurements are thought to be of particular importance in biology. In most biological samples anisotropy is difficult to determine with conventional, macroscopic DP measurements and microscopic variations are of special significance. In this paper, we describe the method of LB imaging. Using magnetically oriented isolated chloroplasts trapped in polyacrylamide gel, we demonstrate that LB can be determined with high sensitivity and good spatial resolution. Granal thylakoid membranes in edge-aligned orientation exhibited strong LB, with large variations in its sign and magnitude. In face-aligned position LB was considerably weaker, and tended to vanish when averaged for the whole image. The strong local variations are attributed to the inherent heterogeneity of the membranes, i.e. to their internal differentiation into multilamellar, stacked membranes (grana), and single thylakoids (stroma membranes). Further details and applications of our DP-LSM will be published elsewhere.

DIY: "Do Imaging Yourself" - Conventional microscopes as powerful tools for in vivo investigation.

PubMed

Antunes, Maísa Mota; Carvalho, Érika de; Menezes, Gustavo Batista

2018-01-01

Intravital imaging has been increasingly employed in cell biology studies and it is becoming one of the most powerful tools for in vivo investigation. Although some protocols can be extremely complex, most intravital imaging procedures can be performed using basic surgery and animal maintenance techniques. More importantly, regular confocal microscopes - the same that are used for imaging immunofluorescence slides - can also acquire high quality intravital images and movies after minor adaptations. Here we propose minimal adaptations in stock microscopes that allow major improvements in different fields of scientific investigation. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2011-03-01

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

Ex vivo confocal microscopy: a new diagnostic technique for mucormycosis.

PubMed

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.

Identification of nodal tissue in the living heart using rapid scanning fiber-optics confocal microscopy and extracellular fluorophores.

PubMed

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; P<0.05). Similar differences of spatial regularities were revealed from second-order image moments (50.0 ± 7.3% for AWM versus 29.3 ± 6.7% for SAN and 27.3 ± 5.5% for AVN; P<0.05). The study demonstrates feasibility of identifying nodal tissue in living heart using extracellular fluorophores and fiber-optics confocal microscopy. Application of the approach in pediatric reconstructive heart surgery may reduce risks of injuring nodal tissues.

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

PubMed

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.

The evolution of structured illumination microscopy in studies of HIV.

PubMed

Marno, Kelly; Al'Zoubi, Lara; Pearson, Matthew; Posch, Markus; McKnight, Áine; Wheeler, Ann P

2015-10-15

The resolution limit of conventional light microscopy has proven to be limiting for many biological structures such as viruses including Human immunodeficiency virus (HIV). Individual HIV virions are impossible to study using confocal microscopy as they are well below the 200 nm resolution limit of conventional light microscopes. Structured illumination microscopy (SIM) allows a twofold enhancement in image resolution compared to standard widefield illumination and so provides an excellent tool for study of HIV. Viral capsids (CAs) vary between 110 and 146 nm so this study challenges the performance of SIM microscopes. SIM microscopy was first developed in 2000, commercialised in 2007 and rapidly developed. Here we present the changes in capabilities of the SIM microscopes for study of HIV localisation as the instrumentation for structured illumination microscopy has evolved over the past 8 years. Copyright © 2015. Published by Elsevier Inc.

Purchase of a Laser Scanning Confocal Microscope at Xavier University of Louisiana

DTIC Science & Technology

2016-05-04

SECURITY CLASSIFICATION OF: The purpose of this grant was to purchase a laser scanning confocal microscope to be used by multiple laboratories at...was being developed for undergraduate education. Over the course of the funding period, the microscope was purchased and installed, multiple training...Distribution Unlimited UU UU UU UU 04-05-2016 1-Feb-2015 31-Jan-2016 Final Report: Purchase of a Laser Scanning Confocal Microscope at Xavier

Rapid Screening of Cancer Margins in Tissue with Multimodal Confocal Microscopy

PubMed Central

Gareau, Daniel S.; Jeon, Hana; Nehal, Kishwer S.; Rajadhyaksha, Milind

2012-01-01

Background Complete and accurate excision of cancer is guided by the examination of histopathology. However, preparation of histopathology is labor intensive and slow, leading to insufficient sampling of tissue and incomplete and/or inaccurate excision of margins. We demonstrate the potential utility of multimodal confocal mosaicing microscopy for rapid screening of cancer margins, directly in fresh surgical excisions, without the need for conventional embedding, sectioning or processing. Materials/Methods A multimodal confocal mosaicing microscope was developed to image basal cell carcinoma margins in surgical skin excisions, with resolution that shows nuclear detail. Multimodal contrast is with fluorescence for imaging nuclei and reflectance for cellular cytoplasm and dermal collagen. Thirtyfive excisions of basal cell carcinomas from Mohs surgery were imaged, and the mosaics analyzed by comparison to the corresponding frozen pathology. Results Confocal mosaics are produced in about 9 minutes, displaying tissue in fields-of-view of 12 mm with 2X magnification. A digital staining algorithm transforms black and white contrast to purple and pink, which simulates the appearance of standard histopathology. Mosaicing enables rapid digital screening, which mimics the examination of histopathology. Conclusions Multimodal confocal mosaicing microscopy offers a technology platform to potentially enable real-time pathology at the bedside. The imaging may serve as an adjunct to conventional histopathology, to expedite screening of margins and guide surgery toward more complete and accurate excision of cancer. PMID:22721570

A handheld MEMS-based line-scanned dual-axis confocal microscope for early cancer detection and surgical guidance (Conference Presentation)

NASA Astrophysics Data System (ADS)

Chen, Ye; Yin, Chengbo; Wei, Linpeng; Glaser, Adam K.; Abeytunge, Sanjee; Peterson, Gary; Mandella, Michael J.; Sanai, Nader; Rajadhyaksha, Milind; Liu, Jonathan T.

2017-02-01

Considerable efforts have been recently undertaken to develop miniature optical-sectioning microscopes for in vivo microendoscopy and point-of-care pathology. These devices enable in vivo interrogation of disease as a real-time and noninvasive alternative to gold-standard histopathology, and therefore could have a transformative impact for the early detection of cancer as well as for guiding tumor-resection procedures. Regardless of the specific modality, various trade-offs in size, speed, field of view, resolution, contrast, and sensitivity are necessary to optimize a device for a particular application. Here, a miniature MEMS-based line-scanned dual-axis confocal (LS-DAC) microscope, with a 12-mm diameter distal tip, has been developed for point-of-care pathology. The dual-axis architecture has demonstrated superior rejection of out-of-focus and multiply scattered photons compared to a conventional single-axis confocal configuration. The use of line scanning enables fast frame rates (≥15 frames/sec), which mitigates motion artifacts of a handheld device during clinical use. We have developed a method to actively align the illumination and collection beams in this miniature LS-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 application, enables the device to achieve an axial 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 that this high-speed LS-DAC microscope can achieve high-contrast imaging of fluorescently labeled tissues with sufficient sensitivity for applications such as oral cancer detection and guiding brain-tumor resections.

In vivo cellular imaging with microscopes enabled by MEMS scanners

NASA Astrophysics Data System (ADS)

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.

EVALUATION OF CONFOCAL MICROSCOPY SYSTEM PERFORMANCE

EPA Science Inventory

BACKGROUND. The confocal laser scanning microscope (CLSM) has enormous potential in many biological fields. Currently there is a subjective nature in the assessment of a confocal microscope's performance by primarily evaluating the system with a specific test slide provided by ea...

Design of small confocal endo-microscopic probe working under multiwavelength environment

NASA Astrophysics Data System (ADS)

Kim, Young-Duk; Ahn, MyoungKi; Gweon, Dae-Gab

2010-02-01

Recently, optical imaging system is widely used in medical purpose. By using optical imaging system specific diseases can be easily diagnosed at early stage because optical imaging system has high resolution performance and various imaging method. These methods are used to get high resolution image of human body and can be used to verify whether the cell is infected by virus. Confocal microscope is one of the famous imaging systems which is used for in-vivo imaging. Because most of diseases are accompanied with cellular level changes, doctors can diagnosis at early stage by observing the cellular image of human organ. Current research is focused in the development of endo-microscope that has great advantage in accessibility to human body. In this research, I designed small probe that is connected to confocal microscope through optical fiber bundle and work as endo-microscope. And this small probe is mainly designed to correct chromatic aberration to use various laser sources for both fluorescence type and reflection type confocal images. By using two kinds of laser sources at the same time we demonstrated multi-modality confocal endo-microscope.

Interference Confocal Microscope Integrated with Spatial Phase Shifter.

PubMed

Wang, Weibo; Gu, Kang; You, Xiaoyu; Tan, Jiubin; Liu, Jian

2016-08-24

We present an interference confocal microscope (ICM) with a new single-body four-step simultaneous phase-shifter device designed to obtain high immunity to vibration. The proposed ICM combines the respective advantages of simultaneous phase shifting interferometry and bipolar differential confocal microscopy to obtain high axis resolution, large dynamic range, and reduce the sensitivity to vibration and reflectance disturbance seamlessly. A compact single body spatial phase shifter is added to capture four phase-shifted interference signals simultaneously without time delay and construct a stable and space-saving simplified interference confocal microscope system. The test result can be obtained by combining the interference phase response and the bipolar property of differential confocal microscopy without phase unwrapping. Experiments prove that the proposed microscope is capable of providing stable measurements with 1 nm of axial depth resolution for either low- or high-numerical aperture objective lenses.

The design and construction of a cost-efficient confocal laser scanning microscope

NASA Astrophysics Data System (ADS)

Xi, Peng; Rajwa, Bartlomiej; Jones, James T.; Robinson, J. Paul

2007-03-01

The optical dissection ability of confocal microscopy makes it a powerful tool for biological materials. However, the cost and complexity of confocal scanning laser microscopy hinders its wide application in education. We describe the construction of a simplified confocal scanning laser microscope and demonstrate three-dimensional projection based on cost-efficient commercial hardware, together with available open source software.

Confined detection volume of fluorescence correlation spectroscopy by bare fiber probes.

PubMed

Lu, Guowei; Lei, Franck H; Angiboust, Jean-François; Manfait, Michel

2010-04-01

A fiber-tip-based near-field fluorescence correlation spectroscopy (FCS) has been developed for confining the detection volume to sub-diffraction-limited dimensions. This near-field FCS is based on near-field illumination by coupling a scanning near-field optical microscope (SNOM) to a conventional confocal FCS. Single-molecule FCS analysis at 100 nM Rhodamine 6G has been achieved by using bare chemically etched, tapered fiber tips. The detection volume under control of the SNOM system has been reduced over one order of magnitude compared to that of the conventional confocal FCS. Related factors influencing the near-field FCS performance are investigated and discussed in detail. In this proof-of-principle study, the preliminary experimental results suggest that the fiber-tip-based near-field FCS might be a good alternative to realize localized analysis at the single-molecule level.

Confocal microscopy patterns in nonmelanoma skin cancer and clinical applications.

PubMed

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.

Fast imaging with inelastically scattered electrons by off-axis chromatic confocal electron microscopy.

PubMed

Zheng, Changlin; Zhu, Ye; Lazar, Sorin; Etheridge, Joanne

2014-04-25

We introduce off-axis chromatic scanning confocal electron microscopy, a technique for fast mapping of inelastically scattered electrons in a scanning transmission electron microscope without a spectrometer. The off-axis confocal mode enables the inelastically scattered electrons to be chromatically dispersed both parallel and perpendicular to the optic axis. This enables electrons with different energy losses to be separated and detected in the image plane, enabling efficient energy filtering in a confocal mode with an integrating detector. We describe the experimental configuration and demonstrate the method with nanoscale core-loss chemical mapping of silver (M4,5) in an aluminium-silver alloy and atomic scale imaging of the low intensity core-loss La (M4,5@840  eV) signal in LaB6. Scan rates up to 2 orders of magnitude faster than conventional methods were used, enabling a corresponding reduction in radiation dose and increase in the field of view. If coupled with the enhanced depth and lateral resolution of the incoherent confocal configuration, this offers an approach for nanoscale three-dimensional chemical mapping.

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

PubMed Central

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

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 mini-microscopy 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. PMID:17465494

Evaluation and purchase of confocal microscopes: Numerous factors to consider

EPA Science Inventory

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...

Ophthalmic applications of confocal microscopy: diagnostics, refractive surgery, and eye banking

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1990-11-01

Confocal microscopy of ocular tissue provides two advantages over traditional imaging techniques: increased range and transverse resolution and increased contrast. The semitransparent cornea and ocular lens in the living eye can be optically sectioned and observed by reflected light confocal microscopy. Within the cornea we observed various cell components nerve fibers nerve cell bodies and fibrous networks. The confocal microscopic images from the in-situ ocular lens show the lens capsule the lens epithelium and the individual lens fibrils. All of the reflected light confocal microscopic images have high contrast and high resolution. Some of the applications of confocal imaging in ophthalmology include: diagnostics of the cornea and the ocular lens examination prior to and after refractive surgery examination of intraocular lenses (IOL) and examination of eye bank material. Other ophthalmic uses of confocal imaging include: studies of wound healing therapeutics and the effects of contact lenses on the cornea. The proposed features of a clinical confocal microscope are reviewed. 2.

Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology

PubMed Central

Sandell, Lisa L.; Kurosaka, Hiroshi; Trainor, Paul A.

2012-01-01

Here we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional widefield fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images of similar specimens produced by Scanning Electron Microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. PMID:22930523

Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology.

PubMed

Sandell, Lisa L; Kurosaka, Hiroshi; Trainor, Paul A

2012-11-01

Here, we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional wide field fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images produced by scanning electron microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. Copyright © 2012 Wiley Periodicals, Inc.

Pupil engineering for a confocal reflectance line-scanning microscope

NASA Astrophysics Data System (ADS)

Patel, Yogesh G.; Rajadhyaksha, Milind; DiMarzio, Charles A.

2011-03-01

Confocal reflectance microscopy may enable screening and diagnosis of skin cancers noninvasively and in real-time, as an adjunct to biopsy and pathology. Current confocal point-scanning systems are large, complex, and expensive. A confocal line-scanning microscope, utilizing a of linear array detector can be simpler, smaller, less expensive, and may accelerate the translation of confocal microscopy in clinical and surgical dermatology. A line scanner may be implemented with a divided-pupil, half used for transmission and half for detection, or with a full-pupil using a beamsplitter. The premise is that a confocal line-scanner with either a divided-pupil or a full-pupil will provide high resolution and optical sectioning that would be competitive to that of the standard confocal point-scanner. We have developed a confocal line-scanner that combines both divided-pupil and full-pupil configurations. This combined-pupil prototype is being evaluated to determine the advantages and limitations of each configuration for imaging skin, and comparison of performance to that of commercially available standard confocal point-scanning microscopes. With the combined configuration, experimental evaluation of line spread functions (LSFs), contrast, signal-to-noise ratio, and imaging performance is in progress under identical optical and skin conditions. Experimental comparisons between divided-pupil and full-pupil LSFs will be used to determine imaging performance. Both results will be compared to theoretical calculations using our previously reported Fourier analysis model and to the confocal point spread function (PSF). These results may lead to a simpler class of confocal reflectance scanning microscopes for clinical and surgical dermatology.

Confocal Fluorescence Microscopy of Mung Beanleaves

NASA Astrophysics Data System (ADS)

Chen, Zhiwei; Liu, Dongwu

Recently, confocal microscope has become a routine technique and indispensable tool for cell biological studies and molecular investigations. The light emitted from the point out-of-focus is blocked by the pinhole and can not reach the detector, which is one of the critical features of the confocal microscope. In present studies, the probes acridine orange (AO) and rhodamine-123 were used to research stoma and mitochondria of mung bean leaves, respectively. The results indicated that the stomatal guard cells and mitochondria were clearly seen in epidermic tissue of mung bean leaves. Taken together, it is a good method to research plant cells with confocal microscope and fluorescence probes.

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

PubMed

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.

In vivo imaging of oral neoplasia using a miniaturized fiber optic confocal reflectance microscope.

PubMed

Maitland, Kristen C; Gillenwater, Ann M; Williams, Michelle D; El-Naggar, Adel K; Descour, Michael R; Richards-Kortum, Rebecca R

2008-11-01

The purpose of this study was to determine whether in vivo images of oral mucosa obtained with a fiber optic confocal reflectance microscope could be used to differentiate normal and neoplastic tissues. We imaged 20 oral sites in eight patients undergoing surgery for squamous cell carcinoma. Normal and abnormal areas within the oral cavity were identified clinically, and real-time videos of each site were obtained in vivo using a fiber optic confocal reflectance microscope. Following imaging, each site was biopsied and submitted for histopathologic examination. We identified distinct features, such as nuclear irregularity and spacing, which can be used to qualitatively differentiate between normal and abnormal tissue. Representative confocal images of normal, pre-neoplastic, and neoplastic oral tissue are presented. Previous work using much larger microscopes has demonstrated the ability of confocal reflectance microscopy to image cellular and tissue architecture in situ. New advances in technology have enabled miniaturization of imaging systems for in vivo use.

Superresolution imaging of Drosophila tissues using expansion microscopy.

PubMed

Jiang, Nan; Kim, Hyeon-Jin; Chozinski, Tyler J; Azpurua, Jorge E; Eaton, Benjamin A; Vaughan, Joshua C; Parrish, Jay Z

2018-06-15

The limited resolving power of conventional diffraction-limited microscopy hinders analysis of small, densely packed structural elements in cells. Expansion microscopy (ExM) provides an elegant solution to this problem, allowing for increased resolution with standard microscopes via physical expansion of the specimen in a swellable polymer hydrogel. Here, we apply, validate, and optimize ExM protocols that enable the study of Drosophila embryos, larval brains, and larval and adult body walls. We achieve a lateral resolution of ∼70 nm in Drosophila tissues using a standard confocal microscope, and we use ExM to analyze fine intracellular structures and intercellular interactions. First, we find that ExM reveals features of presynaptic active zone (AZ) structure that are observable with other superresolution imaging techniques but not with standard confocal microscopy. We further show that synapses known to exhibit age-dependent changes in activity also exhibit age-dependent changes in AZ structure. Finally, we use the significantly improved axial resolution of ExM to show that dendrites of somatosensory neurons are inserted into epithelial cells at a higher frequency than previously reported in confocal microscopy studies. Altogether, our study provides a foundation for the application of ExM to Drosophila tissues and underscores the importance of tissue-specific optimization of ExM procedures.

Re-scan confocal microscopy: scanning twice for better resolution

PubMed Central

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. PMID:24298422

Diffraction-Unlimited Fluorescence Imaging with an EasySTED Retrofitted Confocal Microscope.

PubMed

Klauss, André; Hille, Carsten

2017-01-01

The easySTED technology provides the means to retrofit a confocal microscope to a diffraction-unlimited stimulated emission depletion (STED) microscope.Although commercial STED systems are available today, for many users of confocal laser scanning microscopes the option of retrofitting their confocal system to a STED system ready for diffraction-unlimited imaging may present an attractive option. The easySTED principle allowing for a joint beam path of excitation and depletion light promises some advantages concerning technical complexity and alignment effort for such an STED upgrade. In the one beam path design of easySTED the use of a common laser source, either a supercontinuum source or two separate lasers coupled into the same single-mode fiber, becomes feasible. The alignment of the focal light distribution of the STED beam relative to that of the excitation beam in all three spatial dimensions is therefore omitted respectively reduced to coupling the STED laser into the common single-mode fiber. Thus, only minor modifications need to be applied to the beam path in the confocal microscope to be upgraded. Those comprise adding polarization control elements and the easySTED waveplate, and adapting the beamsplitter to the excitation/STED wavelength combination.

Experiments on terahertz 3D scanning microscopic imaging

NASA Astrophysics Data System (ADS)

Zhou, Yi; Li, Qi

2016-10-01

Compared with the visible light and infrared, terahertz (THz) radiation can penetrate nonpolar and nonmetallic materials. There are many studies on the THz coaxial transmission confocal microscopy currently. But few researches on the THz dual-axis reflective confocal microscopy were reported. In this paper, we utilized a dual-axis reflective confocal scanning microscope working at 2.52 THz. In contrast with the THz coaxial transmission confocal microscope, the microscope adopted in this paper can attain higher axial resolution at the expense of reduced lateral resolution, revealing more satisfying 3D imaging capability. Objects such as Chinese characters "Zhong-Hua" written in paper with a pencil and a combined sheet metal which has three layers were scanned. The experimental results indicate that the system can extract two Chinese characters "Zhong," "Hua" or three layers of the combined sheet metal. It can be predicted that the microscope can be applied to biology, medicine and other fields in the future due to its favorable 3D imaging capability.

Sheet-scanned dual-axis confocal microscopy using Richardson-Lucy deconvolution.

PubMed

Wang, D; Meza, D; Wang, Y; Gao, L; Liu, J T C

2014-09-15

We have previously developed a line-scanned dual-axis confocal (LS-DAC) microscope with subcellular resolution suitable for high-frame-rate diagnostic imaging at shallow depths. Due to the loss of confocality along one dimension, the contrast (signal-to-background ratio) of a LS-DAC microscope is deteriorated compared to a point-scanned DAC microscope. However, by using a sCMOS camera for detection, a short oblique light-sheet is imaged at each scanned position. Therefore, by scanning the light sheet in only one dimension, a thin 3D volume is imaged. Both sequential two-dimensional deconvolution and three-dimensional deconvolution are performed on the thin image volume to improve the resolution and contrast of one en face confocal image section at the center of the volume, a technique we call sheet-scanned dual-axis confocal (SS-DAC) microscopy.

Detection of living Sarcoptes scabiei larvae by reflectance mode confocal microscopy in the skin of a patient with crusted scabies

NASA Astrophysics Data System (ADS)

Levi, Assi; Mumcuoglu, Kosta Y.; Ingber, Arieh; Enk, Claes D.

2012-06-01

Scabies is an intensely pruritic disorder induced by a delayed type hypersensitivity reaction to infestation of the skin by the mite Sarcoptes scabiei. The diagnosis of scabies is established clinically and confirmed by identifying mites or eggs by microscopic examination of scrapings from the skin or by surface microscopy using a dermatoscope. Reflectance-mode confocal microscopy is a novel technique used for noninvasive imaging of skin structures and lesions at a resolution compatible to that of conventional histology. Recently, the technique was employed for the confirmation of the clinical diagnosis of scabies. We demonstrate the first ever documentation of a larva moving freely inside the skin of a patient infected with scabies.

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

NASA Astrophysics Data System (ADS)

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.

Digital differential confocal microscopy based on spatial shift transformation.

PubMed

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.

Fluorescence confocal microscopy for pathologists.

PubMed

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 surgical specimens other than the skin and to evaluate the diagnostic capability of this technology from pathologists' viewpoint.

Evaluation of breast tissue with confocal strip-mosaicking microscopy: a test approach emulating pathology-like examination

PubMed Central

Abeytunge, Sanjee; Larson, Bjorg; Peterson, Gary; Morrow, Monica; Rajadhyaksha, Milind

2017-01-01

Abstract. Confocal microscopy is an emerging technology for rapid imaging of freshly excised tissue without the need for frozen- or fixed-section processing. Initial studies have described imaging of breast tissue using fluorescence confocal microscopy with small regions of interest, typically 750×750  μm2. We present exploration with a microscope, termed confocal strip-mosaicking microscope (CSM microscope), which images an area of 2×2  cm2 of tissue with cellular-level resolution in 10 min of excision. Using the CSM microscope, we imaged 34 fresh, human, large breast tissue specimens from 18 patients, blindly analyzed by a board-certified pathologist and subsequently correlated with the corresponding standard fixed histopathology. Invasive tumors and benign tissue were clearly identified in CSM strip-mosaic images. Thirty specimens were concordant for image-to-histopathology correlation while four were discordant. PMID:28327961

Note: A three-dimensional calibration device for the confocal microscope.

PubMed

Jensen, K E; Weitz, D A; Spaepen, F

2013-01-01

Modern confocal microscopes enable high-precision measurement in three dimensions by collecting stacks of 2D (x-y) images that can be assembled digitally into a 3D image. It is difficult, however, to ensure position accuracy, particularly along the optical (z) axis where scanning is performed by a different physical mechanism than in x-y. We describe a simple device to calibrate simultaneously the x, y, and z pixel-to-micrometer conversion factors for a confocal microscope. By taking a known 2D pattern and positioning it at a precise angle with respect to the microscope axes, we created a 3D reference standard. The device is straightforward to construct and easy to use.

Real-time, non-invasive microscopic confirmation of clinical diagnosis of bullous pemphigoid using in vivo reflectance confocal microscopy.

PubMed

Ardigò, M; Agozzino, M; Amorosi, B; Moscarella, E; Cota, C; de Abreu, L; Berardesca, E

2014-05-01

Bullous pemphigoid is an autoimmune disease affecting prevalently the elder. In vivo reflectance confocal microscopy is a non-invasive technique for real-time imaging of the skin with cellular-level resolution. No previous data has been reported about confocal microscopy of bullous pemphigoid. Aim of this preliminary study is the evaluation of the potential of in vivo reflectance confocal microscopy for real-time, microscopical confirmation of clinical bullous pemphigoid diagnosis. A total of nine lesions from patients affected by pemphigoid underwent in vivo reflectance confocal microscopy before histological examination. In our preliminary study, confocal microscopy showed high grade of correspondence to histopathology. In particular, presence of sub-epidermal cleft and variable amount of oedema of the upper dermis associated with inflammatory cells infiltration were seen as prevalent confocal features in the bullous lesions considered. Differently, in urticarial lesions, no specific features could be appreciated at confocal analysis beside the presence of signs of spongiosis and perivascular inflammation. Confocal microscopy seems to be useful for in vivo, microscopical confirmation of the clinical suspect of bullous pemphigoid and for biopsy site selection in urticarial lesions to obtain a more significant specimen for histopathological examination. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Increased numbers of Demodex in contact lens wearers.

PubMed

Jalbert, Isabelle; Rejab, Shazana

2015-06-01

The aim of this study was to determine if Demodex infestation is more frequent in contact lens wearers than in nonwearers. Secondary aims were to evaluate the effects of Demodex on the ocular surface (symptoms and signs) and to evaluate the ability of confocal laser scanning microscopy to detect and quantify the Demodex infestation compared with the conventional light microscopic technique. Forty Asian female participants (20 nonwearers, 20 lens wearers) with a mean (± SD) age of 27 (± 9) years were recruited. Ocular comfort scores (Ocular Surface Disease Index, Ocular Comfort Index, and Dry Eye Questionnaire), vital staining (corneal, conjunctival, and lid wiper), tear osmolarity, tear breakup time, and meibomian gland evaluation were evaluated. Demodex was detected using in vivo confocal microscopy and conventional light microscopy. The number of Demodex was higher in lens wearers than in nonwearers (7.6 [± 5.8] vs. 5.0 [± 3.1]; p = 0.02). Demodex was observed in a large majority (90%) of lens wearers and in 65% of nonwearers using confocal microscopy (p = 0.06). The detection rate was lower in both groups using conventional light microscopy (p = 0.003) where Demodex could only be confirmed in 70% and 60% of lens wearers and nonwearers, respectively. The number of Demodex tended to increase with age (ρ = 0.28, p = 0.08), but Demodex did not appear to affect ocular comfort or any clinical signs (p > 0.05). Contact lens wearers harbor Demodex as frequently as nonwearers and in higher numbers, which is best detected using in vivo confocal microscopy. The significance of these findings is uncertain because no associations were found with any symptoms and signs of dry eye disease.

Two-photon laser scanning microscopy with electrowetting-based prism scanning

PubMed Central

Supekar, Omkar D.; Ozbay, Baris N.; Zohrabi, Mo; Nystrom, Philip D.; Futia, Gregory L.; Restrepo, Diego; Gibson, Emily A.; Gopinath, Juliet T.; Bright, Victor M.

2017-01-01

Laser scanners are an integral part of high resolution biomedical imaging systems such as confocal or 2-photon excitation (2PE) microscopes. In this work, we demonstrate the utility of electrowetting on dielectric (EWOD) prisms as a lateral laser-scanning element integrated in a conventional 2PE microscope. To the best of our knowledge, this is the first such demonstration for EWOD prisms. EWOD devices provide a transmissive, low power consuming, and compact alternative to conventional adaptive optics, and hence this technology has tremendous potential. We demonstrate 2PE microscope imaging of cultured mouse hippocampal neurons with a FOV of 130 × 130 μm2 using EWOD prism scanning. In addition, we show simulations of the optical system with the EWOD prism, to evaluate the effect of propagating a Gaussian beam through the EWOD prism on the imaging quality. Based on the simulation results a beam size of 0.91 mm full width half max was chosen to conduct the imaging experiments, resulting in a numerical aperture of 0.17 of the imaging system. PMID:29296477

Microscopic Optical Projection Tomography In Vivo

PubMed Central

Meyer, Heiko; Ripoll, Jorge; Tavernarakis, Nektarios

2011-01-01

We describe a versatile optical projection tomography system for rapid three-dimensional imaging of microscopic specimens in vivo. Our tomographic setup eliminates the in xy and z strongly asymmetric resolution, resulting from optical sectioning in conventional confocal microscopy. It allows for robust, high resolution fluorescence as well as absorption imaging of live transparent invertebrate animals such as C. elegans. This system offers considerable advantages over currently available methods when imaging dynamic developmental processes and animal ageing; it permits monitoring of spatio-temporal gene expression and anatomical alterations with single-cell resolution, it utilizes both fluorescence and absorption as a source of contrast, and is easily adaptable for a range of small model organisms. PMID:21559481

Preliminary concept of confocal microscope rotor within the modular cultivation system for the space station

NASA Astrophysics Data System (ADS)

Fruit, Michel; Fuentes, Laure

2018-04-01

This paper, "Preliminary concept of confocal microscope rotor within the modular cultivation system for the space station," was presented as part of International Conference on Space Optics—ICSO 1997, held in Toulouse, France.

Virtual k -Space Modulation Optical Microscopy

NASA Astrophysics Data System (ADS)

Kuang, Cuifang; Ma, Ye; Zhou, Renjie; Zheng, Guoan; Fang, Yue; Xu, Yingke; Liu, Xu; So, Peter T. C.

2016-07-01

We report a novel superresolution microscopy approach for imaging fluorescence samples. The reported approach, termed virtual k -space modulation optical microscopy (VIKMOM), is able to improve the lateral resolution by a factor of 2, reduce the background level, improve the optical sectioning effect and correct for unknown optical aberrations. In the acquisition process of VIKMOM, we used a scanning confocal microscope setup with a 2D detector array to capture sample information at each scanned x -y position. In the recovery process of VIKMOM, we first modulated the captured data by virtual k -space coding and then employed a ptychography-inspired procedure to recover the sample information and correct for unknown optical aberrations. We demonstrated the performance of the reported approach by imaging fluorescent beads, fixed bovine pulmonary artery endothelial (BPAE) cells, and living human astrocytes (HA). As the VIKMOM approach is fully compatible with conventional confocal microscope setups, it may provide a turn-key solution for imaging biological samples with ˜100 nm lateral resolution, in two or three dimensions, with improved optical sectioning capabilities and aberration correcting.

Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems.

PubMed

Wang, E; Babbey, C M; Dunn, K W

2005-05-01

Fluorescence microscopy of the dynamics of living cells presents a special challenge to a microscope imaging system, simultaneously requiring both high spatial resolution and high temporal resolution, but with illumination levels low enough to prevent fluorophore damage and cytotoxicity. We have compared the high-speed Yokogawa CSU10 spinning disc confocal system with several conventional single-point scanning confocal (SPSC) microscopes, using the relationship between image signal-to-noise ratio and fluorophore photobleaching as an index of system efficiency. These studies demonstrate that the efficiency of the CSU10 consistently exceeds that of the SPSC systems. The high efficiency of the CSU10 means that quality images can be collected with much lower levels of illumination; the CSU10 was capable of achieving the maximum signal-to-noise of an SPSC system at illumination levels that incur only at 1/15th of the rate of the photobleaching of the SPSC system. Although some of the relative efficiency of the CSU10 system may be attributed to the use of a CCD rather than a photomultiplier detector system, our analyses indicate that high-speed imaging with the SPSC system is limited by fluorescence saturation at the high levels of illumination frequently needed to collect images at high frame rates. The high speed, high efficiency and freedom from fluorescence saturation combine to make the CSU10 effective for extended imaging of living cells at rates capable of capturing the three-dimensional motion of endosomes moving up to several micrometres per second.

Three-photon fluorescence imaging of melanin with a dual-wedge confocal scanning system

NASA Astrophysics Data System (ADS)

Mega, Yair; Kerimo, Joseph; Robinson, Joseph; Vakili, Ali; Johnson, Nicolette; DiMarzio, Charles

2012-03-01

Confocal microscopy can be used as a practical tool in non-invasive applications in medical diagnostics and evaluation. In particular, it is being used for the early detection of skin cancer to identify pathological cellular components and, potentially, replace conventional biopsies. The detection of melanin and its spatial location and distribution plays a crucial role in the detection and evaluation of skin cancer. Our previous work has shown that the visible emission from melanin is strong and can be easily observed with a near-infrared CW laser using low power. This is due to a unique step-wise, (SW) three-photon excitation of melanin. This paper shows that the same SW, 3-photon fluorescence can also be achieved with an inexpensive, continuous-wave laser using a dual-prism scanning system. This demonstrates that the technology could be integrated into a portable confocal microscope for clinical applications. The results presented here are in agreement with images obtained with the larger and more expensive femtosecond laser system used earlier.

Measurement of the index of refraction of μm crystals by a confocal laser microscope--potential application for the refractive index mapping of μm scale.

PubMed

Kimura, Keisaku; Sato, Seiichi

2014-05-01

A conventional laser microscope can be used to derive the index of refractivity by the ratio of geometrical height of the transparent platelet to the apparent height of the normal incident light for very small crystals in the wide size range. We demonstrate that the simple method is effective for the samples from 100 μm to 16 μm in size using alkali halide crystals as a model system. The method is also applied for the surface fractured micro-crystals and an inclined crystal with microscopic size regime. Furthermore, we present two-dimensional refractive index mapping as well as two-dimensional height profile for the mixture of three alkali halides, KCl, KI, and NaCl, all are μm in size.

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

PubMed

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.

Laser scanning confocal microscopy: history, applications, and related optical sectioning techniques.

PubMed

Paddock, Stephen W; Eliceiri, Kevin W

2014-01-01

Confocal microscopy is an established light microscopical technique for imaging fluorescently labeled specimens with significant three-dimensional structure. Applications of confocal microscopy in the biomedical sciences include the imaging of the spatial distribution of macromolecules in either fixed or living cells, the automated collection of 3D data, the imaging of multiple labeled specimens and the measurement of physiological events in living cells. The laser scanning confocal microscope continues to be chosen for most routine work although a number of instruments have been developed for more specific applications. Significant improvements have been made to all areas of the confocal approach, not only to the instruments themselves, but also to the protocols of specimen preparation, to the analysis, the display, the reproduction, sharing and management of confocal images using bioinformatics techniques.

A combined confocal and magnetic resonance microscope for biological studies

NASA Astrophysics Data System (ADS)

Majors, Paul D.; Minard, Kevin R.; Ackerman, Eric J.; Holtom, Gary R.; Hopkins, Derek F.; Parkinson, Christopher I.; Weber, Thomas J.; Wind, Robert A.

2002-12-01

Complementary data acquired with different microscopy techniques provide a basis for establishing a more comprehensive understanding of cell function in health and disease, particularly when results acquired with different methodologies can be correlated in time and space. In this article, a novel microscope is described for studying live cells simultaneously with both confocal scanning laser fluorescence optical microscopy and magnetic resonance microscopy. The various design considerations necessary for integrating these two complementary techniques are discussed, the layout and specifications of the instrument are given, and examples of confocal and magnetic resonance images of large frog cells and model tumor spheroids obtained with the compound microscope are presented.

In vivo fiber-optic confocal reflectance microscope with an injection-molded plastic miniature objective lens.

PubMed

Carlson, Kristen; Chidley, Matthew; Sung, Kung-Bin; Descour, Michael; Gillenwater, Ann; Follen, Michele; Richards-Kortum, Rebecca

2005-04-01

For in vivo optical diagnostic technologies to be distributed to the developed and developing worlds, optical imaging systems must be constructed of inexpensive components. We present a fiber-optic confocal reflectance microscope with a cost-effective injection-molded plastic miniature objective lens for in vivo imaging of human tissues in near real time. The measured lateral resolution is less than 2.2 microm, and the measured axial resolution is 10 microm. Confocal images of ex vivo cervical tissue biopsies and in vivo human lip taken at 15 frames/s demonstrate the microscope's capability of imaging cell morphology and tissue architecture.

Fiber-optic confocal reflectance microscope with miniature objective for in vivo imaging of human tissues.

PubMed

Sung, Kung-Bin; Liang, Chen; Descour, Michael; Collier, Tom; Follen, Michele; Richards-Kortum, Rebecca

2002-10-01

We have built a fiber-optic confocal reflectance microscope capable of imaging human tissues in near real time. Miniaturization of the objective lens and the mechanical components for positioning and axially scanning the objective enables the device to be used in inner organs of the human body. The lateral resolution is 2 micrometers and axial resolution is 10 micrometers. Confocal images of fixed tissue biopsies and the human lip in vivo have been obtained at 15 frames/s without any fluorescent stains. Both cell morphology and tissue architecture can be appreciated from images obtained with this microscope.

Laser excited confocal microscope fluorescence scanner and method

DOEpatents

Mathies, Richard A.; Peck, Konan

1992-01-01

A fluorescent scanner for scanning the fluorescence from a fluorescence labeled separated sample on a sample carrier including a confocal microscope for illuminating a predetermined volume of the sample carrier and/or receiving and processing fluorescence emissions from said volume to provide a display of the separated sample.

Laser scanning confocal microscope with programmable amplitude, phase, and polarization of the illumination beam.

PubMed

Boruah, B R; Neil, M A A

2009-01-01

We describe the design and construction of a laser scanning confocal microscope with programmable beam forming optics. The amplitude, phase, and polarization of the laser beam used in the microscope can be controlled in real time with the help of a liquid crystal spatial light modulator, acting as a computer generated hologram, in conjunction with a polarizing beam splitter and two right angled prisms assembly. Two scan mirrors, comprising an on-axis fast moving scan mirror for line scanning and an off-axis slow moving scan mirror for frame scanning, configured in a way to minimize the movement of the scanned beam over the pupil plane of the microscope objective, form the XY scan unit. The confocal system, that incorporates the programmable beam forming unit and the scan unit, has been implemented to image in both reflected and fluorescence light from the specimen. Efficiency of the system to programmably generate custom defined vector beams has been demonstrated by generating a bottle structured focal volume, which in fact is the overlap of two cross polarized beams, that can simultaneously improve both the lateral and axial resolutions if used as the de-excitation beam in a stimulated emission depletion confocal microscope.

Evaluation of penetration depth of 2% chlorhexidine digluconate into root dentinal tubules using confocal laser scanning microscope.

PubMed

Vadhana, Sekar; Latha, Jothi; Velmurugan, Natanasabapathy

2015-05-01

This study evaluated the penetration depth of 2% chlorhexidine digluconate (CHX) into root dentinal tubules and the influence of passive ultrasonic irrigation (PUI) using a confocal laser scanning microscope (CLSM). Twenty freshly extracted anterior teeth were decoronated and instrumented using Mtwo rotary files up to size 40, 4% taper. The samples were randomly divided into two groups (n = 10), that is, conventional syringe irrigation (CSI) and PUI. CHX was mixed with Rhodamine B dye and was used as the final irrigant. The teeth were sectioned at coronal, middle and apical levels and viewed under CLSM to record the penetration depth of CHX. The data were statistically analyzed using Kruskal-Wallis and Mann-Whitney U tests. The mean penetration depths of 2% CHX in coronal, middle and apical thirds were 138 µm, 80 µm and 44 µm in CSI group, respectively, whereas the mean penetration depths were 209 µm, 138 µm and 72 µm respectively in PUI group. Statistically significant difference was present between CSI group and PUI group at all three levels (p < 0.01 for coronal third and p < 0.001 for middle and apical thirds). On intragroup analysis, both groups showed statistically significant difference among three levels (p < 0.001). Penetration depth of 2% CHX into root dentinal tubules is deeper in coronal third when compared to middle and apical third. PUI aided in deeper penetration of 2% CHX into dentinal tubules when compared to conventional syringe irrigation at all three levels.

To boldly glow ... applications of laser scanning confocal microscopy in developmental biology.

PubMed

Paddock, S W

1994-05-01

The laser scanning confocal microscope (LSCM) is now established as an invaluable tool in developmental biology for improved light microscope imaging of fluorescently labelled eggs, embryos and developing tissues. The universal application of the LSCM in biomedical research has stimulated improvements to the microscopes themselves and the synthesis of novel probes for imaging biological structures and physiological processes. Moreover the ability of the LSCM to produce an optical series in perfect register has made computer 3-D reconstruction and analysis of light microscope images a practical option.

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

NASA Astrophysics Data System (ADS)

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.

Laser excited confocal microscope fluorescence scanner and method

DOEpatents

Mathies, R.A.; Peck, K.

1992-02-25

A fluorescent scanner is designed for scanning the fluorescence from a fluorescence labeled separated sample on a sample carrier. The scanner includes a confocal microscope for illuminating a predetermined volume of the sample carrier and/or receiving and processing fluorescence emissions from the volume to provide a display of the separated sample. 8 figs.

Fluorescence (Multiwave) Confocal Microscopy.

PubMed

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.

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

NASA Astrophysics Data System (ADS)

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.

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

NASA Astrophysics Data System (ADS)

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.

Design considerations of a real-time clinical confocal microscope

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1991-06-01

A real-time clinical confocal light microscope provides the ophthalmologist with a new tool for the observation of the cornea and the ocular lens. In addition, the ciliary body, the iris, and the sclera can be observed. The real-time light microscopic images have high contrast and resolution. The transverse resolution is about one half micron and the range resolution is one micron. The following observations were made with visible light: corneal epithelial cells, wing cells, basal cells, Bowman's membrane, nerve fibers, basal lamina, fibroblast nuclei, Descemet's membrane, endothelial cells. Observation of the in situ ocular lens showed lens capsule, lens epithelium, lens fibrils, the interior of lens fibrils. The applications of the confocal microscope include: eye banking, laser refractive surgery, observation of wound healing, observation of the iris, the sciera, the ciliary body, the ocular lens, and the intraocular lens. Digital image processing can produce three-dimensional reconstructions of the cornea and the ocular lens.

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

PubMed

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.

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

NASA Astrophysics Data System (ADS)

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.

Model wavefront sensor for adaptive confocal microscopy

NASA Astrophysics Data System (ADS)

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.

Wide spectral range confocal microscope based on endlessly single-mode fiber.

PubMed

Hubbard, R; Ovchinnikov, Yu B; Hayes, J; Richardson, D J; Fu, Y J; Lin, S D; See, P; Sinclair, A G

2010-08-30

We report an endlessly single mode, fiber-optic confocal microscope, based on a large mode area photonic crystal fiber. The microscope confines a very broad spectral range of excitation and emission wavelengths to a single spatial mode in the fiber. Single-mode operation over an optical octave is feasible. At a magnification of 10 and λ = 900 nm, its resolution was measured to be 1.0 μm (lateral) and 2.5 μm (axial). The microscope's use is demonstrated by imaging single photons emitted by individual InAs quantum dots in a pillar microcavity.

A high-resolution, confocal laser-scanning microscope and flash photolysis system for physiological studies.

PubMed

Parker, I; Callamaras, N; Wier, W G

1997-06-01

We describe the construction of a high-resolution confocal laser-scanning microscope, and illustrate its use for studying elementary Ca2+ signalling events in cells. An avalanche photodiode module and simple optical path provide a high efficiency system for detection of fluorescence signals, allowing use of a small confocal aperture giving near diffraction-limited spatial resolution (< 300 nm lateral and < 400 nm axial). When operated in line-scan mode, the maximum temporal resolution is 1 ms, and the associated computer software allows complete flexibility to record line-scans continuously for long (minutes) periods or to obtain any desired pixel resolution in x-y scans. An independent UV irradiation system permits simultaneous photolysis of caged compounds over either a uniform, wide field (arc lamp source) or at a tightly focussed spot (frequency-tripled Nd:YAG laser). The microscope thus provides a versatile tool for optical studies of dynamic cellular processes, as well as excellent resolution for morphological studies. The confocal scanner can be added to virtually any inverted microscope for a component cost that is only a small fraction of that of comparable commercial instruments, yet offers better performance and greater versatility.

CONFOCAL LASER SCANNING MICROSCOPY OF RAT FOLLICLE DEVELOPMENT

EPA Science Inventory

This study used confocal laser scanning microscopy (CLSM) to study follicular development in millimeter pieces of rat ovary. To use this technology, it is essential to stain the tissue before laser excitation with the confocal microscope. Various fluorescent stains (Yo-Pro, Bo-Pr...

Miniaturized fiber-coupled confocal fluorescence microscope with an electrowetting variable focus lens using no moving parts

PubMed Central

Ozbay, Baris N.; Losacco, Justin T.; Cormack, Robert; Weir, Richard; Bright, Victor M.; Gopinath, Juliet T.; Restrepo, Diego; Gibson, Emily A.

2015-01-01

We report a miniature, lightweight fiber-coupled confocal fluorescence microscope that incorporates an electrowetting variable focus lens to provide axial scanning for full three-dimensional (3D) imaging. Lateral scanning is accomplished by coupling our device to a laser-scanning confocal microscope through a coherent imaging fiber-bundle. The optical components of the device are combined in a custom 3D-printed adapter with an assembled weight of <2 g that can be mounted onto the head of a mouse. Confocal sectioning provides an axial resolution of ~12 µm and an axial scan range of ~80 µm. The lateral field-of-view is 300 µm, and the lateral resolution is 1.8 µm. We determined these parameters by imaging fixed sections of mouse neuronal tissue labeled with green fluorescent protein (GFP) and fluorescent bead samples in agarose gel. To demonstrate viability for imaging intact tissue, we resolved multiple optical sections of ex vivo mouse olfactory nerve fibers expressing yellow fluorescent protein (YFP). PMID:26030555

Towards real-time image deconvolution: application to confocal and STED microscopy

PubMed Central

Zanella, R.; Zanghirati, G.; Cavicchioli, R.; Zanni, L.; Boccacci, P.; Bertero, M.; Vicidomini, G.

2013-01-01

Although deconvolution can improve the quality of any type of microscope, the high computational time required has so far limited its massive spreading. Here we demonstrate the ability of the scaled-gradient-projection (SGP) method to provide accelerated versions of the most used algorithms in microscopy. To achieve further increases in efficiency, we also consider implementations on graphic processing units (GPUs). We test the proposed algorithms both on synthetic and real data of confocal and STED microscopy. Combining the SGP method with the GPU implementation we achieve a speed-up factor from about a factor 25 to 690 (with respect the conventional algorithm). The excellent results obtained on STED microscopy images demonstrate the synergy between super-resolution techniques and image-deconvolution. Further, the real-time processing allows conserving one of the most important property of STED microscopy, i.e the ability to provide fast sub-diffraction resolution recordings. PMID:23982127

Analysis of protein and lipid dynamics using confocal fluorescence recovery after photobleaching (FRAP)

PubMed Central

Day, Charles A.; Kraft, Lewis J.; Kang, Minchul; Kenworthy, Anne K.

2012-01-01

Fluorescence recovery after photobleaching (FRAP) is a powerful, versatile and widely accessible tool to monitor molecular dynamics in living cells that can be performed using modern confocal microscopes. Although the basic principles of FRAP are simple, quantitative FRAP analysis requires careful experimental design, data collection and analysis. In this review we discuss the theoretical basis for confocal FRAP, followed by step-by-step protocols for FRAP data acquisition using a laser scanning confocal microscope for (1) measuring the diffusion of a membrane protein, (2) measuring the diffusion of a soluble protein, and (3) analysis of intracellular trafficking. Finally, data analysis procedures are discussed and an equation for determining the diffusion coefficient of a molecular species undergoing pure diffusion is presented. PMID:23042527

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

PubMed

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.

Fiber-based confocal microscope for cryogenic spectroscopy.

PubMed

Högele, Alexander; Seidl, Stefan; Kroner, Martin; Karrai, Khaled; Schulhauser, Christian; Sqalli, Omar; Scrimgeour, Jan; Warburton, Richard J

2008-02-01

We describe the design and performance of a fiber-based confocal microscope for cryogenic operation. The microscope combines positioning at low temperatures along three space coordinates of millimeter translation and nanometer precision with high stability and optical performance at the diffraction limit. It was successfully tested under ambient conditions as well as at liquid nitrogen (77 K) and liquid helium (4 K) temperatures. The compact nonmagnetic design provides for long term position stability against helium refilling transfers, temperature sweeps, as well as magnetic field variation between -9 and 9 T. As a demonstration of the microscope performance, applications in the spectroscopy of single semiconductor quantum dots are presented.

Conjugation of both on-axis and off-axis light in Nipkow disk confocal microscope to increase availability of incoherent light source.

PubMed

Saito, Kenta; Arai, Yoshiyuki; Zhang, Jize; Kobayashi, Kentaro; Tani, Tomomi; Nagai, Takeharu

2011-01-01

Laser-scanning confocal microscopy has been employed for exploring structures at subcellular, cellular and tissue level in three dimensions. To acquire the confocal image, a coherent light source, such as laser, is generally required in conventional single-point scanning microscopy. The illuminating beam must be focused onto a small spot with diffraction-limited size, and this determines the spatial resolution of the microscopy system. In contrast, multipoint scanning confocal microscopy using a Nipkow disk enables the use of an incoherent light source. We previously demonstrated successful application of a 100 W mercury arc lamp as a light source for the Yokogawa confocal scanner unit in which a microlens array was coupled with a Nipkow disk to focus the collimated incident light onto a pinhole (Saito et al., Cell Struct. Funct., 33: 133-141, 2008). However, transmission efficiency of incident light through the pinhole array was low because off-axis light, the major component of the incident light, was blocked by the non-aperture area of the disk. To improve transmission efficiency, we propose an optical system in which off-axis light is able to be transmitted through pinholes surrounding the pinhole located on the optical axis of the collimator lens. This optical system facilitates the use of not only the on-axis but also the off-axis light such that the available incident light is considerably improved. As a result, we apply the proposed system to high-speed confocal and multicolor imaging both with a satisfactory signal-to-noise ratio.

Noninvasive, label-free, three-dimensional imaging of melanoma with confocal photothermal microscopy: Differentiate malignant melanoma from benign tumor tissue

NASA Astrophysics Data System (ADS)

He, Jinping; Wang, Nan; Tsurui, Hiromichi; Kato, Masashi; Iida, Machiko; Kobayashi, Takayoshi

2016-07-01

Skin cancer is one of the most common cancers. Melanoma accounts for less than 2% of skin cancer cases but causes a large majority of skin cancer deaths. Early detection of malignant melanoma remains the key factor in saving lives. However, the melanoma diagnosis is still clinically challenging. Here, we developed a confocal photothermal microscope for noninvasive, label-free, three-dimensional imaging of melanoma. The axial resolution of confocal photothermal microscope is ~3 times higher than that of commonly used photothermal microscope. Three-dimensional microscopic distribution of melanin in pigmented lesions of mouse skin is obtained directly with this setup. Classic morphometric and fractal analysis of sixteen 3D images (eight for benign melanoma and eight for malignant) showed a capability of pathology of melanoma: melanin density and size become larger during the melanoma growth, and the melanin distribution also becomes more chaotic and unregulated. The results suggested new options for monitoring the melanoma growth and also for the melanoma diagnosis.

Effect of photon-initiated photoacoustic streaming, passive ultrasonic, and sonic irrigation techniques on dentinal tubule penetration of irrigation solution: a confocal microscopic study.

PubMed

Akcay, Merve; Arslan, Hakan; Mese, Merve; Durmus, Nazlı; Capar, Ismail Davut

2017-09-01

The aim of this in vitro study was to evaluate the efficacy of different irrigation techniques including laser-activated irrigation using an erbium:yttrium-aluminum-garnet (Er:YAG) laser with a novel tip design (photon-induced photoacoustic streaming (PIPS)), Er:YAG laser with Preciso tip, sonic activation, and passive ultrasonic activation on the final irrigation solution penetration into dentinal tubules by using a laser scanning confocal microscope. In this study, 65 extracted single-rooted human mandibular premolars were instrumented up to size 40 and randomly divided into 5 groups (n = 13) based on the activation technique of the final irrigation solution as follows: conventional irrigation (control group), sonic activation, passive ultrasonic activation, Er:YAG-PIPS tip activation, and Er:YAG-Preciso tip activation. In each group, 5 mL of 5% NaOCl labeled with fluorescent dye was used during the activation as the final irrigation solution. Specimens were sectioned at 2.5 and 8 mm from the apex and then examined under a confocal microscope to calculate the dentinal tubule penetration area. Data were analyzed using two-way analysis of variance (ANOVA) and Tukey's post hoc tests (P = 0.05). Both Er:YAG laser (Preciso/PIPS) activations exhibited a significantly higher penetration area than the other groups (P < 0.05). Additionally, passive ultrasonic activation had significantly higher penetration than the sonic activation group and the control group. Statistically significant differences were also found between each root canal third (coronal > middle > apical) (P < 0.001). The results from the present study support the use of Er:YAG laser activation (Preciso/PIPS) to improve the effectiveness of the final irrigation procedure by increasing the irrigant penetration area into the dentinal tubules. The activation of the irrigant and the creation of the streaming with the Er:YAG laser have a positive effect on the irrigant penetration.

High-resolution confocal Raman microscopy using pixel reassignment.

PubMed

Roider, Clemens; Ritsch-Marte, Monika; Jesacher, Alexander

2016-08-15

We present a practical modification of fiber-coupled confocal Raman scanning microscopes that is able to provide high confocal resolution in conjunction with high light collection efficiency. For this purpose, the single detection fiber is replaced by a hexagonal lenslet array in combination with a hexagonally packed round-to-linear multimode fiber bundle. A multiline detector is used to collect individual Raman spectra for each fiber. Data post-processing based on pixel reassignment allows one to improve the lateral resolution by up to 41% compared to a single fiber of equal light collection efficiency. We present results from an experimental implementation featuring seven collection fibers, yielding a resolution improvement of about 30%. We believe that our implementation represents an attractive upgrade for existing confocal Raman microscopes that employ multi-line detectors.

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

PubMed Central

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

Using Cell-ID 1.4 with R for Microscope-Based Cytometry

PubMed Central

Bush, Alan; Chernomoretz, Ariel; Yu, Richard; Gordon, Andrew

2012-01-01

This unit describes a method for quantifying various cellular features (e.g., volume, total and subcellular fluorescence localization) from sets of microscope images of individual cells. It includes procedures for tracking cells over time. One purposefully defocused transmission image (sometimes referred to as bright-field or BF) is acquired to segment the image and locate each cell. Fluorescent images (one for each of the color channels to be analyzed) are then acquired by conventional wide-field epifluorescence or confocal microscopy. This method uses the image processing capabilities of Cell-ID (Gordon et al., 2007, as updated here) and data analysis by the statistical programming framework R (R-Development-Team, 2008), which we have supplemented with a package of routines for analyzing Cell-ID output. Both Cell-ID and the analysis package are open-source. PMID:23026908

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

PubMed Central

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. PMID:21280920

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy.

PubMed

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.

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

NASA Astrophysics Data System (ADS)

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.

Use of digital micromirror devices as dynamic pinhole arrays for adaptive confocal fluorescence microscopy

NASA Astrophysics Data System (ADS)

Pozzi, Paolo; Wilding, Dean; Soloviev, Oleg; Vdovin, Gleb; Verhaegen, Michel

2018-02-01

In this work, we present a new confocal laser scanning microscope capable to perform sensorless wavefront optimization in real time. The device is a parallelized laser scanning microscope in which the excitation light is structured in a lattice of spots by a spatial light modulator, while a deformable mirror provides aberration correction and scanning. A binary DMD is positioned in an image plane of the detection optical path, acting as a dynamic array of reflective confocal pinholes, images by a high performance cmos camera. A second camera detects images of the light rejected by the pinholes for sensorless aberration correction.

How Confocal Is Confocal Raman Microspectroscopy on the Skin? Impact of Microscope Configuration and Sample Preparation on Penetration Depth Profiles.

PubMed

Lunter, Dominique Jasmin

2016-01-01

The aim of the study was to elucidate the effect of sample preparation and microscope configuration on the results of confocal Raman microspectroscopic evaluation of the penetration of a pharmaceutical active into the skin (depth profiling). Pig ear skin and a hydrophilic formulation containing procaine HCl were used as a model system. The formulation was either left on the skin during the measurement, or was wiped off or washed off prior to the analysis. The microscope configuration was varied with respect to objectives and pinholes used. Sample preparation and microscope configuration had a tremendous effect on the results of depth profiling. Regarding sample preparation, the best results could be observed when the formulation was washed off the skin prior to the analysis. Concerning microscope configuration, the use of a 40 × 0.6 numerical aperture (NA) objective in combination with a 25-µm pinhole or a 100 × 1.25 NA objective in combination with a 50-µm pinhole was found to be advantageous. Complete removal of the sample from the skin before the analysis was found to be crucial. A thorough analysis of the suitability of the chosen microscope configuration should be performed before acquiring concentration depth profiles. © 2016 S. Karger AG, Basel.

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

PubMed

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.

ConfocalCheck - A Software Tool for the Automated Monitoring of Confocal Microscope Performance

PubMed Central

Hng, Keng Imm; Dormann, Dirk

2013-01-01

Laser scanning confocal microscopy has become an invaluable tool in biomedical research but regular quality testing is vital to maintain the system’s performance for diagnostic and research purposes. Although many methods have been devised over the years to characterise specific aspects of a confocal microscope like measuring the optical point spread function or the field illumination, only very few analysis tools are available. Our aim was to develop a comprehensive quality assurance framework ranging from image acquisition to automated analysis and documentation. We created standardised test data to assess the performance of the lasers, the objective lenses and other key components required for optimum confocal operation. The ConfocalCheck software presented here analyses the data fully automatically. It creates numerous visual outputs indicating potential issues requiring further investigation. By storing results in a web browser compatible file format the software greatly simplifies record keeping allowing the operator to quickly compare old and new data and to spot developing trends. We demonstrate that the systematic monitoring of confocal performance is essential in a core facility environment and how the quantitative measurements obtained can be used for the detailed characterisation of system components as well as for comparisons across multiple instruments. PMID:24224017

Real-Time Confocal Imaging Of The Living Eye

NASA Astrophysics Data System (ADS)

Jester, James V.; Cavanagh, H. Dwight; Essepian, John; Shields, William J.; Lemp, Michael A.

1989-12-01

In 1986, we adapted the Tandem Scanning Reflected Light Microscope of Petran and Hadraysky to permit non-invasive, confocal imaging of the living eye in real-time. We were first to obtain stable, confocal optical sections in vivo, from human and animal eyes. Using confocal imaging systems we have now studied living, normal volunteers, rabbits, cats and primates sequentially, non-invasively, and in real-time. The continued development of real-time confocal imaging systems will unlock the door to a new field of cell biology involving for the first time the study of dynamic cellular processes in living organ systems. Towards this end we have concentrated our initial studies on three areas (1) evaluation of confocal microscope systems for real-time image acquisition, (2) studies of the living normal cornea (epithelium, stroma, endothelium) in human and other species; and (3) sequential wound-healing responses in the cornea in single animals to lamellar-keratectomy injury (cellular migration, inflammation, scarring). We believe that this instrument represents an important, new paradigm for research in cell biology and pathology and that it will fundamentally alter all experimental and clinical approaches in future years.

Swept Field Laser Confocal Microscopy for Enhanced Spatial and Temporal Resolution in Live-Cell Imaging

PubMed Central

Castellano-Muñoz, Manuel; Peng, Anthony Wei; Salles, Felipe T.; Ricci, Anthony J.

2013-01-01

Confocal fluorescence microscopy is a broadly used imaging technique that enhances the signal-to-noise ratio by removing out of focal plane fluorescence. Confocal microscopes come with a variety of modifications depending on the particular experimental goals. Microscopes, illumination pathways, and light collection were originally focused upon obtaining the highest resolution image possible, typically on fixed tissue. More recently, live-cell confocal imaging has gained importance. Since measured signals are often rapid or transient, thus requiring higher sampling rates, specializations are included to enhance spatial and temporal resolution while maintaining tissue viability. Thus, a balance between image quality, temporal resolution, and tissue viability is needed. A subtype of confocal imaging, termed swept field confocal (SFC) microscopy, can image live cells at high rates while maintaining confocality. SFC systems can use a pinhole array to obtain high spatial resolution, similar to spinning disc systems. In addition, SFC imaging can achieve faster rates by using a slit to sweep the light across the entire image plane, thus requiring a single scan to generate an image. Coupled to a high-speed charge-coupled device camera and a laser illumination source, images can be obtained at greater than 1,000 frames per second while maintaining confocality. PMID:22831554

Fast parallel 3D profilometer with DMD technology

NASA Astrophysics Data System (ADS)

Hou, Wenmei; Zhang, Yunbo

2011-12-01

Confocal microscope has been a powerful tool for three-dimensional profile analysis. Single mode confocal microscope is limited by scanning speed. This paper presents a 3D profilometer prototype of parallel confocal microscope based on DMD (Digital Micromirror Device). In this system the DMD takes the place of Nipkow Disk which is a classical parallel scanning scheme to realize parallel lateral scanning technique. Operated with certain pattern, the DMD generates a virtual pinholes array which separates the light into multi-beams. The key parameters that affect the measurement (pinhole size and the lateral scanning distance) can be configured conveniently by different patterns sent to DMD chip. To avoid disturbance between two virtual pinholes working at the same time, a scanning strategy is adopted. Depth response curve both axial and abaxial were extract. Measurement experiments have been carried out on silicon structured sample, and axial resolution of 55nm is achieved.

Confocal fluorescence microscope with dual-axis architecture and biaxial postobjective scanning

PubMed Central

Wang, Thomas D.; Contag, Christopher H.; Mandella, Michael J.; Chan, Ning Y.; Kino, Gordon S.

2007-01-01

We present a novel confocal microscope that has dual-axis architecture and biaxial postobjective scanning for the collection of fluorescence images from biological specimens. This design uses two low-numerical-aperture lenses to achieve high axial resolution and long working distance, and the scanning mirror located distal to the lenses rotates along the orthogonal axes to produce arc-surface images over a large field of view (FOV). With fiber optic coupling, this microscope can potentially be scaled down to millimeter dimensions via microelectromechanical systems (MEMS) technology. We demonstrate a benchtop prototype with a spatial resolution ≤4.4 μm that collects fluorescence images with a high SNR and a good contrast ratio from specimens expressing GFP. Furthermore, the scanning mechanism produces only small differences in aberrations over the image FOV. These results demonstrate proof of concept of the dual-axis confocal architecture for in vivo molecular and cellular imaging. PMID:15250760

Imaging inflammation in mouse colon using a rapid stage-scanning confocal fluorescence microscope

NASA Astrophysics Data System (ADS)

Saldua, Meagan A.; Olsovsky, Cory A.; Callaway, Evelyn S.; Chapkin, Robert S.; Maitland, Kristen C.

2012-01-01

Large area confocal microscopy may provide fast, high-resolution image acquisition for evaluation of tissue in pre-clinical studies with reduced tissue processing in comparison to histology. We present a rapid beam and stage-scanning confocal fluorescence microscope to image cellular and tissue features along the length of the entire excised mouse colon. The beam is scanned at 8,333 lines/sec by a polygon scanning mirror while the specimen is scanned in the orthogonal axis by a motorized translation stage with a maximum speed of 7 mm/sec. A single 1×60 mm2 field of view image spanning the length of the mouse colon is acquired in 10 s. Z-projection images generated from axial image stacks allow high resolution imaging of the surface of non-flat specimens. In contrast to the uniform size, shape, and distribution of colon crypts in confocal images of normal colon, confocal images of chronic bowel inflammation exhibit heterogeneous tissue structure with localized severe crypt distortion.

Effects of benoxinate hydrochloride 0.4% on the morphological appearance of the cornea using confocal microscopy.

PubMed

Perez-Gomez, Inma; Hollingsworth, Jo; Efron, Nathan

2004-03-01

To investigate whether benoxinate hydrochloride 0.4% used to make confocal microscopy more comfortable alters the morphology of the cornea as viewed with the confocal microscope. Confocal microscopy was performed on both eyes of 10 subjects prior to instillation of either topical anaesthetic or non-preserved sterile saline, on two randomly ordered occasions. Images of all corneal layers were analysed quantitatively and qualitatively in a masked fashion. The images were similar in appearance in 5/10 subjects, there was greater clarity when anaesthetic was instilled in 4/10 subjects, and in the remaining subject there was greater clarity when saline was used. Anaesthetic had no influence on anterior keratocyte density (AKD), posterior keratocyte density (PKD) or endothelial cell density (ECD). Local anaesthetic does not affect corneal morphology as imaged using the confocal microscope. However, failure to use anaesthetic may lead to a degradation of image quality due to patient discomfort and excessive eye movements.

A Case of Bilateral Descemet's Membrane and Subepithelial Opacity: In vivo Laser Confocal Microscopic Study.

PubMed

Hatta, Yukiko; Yokogawa, Hideaki; Kobayashi, Akira; Torisaki, Makoto; Sugiyama, Kazuhisa

2013-01-01

To report the in vivo laser confocal microscopy findings from a patient with Descemet's membrane and subepithelial opacity OU. A healthy 41-year-old male with Descemet's membrane and subepithelial opacity OU was studied. Routine ophthalmic examination, standard slit-lamp biomicroscopy, and in vivo laser confocal microscopic analysis of the entire corneal layer were performed. Slit-lamp biomicroscopy revealed subepithelial opacity in the mid-peripheral to peripheral cornea and numerous opacities located at the level of Descemet's membrane. It was difficult to distinguish the precise histological location of the opacity. In vivo laser confocal microscopy showed numerous hyperreflective particles in the subepithelium to superficial stroma and hyperreflectivity of Descemet's membrane. No abnormalities could be detected in the epithelial cell layer, midstromal layer, deep stromal layer, or endothelial cell layer. Although the origin of the corneal opacities was unclear, in vivo laser confocal microscopy was useful for observing microstructural abnormalities in a case of Descemet's membrane and subepithelial opacity.

A Case of Bilateral Descemet's Membrane and Subepithelial Opacity: In vivo Laser Confocal Microscopic Study

PubMed Central

Hatta, Yukiko; Yokogawa, Hideaki; Kobayashi, Akira; Torisaki, Makoto; Sugiyama, Kazuhisa

2013-01-01

Purpose To report the in vivo laser confocal microscopy findings from a patient with Descemet's membrane and subepithelial opacity OU. Case Report A healthy 41-year-old male with Descemet's membrane and subepithelial opacity OU was studied. Routine ophthalmic examination, standard slit-lamp biomicroscopy, and in vivo laser confocal microscopic analysis of the entire corneal layer were performed. Slit-lamp biomicroscopy revealed subepithelial opacity in the mid-peripheral to peripheral cornea and numerous opacities located at the level of Descemet's membrane. It was difficult to distinguish the precise histological location of the opacity. In vivo laser confocal microscopy showed numerous hyperreflective particles in the subepithelium to superficial stroma and hyperreflectivity of Descemet's membrane. No abnormalities could be detected in the epithelial cell layer, midstromal layer, deep stromal layer, or endothelial cell layer. Conclusion Although the origin of the corneal opacities was unclear, in vivo laser confocal microscopy was useful for observing microstructural abnormalities in a case of Descemet's membrane and subepithelial opacity. PMID:23626574

Immunogold labeling reveals subcellular localisation of silica nanoparticles in a human blood-brain barrier model

NASA Astrophysics Data System (ADS)

Ye, Dong; Anguissola, Sergio; O'Neill, Tiina; Dawson, Kenneth A.

2015-05-01

Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles.Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles. Electronic supplementary information (ESI) available: Nanoparticle characterisation data, preservation of cellular structures, staining controls, optimisation of size amplification via the silver enhancement, and more imaging results from anti-clathrin and anti-caveolin 1 immunolabeling. See DOI: 10.1039/c5nr01539a

Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry.

PubMed

Schwertner, M; Booth, M J; Neil, M A A; Wilson, T

2004-01-01

Confocal or multiphoton microscopes, which deliver optical sections and three-dimensional (3D) images of thick specimens, are widely used in biology. These techniques, however, are sensitive to aberrations that may originate from the refractive index structure of the specimen itself. The aberrations cause reduced signal intensity and the 3D resolution of the instrument is compromised. It has been suggested to correct for aberrations in confocal microscopes using adaptive optics. In order to define the design specifications for such adaptive optics systems, one has to know the amount of aberrations present for typical applications such as with biological samples. We have built a phase stepping interferometer microscope that directly measures the aberration of the wavefront. The modal content of the wavefront is extracted by employing Zernike mode decomposition. Results for typical biological specimens are presented. It was found for all samples investigated that higher order Zernike modes give only a small contribution to the overall aberration. Therefore, these higher order modes can be neglected in future adaptive optics sensing and correction schemes implemented into confocal or multiphoton microscopes, leading to more efficient designs.

Development of confocal laser microscope system for examination of microscopic characteristics of radiophotoluminescence glass dosemeters.

PubMed

Maki, Daisuke; Ishii, Tetsuya; Sato, Fuminobu; Kato, Yushi; Yamamoto, Takayoshi; Iida, Toshiyuki

2011-03-01

A confocal laser microscope system was developed for the measurement of radiophotoluminescence (RPL) photons emitted from a minute alpha-ray-irradiated area in an RPL glass dosemeter. The system was composed mainly of an inverted-type microscope, an ultraviolet laser, an XY movable stage and photon-counting circuits. The photon-counting circuits were effective in the reduction of the background noise level in the measurement of RPL photons. The performance of this microscope system was examined by the observation of standard RPL glass samples irradiated using (241)Am alpha rays. The spatial resolution of this system was ∼ 3 μm, and with regard to the sensitivity of this system, a hit of more than four to five alpha rays in unit area produced enough amount of RPL photons to construct the image.

Chromatic confocal microscope using hybrid aspheric diffractive lenses

NASA Astrophysics Data System (ADS)

Rayer, Mathieu; Mansfield, Daniel

2014-05-01

A chromatic confocal microscope is a single point non-contact distance measurement sensor. For three decades the vast majority of the chromatic confocal microscope use refractive-based lenses to code the measurement axis chromatically. However, such an approach is limiting the range of applications. In this paper the performance of refractive, diffractive and Hybrid aspheric diffractive are compared. Hybrid aspheric diffractive lenses combine the low geometric aberration of a diffractive lens with the high optical power of an aspheric lens. Hybrid aspheric diffractive lenses can reduce the number of elements in an imaging system significantly or create large hyper- chromatic lenses for sensing applications. In addition, diffractive lenses can improve the resolution and the dynamic range of a chromatic confocal microscope. However, to be suitable for commercial applications, the diffractive optical power must be significant. Therefore, manufacturing such lenses is a challenge. We show in this paper how a theoretical manufacturing model can demonstrate that the hybrid aspheric diffractive configuration with the best performances is achieved by step diffractive surface. The high optical quality of step diffractive surface is then demonstrated experimentally. Publisher's Note: This paper, originally published on 5/10/14, was replaced with a corrected/revised version on 5/19/14. If you downloaded the original PDF but are unable to access the revision, please contact SPIE Digital Library Customer Service for assistance.

Inhibition of biofilm development of uropathogens by curcumin - an anti-quorum sensing agent from Curcuma longa.

PubMed

Packiavathy, Issac Abraham Sybiya Vasantha; Priya, Selvam; Pandian, Shunmugiah Karutha; Ravi, Arumugam Veera

2014-04-01

Urinary tract infection is caused primarily by the quorum sensing (QS)-dependent biofilm forming ability of uropathogens. In the present investigation, an anti-quorum sensing (anti-QS) agent curcumin from Curcuma longa (turmeric) was shown to inhibit the biofilm formation of uropathogens, such as Escherichia coli, Pseudomonas aeruginosa PAO1, Proteus mirabilis and Serratia marcescens, possibly by interfering with their QS systems. The antibiofilm potential of curcumin on uropathogens as well as its efficacy in disturbing the mature biofilms was examined under light microscope and confocal laser scanning microscope. The treatment with curcumin was also found to attenuate the QS-dependent factors, such as exopolysaccharide production, alginate production, swimming and swarming motility of uropathogens. Furthermore, it was documented that curcumin enhanced the susceptibility of a marker strain and uropathogens to conventional antibiotics. Copyright © 2012 Elsevier Ltd. All rights reserved.

A novel system for water soluble protein encapsulation with high efficiency: "micelles enhanced" polyelectrolyte capsules.

PubMed

Li, Xiaodong; Li, Xiaohui; Zhang, Jianxiang; Zhao, Shifang; Shen, Jiacong

2008-06-01

Novel "micelles enhanced" polyelectrolyte (PE) capsules based on functional templates of hybrid calcium carbonate were fabricated. Evidences suggested that the structure of capsule wall was different from that of conventional PE capsules, and the wall permeability of these PE capsules changed significantly. Lysozyme, a positively charged protein in neutral solution, was studied as a model protein to be encapsulated into the "micelles enhanced" PE capsules. Confocal laser scanning microscope was used to observe the entrapping process in real time, while UV-Vis spectroscope and scanning force microscope measurements suggested the high efficiency of encapsulation. In addition, the fluorescence recovery after photobleaching technique was employed to determine the existence form of deposited molecules. Further studies showed even negatively charged water-soluble peptides or proteins can be encapsulated into these hybrid capsules by modulating the pH value in bulk solution under its isoelectronic point as well. Copyright 2007 Wiley Periodicals, Inc.

Zone-specific cell biosynthetic activity in mature bovine articular cartilage: a new method using confocal microscopic stereology and quantitative autoradiography.

PubMed

Wong, M; Wuethrich, P; Eggli, P; Hunziker, E

1996-05-01

A new methodology was developed to measure spatial variations in chondrocyte/matrix structural parameters and chondrocyte biosynthetic activity in articular cartilage. This technique is based on the use of a laser scanning confocal microscope that can "optically" section chemically fixed, unembedded tissue. The confocal images are used for morphometric measurement of stereologic parameters such as cell density (cells/mm3), cell volume fraction (%), surface density (l/cm), mean cell volume (micron3), and mean cell surface area (micron2). Adjacent pieces of tissue are simultaneously processed for conventional liquid emulsion autoradiography, and a semiautomated grain counting program is used to measure the silver grain density at regions corresponding to the same sites used for structural measurements. An estimate of chondrocyte biosynthetic activity in terms of grains per cell is obtained by dividing the value for grain density by that for cell density. In this paper, the newly developed methodology was applied to characterize the zone-specific behavior of adult articular cartilage in the free-swelling state. Cylinders of young adult bovine articular cartilage were labelled with either [3H]proline or [35S]sulfate, and chondrocyte biosynthesis and structural parameters were measured from the articular surface to the tidemark. The results showed that chondrocytes of the radial zone occupied twice the volume and surface area of the chondrocytes of the superficial zone but were 10 times more synthetically active. This efficient and unbiased technique may prove useful in studying the correlation between mechanically induced changes in cell form and biosynthetic activity within inhomogeneous tissue as well as metabolic changes in cartilage due to ageing and disease.

Validating Intravascular Imaging with Serial Optical Coherence Tomography and Confocal Fluorescence Microscopy.

PubMed

Tardif, Pier-Luc; Bertrand, Marie-Jeanne; Abran, Maxime; Castonguay, Alexandre; Lefebvre, Joël; Stähli, Barbara E; Merlet, Nolwenn; Mihalache-Avram, Teodora; Geoffroy, Pascale; Mecteau, Mélanie; Busseuil, David; Ni, Feng; Abulrob, Abedelnasser; Rhéaume, Éric; L'Allier, Philippe; Tardif, Jean-Claude; Lesage, Frédéric

2016-12-15

Atherosclerotic cardiovascular diseases are characterized by the formation of a plaque in the arterial wall. Intravascular ultrasound (IVUS) provides high-resolution images allowing delineation of atherosclerotic plaques. When combined with near infrared fluorescence (NIRF), the plaque can also be studied at a molecular level with a large variety of biomarkers. In this work, we present a system enabling automated volumetric histology imaging of excised aortas that can spatially correlate results with combined IVUS/NIRF imaging of lipid-rich atheroma in cholesterol-fed rabbits. Pullbacks in the rabbit aortas were performed with a dual modality IVUS/NIRF catheter developed by our group. Ex vivo three-dimensional (3D) histology was performed combining optical coherence tomography (OCT) and confocal fluorescence microscopy, providing high-resolution anatomical and molecular information, respectively, to validate in vivo findings. The microscope was combined with a serial slicer allowing for the imaging of the whole vessel automatically. Colocalization of in vivo and ex vivo results is demonstrated. Slices can then be recovered to be tested in conventional histology.

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

PubMed Central

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

2016-01-01

Objectives 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. Methods We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. Results 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. Conclusions 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. PMID:27525165

Image formation of volume holographic microscopy using point spread functions

NASA Astrophysics Data System (ADS)

Luo, Yuan; Oh, Se Baek; Kou, Shan Shan; Lee, Justin; Sheppard, Colin J. R.; Barbastathis, George

2010-04-01

We present a theoretical formulation to quantify the imaging properties of volume holographic microscopy (VHM). Volume holograms are formed by exposure of a photosensitive recording material to the interference of two mutually coherent optical fields. Recently, it has been shown that a volume holographic pupil has spatial and spectral sectioning capability for fluorescent samples. Here, we analyze the point spread function (PSF) to assess the imaging behavior of the VHM with a point source and detector. The coherent PSF of the VHM is derived, and the results are compared with those from conventional microscopy, and confocal microscopy with point and slit apertures. According to our analysis, the PSF of the VHM can be controlled in the lateral direction by adjusting the parameters of the VH. Compared with confocal microscopes, the performance of the VHM is comparable or even potentially better, and the VHM is also able to achieve real-time and three-dimensional (3D) imaging due to its multiplexing ability.

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

PubMed

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.

CALIBRATION AND VALIDATION OF CONFOCAL SPECTRAL IMAGING SYSTEMS

EPA Science Inventory

Confocal spectral imaging (CSI) microscope systems now on the market can perform spectral characterization of biological specimens containing fluorescent proteins, labels or dyes. Some CSI have been found to present inconsistent spectral characterizations within a particular syst...

Second-harmonic patterned polarization-analyzed reflection confocal microscope

NASA Astrophysics Data System (ADS)

Okoro, Chukwuemeka; Toussaint, Kimani C.

2017-08-01

We introduce the second-harmonic patterned polarization-analyzed reflection confocal (SPPARC) microscope-a multimodal imaging platform that integrates Mueller matrix polarimetry with reflection confocal and second-harmonic generation (SHG) microscopy. SPPARC microscopy provides label-free three-dimensional (3-D), SHG-patterned confocal images that lend themselves to spatially dependent, linear polarimetric analysis for extraction of rich polarization information based on the Mueller calculus. To demonstrate its capabilities, we use SPPARC microscopy to analyze both porcine tendon and ligament samples and find differences in both circular degree-of-polarization and depolarization parameters. Moreover, using the collagen-generated SHG signal as an endogenous counterstain, we show that the technique can be used to provide 3-D polarimetric information of the surrounding extrafibrillar matrix plus cells or EFMC region. The unique characteristics of SPPARC microscopy holds strong potential for it to more accurately and quantitatively describe microstructural changes in collagen-rich samples in three spatial dimensions.

Design, assembly, and optical bench testing of a high-numerical-aperture miniature injection-molded objective for fiber-optic confocal reflectance microscopy.

PubMed

Chidley, Matthew D; Carlson, Kristen D; Richards-Kortum, Rebecca R; Descour, Michael R

2006-04-10

The design, analysis, assembly methods, and optical-bench test results for a miniature injection-molded plastic objective lens used in a fiber-optic confocal reflectance microscope are presented. The five-lens plastic objective was tested as a stand-alone optical system before its integration into a confocal microscope for in vivo imaging of cells and tissue. Changing the spacing and rotation of the individual optical elements can compensate for fabrication inaccuracies and improve performance. The system performance of the miniature objective lens is measured by use of an industry-accepted slanted-edge modulation transfer function (MTF) metric. An estimated Strehl ratio of 0.61 and a MTF value of 0.66 at the fiber-optic bundle Nyquist frequency have been obtained. The optical bench testing system is configured to permit interactive optical alignment during testing to optimize performance. These results are part of an effort to demonstrate the manufacturability of low-cost, high-performance biomedical optics for high-resolution in vivo imaging. Disposable endoscopic microscope objectives could help in vivo confocal microscopy technology mature to permit wide-scale clinical screening and detection of early cancers and precancerous lesions.

Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

PubMed

Ma, Qian; Khademhosseinieh, Bahar; Huang, Eric; Qian, Haoliang; Bakowski, Malina A; Troemel, Emily R; Liu, Zhaowei

2016-08-16

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume.

First identification of the herpes simplex virus by skin-dedicated ex vivo fluorescence confocal microscopy during herpetic skin infections.

PubMed

Cinotti, E; Perrot, J L; Labeille, B; Campolmi, N; Thuret, G; Naigeon, N; Bourlet, T; Pillet, S; Cambazard, F

2015-06-01

Skin-dedicated ex vivo fluorescence confocal microscopy (FCM) has so far been used to identify cutaneous tumours on freshly excised samples using acridine orange as fluorochrome. To use FCM for a new indication, namely, the identification of the herpes simplex virus (HSV) in skin lesions, using fluorescent antibodies. Six roof samples from skin vesicles suspicious for HSV lesions were incubated with anti-HSV-1 and anti-HSV-2 antibodies coupled with fluorescein isothiocyanate, and examined under skin-dedicated ex vivo FCM. The positive controls were swabs taken from the floor of each vesicle and observed under conventional direct fluorescence assay (DFA) and by viral cultures. Roof samples from three bullae of bullous pemphigoid were the negative controls. Using ex vivo FCM, the samples from the lesions clinically suspicious for HSV infection were seen to be fluorescent after incubation with anti-HSV-1, and were negative after incubation with anti-HSV-2 antibodies. Conventional DFA with an optical microscope and cultures confirmed the presence of HSV-1 infection. By using fluorescent antibodies to identify precise structures, ex vivo FCM can be used for indications other than tumour identification. More specifically, it can be an additional diagnostic tool for HSV infection. © 2014 British Association of Dermatologists.

Improving confocal microscopy with solid-state semiconductor excitation sources

NASA Astrophysics Data System (ADS)

Sivers, Nelson L.

To efficiently excite the fluorescent dyes used in imaging biological samples with a confocal microscope, the wavelengths of the exciting laser must be near the fluorochrome absorption peak. However, this causes imaging problems when the fluorochrome absorption and emission spectra overlap significantly, i.e. have small Stokes shifts, which is the case for most fluorochromes that emit in the red to infrared. As a result, the reflected laser excitation cannot be distinguished from the information-containing fluorescence signal. However, cryogenically cooling the exciting laser diode enabled the laser emission wavelengths to be tuned to shorter wavelengths, decreasing the interference between the laser and the fluorochrome's fluorescence. This reduced the amount of reflected laser light in the confocal image. However, the cooled laser diode's shorter wavelength signal resulted in slightly less efficient fluorochrome excitation. Spectrophotometric analysis showed that as the laser diodes were cooled, their output power increased, which more than compensated for the lower fluorochrome excitation and resulted in significantly more intense fluorescence. Thus, by tuning the laser diode emission wavelengths away from the fluorescence signal, less reflected laser light and more fluorescence information reached the detector, creating images with better signal to noise ratios. Additionally, new, high, luminous flux, light-emitting diodes (LEDs) are now powerful enough to create confocal fluorescence signals comparable to those produced by the traditional laser excitation sources in fluorescence confocal microscopes. The broader LED spectral response effectively excited the fluorochrome, yet was spectrally limited enough for standard filter sets to separate the LED excitation from the fluorochrome fluorescence signal. Spectrophotometric analysis of the excitation and fluorescence spectra of several fluorochromes showed that high-powered, LED-induced fluorescence contained the same spectral information and could be more intense than that produced by lasers. An alternative, LED-based, confocal microscope is proposed in this thesis that would be capable of exciting multiple fluorochromes in a single specimen, producing images of several distinct cellular components simultaneously. The inexpensive, LED-based, confocal microscope would require lower peak excitation intensities to produce fluorescence signals equal to those produced by laser excitation, reducing cellular damage and slowing fluorochrome photobleaching.

Optimal pupil design for confocal microscopy

NASA Astrophysics Data System (ADS)

Patel, Yogesh G.; Rajadhyaksha, Milind; DiMarzio, Charles A.

2010-02-01

Confocal reflectance microscopy may enable screening and diagnosis of skin cancers noninvasively and in real-time, as an adjunct to biopsy and pathology. Current instruments are large, complex, and expensive. A simpler, confocal line-scanning microscope may accelerate the translation of confocal microscopy in clinical and surgical dermatology. A confocal reflectance microscope may use a beamsplitter, transmitting and detecting through the pupil, or a divided pupil, or theta configuration, with half used for transmission and half for detection. The divided pupil may offer better sectioning and contrast. We present a Fourier optics model and compare the on-axis irradiance of a confocal point-scanning microscope in both pupil configurations, optimizing the profile of a Gaussian beam in a circular or semicircular aperture. We repeat both calculations with a cylindrical lens which focuses the source to a line. The variable parameter is the fillfactor, h, the ratio of the 1/e2 diameter of the Gaussian beam to the diameter of the full aperture. The optimal values of h, for point scanning are 0.90 (full) and 0.66 for the half-aperture. For line-scanning, the fill-factors are 1.02 (full) and 0.52 (half). Additional parameters to consider are the optimal location of the point-source beam in the divided-pupil configuration, the optimal line width for the line-source, and the width of the aperture in the divided-pupil configuration. Additional figures of merit are field-of-view and sectioning. Use of optimal designs is critical in comparing the experimental performance of the different configurations.

CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: SPECTROSCOPY

EPA Science Inventory

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...

WAVELENGTH AND ALIGNMENT TESTS FOR CONFOCAL SPECTRAL IMAGING SYSTEMS

EPA Science Inventory

Confocal spectral imaging (CSI) microscope systems now on the market delineate multiple fluorescent proteins, labels, or dyes within biological specimens by performing spectral characterizations. However, we find that some CSI present inconsistent spectral profiles of reference s...

A light sheet confocal microscope for image cytometry with a variable linear slit detector

NASA Astrophysics Data System (ADS)

Hutcheson, Joshua A.; Khan, Foysal Z.; Powless, Amy J.; Benson, Devin; Hunter, Courtney; Fritsch, Ingrid; Muldoon, Timothy J.

2016-03-01

We present a light sheet confocal microscope (LSCM) capable of high-resolution imaging of cell suspensions in a microfluidic environment. In lieu of conventional pressure-driven flow or mechanical translation of the samples, we have employed a novel method of fluid transport, redox-magnetohydrodynamics (redox-MHD). This method achieves fluid motion by inducing a small current into the suspension in the presence of a magnetic field via electrodes patterned onto a silicon chip. This on-chip transportation requires no moving parts, and is coupled to the remainder of the imaging system. The microscopy system comprises a 450 nm diode 20 mW laser coupled to a single mode fiber and a cylindrical lens that converges the light sheet into the back aperture of a 10x, 0.3 NA objective lens in an epi-illumination configuration. The emission pathway contains a 150 mm tube lens that focuses the light onto the linear sensor at the conjugate image plane. The linear sensor (ELiiXA+ 8k/4k) has three lateral binning modes which enables variable detection aperture widths between 5, 10, or 20 μm, which can be used to vary axial resolution. We have demonstrated redox-MHD-enabled light sheet microscopy in suspension of fluorescent polystyrene beads. This approach has potential as a high-throughput image cytometer with myriad cellular diagnostic applications.

Visualizing substructure of Ca2+ waves by total internal reflection fluorescence microscopy

NASA Astrophysics Data System (ADS)

Bai, Yongqiang; Tang, Aihui; Wang, Shiqiang; Zhu, Xing

2005-02-01

Total internal reflection fluorescence microscope is a new optical microscopic system based on near-field optical theory. Its character of illumination by evanescent wave, together with the great signal-to-noise ratio and temporal resolution achieved by high quality CCD, allows us to analyze the spatiotemporal details of local Ca2+ dynamics within the nanoscale microdomain surrounding different Ca2+ channels. We have recently constructed a versatile objective TIRFM equipped with a high numerical aperture (NA=1.45) objective. Using fluo-4 as the Ca2+ indicator, we visualized the near-membrane profiles of Ca2+ waves and elementary Ca2+ sparks generated by Ca2+ release channels in rat ventricular myocytes. Different from those detected using conventional and confocal microscopy, Ca2+ waves observed with TIRFM exhibited fine inhomogenous substructures composed of fluctuating Ca2+ sparks. The anfractuous routes of spark recruitment suggested that the propagation of Ca2+ waves is much more complicated than previously imagined. We believe that TIRFM will provide a unique tool for dissecting the microscopic mechanisms of intracellular Ca2+ signaling.

Intracellular Protein Delivery for Treating Breast Cancer

DTIC Science & Technology

2012-06-01

characterized by confocal microscopy, and rhodamine-labeled apoptin can be observed in the nuclei of cancer cells only. Released apoptin induced tumor...acquired on a Yokogawa spinning-disk confocal scanner system (Solamere Technology Group, Salt Lake City, UT) using a Nikon eclipse Ti-E microscope...protein localization using confocal microscopy, two cancer cell lines HeLa and MCF-7, together with the noncancerous human foreskin fibroblast (HFF), were

CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: LASER POWER MEASUREMENTS

EPA Science Inventory

Laser power abstract
The reliability of the confocal laser-scanning microscope (CLSM) to obtain intensity measurements and quantify fluorescence data is dependent on using a correctly aligned machine that contains a stable laser power. The laser power test appears to be one ...

CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: SPECTROSCOPY AND FOUNDATIONS FOR QUANTITATION

EPA Science Inventory

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...

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

EPA Science Inventory

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...

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

EPA Science Inventory

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. ...

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

NASA Astrophysics Data System (ADS)

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 (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.

High temporal and spatial resolution studies of bone cells using real-time confocal reflection microscopy.

PubMed

Boyde, A; Vesely, P; Gray, C; Jones, S J

1994-01-01

Chick and rat bone-derived cells were mounted in sealed coverslip-covered chambers; individual osteoclasts (but also osteoblasts) were selected and studied at 37 degrees C using three different types of high-speed scanning confocal microscopes: (1) A Noran Tandem Scanning Microscope (TSM) was used with a low light level, cooled CCD camera for image transfer to a Noran TN8502 frame store-based image analysing computer to make time lapse movie sequences using 0.1 s exposure periods, thus losing some of the advantage of the high frame rate of the TSM. Rapid focus adjustment using computer controlled piezo drivers permitted two or more focus planes to be imaged sequentially: thus (with additional light-source shuttering) the reflection confocal image could be alternated with the phase contrast image at a different focus. Individual cells were followed for up to 5 days, suggesting no significant irradiation problem. (2) Exceptional temporal and spatial resolution is available in video rate laser confocal scanning microscopes (VRCSLMs). We used the Noran Odyssey unitary beam VRCSLM with an argon ion laser at 488 nm and acousto-optic deflection (AOD) on the line axis: this instrument is truly and adjustably confocal in the reflection mode. (3) We also used the Lasertec 1LM11 line scan instrument, with an He-Ne laser at 633 nm, and AOD for the frame scan. We discuss the technical problems and merits of the different approaches. The VRCSLMs documented rapid, real-time oscillatory motion: all the methods used show rapid net movement of organelles within bone cells. The interference reflection mode gives particularly strong contrasts in confocal instruments. Phase contrast and other interference methods used in the microscopy of living cells can be used simultaneously in the TSM.

Confocal laser induced fluorescence with comparable spatial localization to the conventional method

NASA Astrophysics Data System (ADS)

Thompson, Derek S.; Henriquez, Miguel F.; Scime, Earl E.; Good, Timothy N.

2017-10-01

We present measurements of ion velocity distributions obtained by laser induced fluorescence (LIF) using a single viewport in an argon plasma. A patent pending design, which we refer to as the confocal fluorescence telescope, combines large objective lenses with a large central obscuration and a spatial filter to achieve high spatial localization along the laser injection direction. Models of the injection and collection optics of the two assemblies are used to provide a theoretical estimate of the spatial localization of the confocal arrangement, which is taken to be the full width at half maximum of the spatial optical response. The new design achieves approximately 1.4 mm localization at a focal length of 148.7 mm, improving on previously published designs by an order of magnitude and approaching the localization achieved by the conventional method. The confocal method, however, does so without requiring a pair of separated, perpendicular optical paths. The confocal technique therefore eases the two window access requirement of the conventional method, extending the application of LIF to experiments where conventional LIF measurements have been impossible or difficult, or where multiple viewports are scarce.

Potential application of a handheld confocal endomicroscope imaging system using a variety of fluorophores in experimental gliomas and normal brain.

PubMed

Martirosyan, Nikolay L; Georges, Joseph; Eschbacher, Jennifer M; Cavalcanti, Daniel D; Elhadi, Ali M; Abdelwahab, Mohammed G; Scheck, Adrienne C; Nakaji, Peter; Spetzler, Robert F; Preul, Mark C

2014-02-01

The authors sought to assess the feasibility of a handheld visible-wavelength confocal endomicroscope imaging system (Optiscan 5.1, Optiscan Pty., Ltd.) using a variety of rapid-acting fluorophores to provide histological information on gliomas, tumor margins, and normal brain in animal models. Mice (n = 25) implanted with GL261 cells were used to image fluorescein sodium (FNa), 5-aminolevulinic acid (5-ALA), acridine orange (AO), acriflavine (AF), and cresyl violet (CV). A U251 glioma xenograft model in rats (n = 5) was used to image sulforhodamine 101 (SR101). A swine (n = 3) model with AO was used to identify confocal features of normal brain. Images of normal brain, obvious tumor, and peritumoral zones were collected using the handheld confocal endomicroscope. Histological samples were acquired through biopsies from matched imaging areas. Samples were visualized with a benchtop confocal microscope. Histopathological features in corresponding confocal images and photomicrographs of H & E-stained tissues were reviewed. Fluorescence induced by FNa, 5-ALA, AO, AF, CV, and SR101 and detected with the confocal endomicroscope allowed interpretation of histological features. Confocal endomicroscopy revealed satellite tumor cells within peritumoral tissue, a definitive tumor border, and striking fluorescent cellular and subcellular structures. Fluorescence in various tumor regions correlated with standard histology and known tissue architecture. Characteristic features of different areas of normal brain were identified as well. Confocal endomicroscopy provided rapid histological information precisely related to the site of microscopic imaging with imaging characteristics of cells related to the unique labeling features of the fluorophores. Although experimental with further clinical trial validation required, these data suggest that intraoperative confocal imaging can help to distinguish normal brain from tumor and tumor margin and may have application in improving intraoperative decisions during resection of brain tumors.

Fluorescence microscopy.

PubMed

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.

Miniature near-infrared dual-axes confocal microscope utilizing a two-dimensional microelectromechanical systems scanner

PubMed Central

Liu, Jonathan T. C.; Mandella, Michael J.; Ra, Hyejun; Wong, Larry K.; Solgaard, Olav; Kino, Gordon S.; Piyawattanametha, Wibool; Contag, Christopher H.; Wang, Thomas D.

2007-01-01

The first, to our knowledge, miniature dual-axes confocal microscope has been developed, with an outer diameter of 10 mm, for subsurface imaging of biological tissues with 5–7 μm resolution. Depth-resolved en face images are obtained at 30 frames per second, with a field of view of 800 × 100 μm, by employing a two-dimensional scanning microelectromechanical systems mirror. Reflectance and fluorescence images are obtained with a laser source at 785 nm, demonstrating the ability to perform real-time optical biopsy. PMID:17215937

Fluorescence Microscopy

PubMed Central

Sanderson, Michael J.; Smith, Ian; Parker, Ian; Bootman, Martin D.

2016-01-01

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. PMID:25275114

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

PubMed

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.

Confocal Raman spectroscopic analysis of cross-linked ultra-high molecular weight polyethylene for application in artificial hip joints.

PubMed

Pezzotti, Giuseppe; Kumakura, Tsuyoshi; Yamada, Kiyotaka; Tateiwa, Toshiyuki; Puppulin, Leonardo; Zhu, Wenliang; Yamamoto, Kengo

2007-01-01

Confocal spectroscopic techniques are applied to selected Raman bands to study the microscopic features of acetabular cups made of ultra-high molecular weight polyethylene (UHMWPE) before and after implantation in vivo. The micrometric lateral resolution of a laser beam focused on the polymeric surface (or subsurface) enables a highly resolved visualization of 2-D conformational population patterns, including crystalline, amorphous, orthorhombic phase fractions, and oxidation index. An optimized confocal probe configuration, aided by a computational deconvolution of the optical probe, allows minimization of the probe size along the in-depth direction and a nondestructive evaluation of microstructural properties along the material subsurface. Computational deconvolution is also attempted, based on an experimental assessment of the probe response function of the polyethylene Raman spectrum, according to a defocusing technique. A statistical set of high-resolution microstructural data are collected on a fully 3-D level on gamma-ray irradiated UHMWPE acetabular cups both as-received from the maker and after retrieval from a human body. Microstructural properties reveal significant gradients along the immediate material subsurface and distinct differences are found due to the loading history in vivo, which cannot be revealed by conventional optical spectroscopy. The applicability of the confocal spectroscopic technique is valid beyond the particular retrieval cases examined in this study, and can be easily extended to evaluate in-vitro tested components or to quality control of new polyethylene brands. Confocal Raman spectroscopy may also contribute to rationalize the complex effects of gamma-ray irradiation on the surface of medical grade UHMWPE for total joint replacement and, ultimately, to predict their actual lifetime in vivo.

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

PubMed Central

Bertani, Francesca R.; Ferrari, Luisa; Mussi, Valentina; Botti, Elisabetta; Costanzo, Antonio; Selci, Stefano

2013-01-01

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. PMID:24233077

Semi-automated confocal imaging of fungal pathogenesis on plants: microscopic analysis of macroscopic specimens

USDA-ARS?s Scientific Manuscript database

Contextualizing natural genetic variation in plant disease resistance in terms of pathogenesis can provide information about the function of causal genes. Cellular mechanisms associated with pathogenesis can be elucidated with confocal microscopy, but systematic phenotyping platforms—from sample pro...

Target-locking acquisition with real-time confocal (TARC) microscopy.

PubMed

Lu, Peter J; Sims, Peter A; Oki, Hidekazu; Macarthur, James B; Weitz, David A

2007-07-09

We present a real-time target-locking confocal microscope that follows an object moving along an arbitrary path, even as it simultaneously changes its shape, size and orientation. This Target-locking Acquisition with Realtime Confocal (TARC) microscopy system integrates fast image processing and rapid image acquisition using a Nipkow spinning-disk confocal microscope. The system acquires a 3D stack of images, performs a full structural analysis to locate a feature of interest, moves the sample in response, and then collects the next 3D image stack. In this way, data collection is dynamically adjusted to keep a moving object centered in the field of view. We demonstrate the system's capabilities by target-locking freely-diffusing clusters of attractive colloidal particles, and activelytransported quantum dots (QDs) endocytosed into live cells free to move in three dimensions, for several hours. During this time, both the colloidal clusters and live cells move distances several times the length of the imaging volume.

Reflectance confocal microscopy of cutaneous melanoma. Correlation with dermoscopy and histopathology*

PubMed Central

Rstom, Silvia Arroyo; Libório, Lorena Silva; Paschoal, Francisco Macedo

2015-01-01

In vivo Confocal Microscopy is a method for non-invasive, real-time visualization of microscopic structures and cellular details of the epidermis and dermis, which has a degree of resolution similar to that obtained with histology. We present a case of cutaneous melanoma in which diagnosis was aided by confocal microscopy examination. We also correlate the observed features with the dermoscopic and histopathological findings. Confocal microscopy proved to be an useful adjunct to dermoscopy, playing an important role as a method 'between clinical evaluation and histopathology'. PMID:26131877

Microscopic and macroscopic spectral peculiarities of cutaneous tumours

NASA Astrophysics Data System (ADS)

Borisova, Ekaterina; Genova, Tsanislava; Troyanova, Petranka; Terziev, Ivan; Zakharov, Valery; Bratchenko, Ivan; Lomova, Maria; Gorin, Dmitry; Avramov, Latchezar

2017-12-01

Autofluorescence spectral and confocal microscopic measurements were made on different cutaneous neoplastic lesions, namely basal cell carcinoma, squamous cell carcinoma, malignant melanoma, and their dysplastic forms - keratoacantoma, dysplastic nevi and benign lesions related - basal cell papiloma, seboreic keratosa and compound nevi using excitation at 405 nm. Spectroscopic investigations were made on in vivo and ex vivo tissue samples, and confocal microscopy investigations were made on ex vivo and eosin-haematoxilin stained thin slices of the tumours detected. Correlation between the spectral data received and the microscopic features observed was found, related to the morphological and biochemical alterations during neoplasia growth. Specific spectral features observed in each type of lesion investigated on micro and macro level would be presented and discussed.

In-vivo immunofluorescence confocal microscopy of herpes simplex virus type 1 keratitis

NASA Astrophysics Data System (ADS)

Kaufman, Stephen C.; Laird, Jeffery A.; Beuerman, Roger W.

1996-05-01

The white-light confocal microscope offers an in vivo, cellular-level resolution view of the cornea. This instrument has proven to be a valuable research and diagnostic tool for the study of infectious keratitis. In this study, we investigate the direct visualization of herpes simplex virus type 1 (HSV-1)-infected corneal epithelium, with in vivo confocal microscopy, using HSV-1 immunofluorescent antibodies. New Zealand white rabbits were infected with McKrae strain of HSV-1 in one eye; the other eye of each rabbit was used as an uninfected control. Four days later, the rabbits were anesthetized and a cellulose sponge was applied to each cornea, and a drop of direct HSV fluorescein-tagged antibody was placed on each sponge every 3 to 5 minutes for 1 hour. Fluorescence confocal microscopy was then performed. The HSV-infected corneas showed broad regions of hyperfluorescent epithelial cells. The uninfected corneas revealed no background fluorescence. Thus, using the confocal microscope with a fluorescent cube, we were able to visualize HSV-infected corneal epithelial cells tagged with a direct fluorescent antibody. This process may prove to be a useful clinical tool for the in vivo diagnosis of HSV keratitis.

DURIP: A Confocal Imaging System for Ultra-Fast Three-Dimensional Transport Studies in Thermal Management Applications

DTIC Science & Technology

2011-12-01

Transport Phenomena and Thermal Management Applications,” Proceedings of the XXVIII UIT Heat Transfer Conference, Brescia, Italy, June 21-23, 2010...measurements in microscale systems. The integrated confocal microscope system is a critical component to obtain understanding of fluid- heat ...objective of this work was to develop a high speed three-dimensional (3D) confocal imaging system to study coupled fluidic and heat transport

Automatic analysis for neuron by confocal laser scanning microscope

NASA Astrophysics Data System (ADS)

Satou, Kouhei; Aoki, Yoshimitsu; Mataga, Nobuko; Hensh, Takao K.; Taki, Katuhiko

2005-12-01

The aim of this study is to develop a system that recognizes both the macro- and microscopic configurations of nerve cells and automatically performs the necessary 3-D measurements and functional classification of spines. The acquisition of 3-D images of cranial nerves has been enabled by the use of a confocal laser scanning microscope, although the highly accurate 3-D measurements of the microscopic structures of cranial nerves and their classification based on their configurations have not yet been accomplished. In this study, in order to obtain highly accurate measurements of the microscopic structures of cranial nerves, existing positions of spines were predicted by the 2-D image processing of tomographic images. Next, based on the positions that were predicted on the 2-D images, the positions and configurations of the spines were determined more accurately by 3-D image processing of the volume data. We report the successful construction of an automatic analysis system that uses a coarse-to-fine technique to analyze the microscopic structures of cranial nerves with high speed and accuracy by combining 2-D and 3-D image analyses.

Line-scanning, stage scanning confocal microscope

NASA Astrophysics Data System (ADS)

Carucci, John A.; Stevenson, Mary; Gareau, Daniel

2016-03-01

We created a line-scanning, stage scanning confocal microscope as part of a new procedure: video assisted micrographic surgery (VAMS). The need for rapid pathological assessment of the tissue on the surface of skin excisions very large since there are 3.5 million new skin cancers diagnosed annually in the United States. The new design presented here is a confocal microscope without any scanning optics. Instead, a line is focused in space and the sample, which is flattened, is physically translated such that the line scans across its face in a direction perpendicular to the line its self. The line is 6mm long and the stage is capable of scanning 50 mm, hence the field of view is quite large. The theoretical diffraction-limited resolution is 0.7um lateral and 3.7um axial. However, in this preliminary report, we present initial results that are a factor of 5-7 poorer in resolution. The results are encouraging because they demonstrate that the linear array detector measures sufficient signal from fluorescently labeled tissue and also demonstrate the large field of view achievable with VAMS.

Low-power laser effects at the single-cell level: a confocal microscopy study

NASA Astrophysics Data System (ADS)

Alexandratou, Eleni; Yova, Dido M.; Atlamazoglou, Vassilis; Handris, Panagiotis; Kletsas, Dimitris; Loukas, Spyros

2000-11-01

Confocal microscopy was used for irradiation and observation of the same area of interest, allowing the imaging of low power laser effects in subcellular components and functions, at the single cell level. Coverslips cultures of human fetal foreskin fibroblasts (HFFF2) were placed in a small incubation chamber for in vivo microscopic observation. Cells were stimulated by the 647 nm line of the Argon- Krypton laser of the confocal microscope (0.1 mW/cm2). Membrane permeability, mitochondrial membrane potential ((delta) Psim), intracellular pHi, calcium alterations and nuclear chromatin accessibility were monitored, at different times after irradiation, using specific fluorescent vital probes. Images were stored to the computer and quantitative evaluation was performed using image- processing software. After irradiation, influx and efflux of the appropriate dyes monitored changes in cell membrane permeability. Laser irradiation caused alkalizatoin of the cytosolic pHi and increase of the mitochondrial membrane potential ((delta) Psim). Temporary global Ca2+ responses were also observed. No such effects were noted in microscopic fields other than the irradiated ones. No toxic effects were observed, during time course of the experiment.

Improved resolution in practical light microscopy by means of a glass-fiber 2 π-tilting device

NASA Astrophysics Data System (ADS)

Bradl, Joachim; Rinke, Bernd; Schneider, Bernhard; Hausmann, Michael; Cremer, Christoph G.

1996-01-01

The spatial resolution of a conventional light microscope or a confocal laser scanning microscope can be determined by calculating the point spread function for the objective used. Normally, ideal conditions are assumed for these calculations. Such conditions, however, are often not fulfilled in biological applications especially in those cases where biochemical requirements (e.g. buffer conditions) influence the specimen preparation on the microscope slide (i.e. 'practical' light microscopy). It has been shown that the problem of a reduced z- resolution in 3D-microscopy (optical sectioning) can be overcome by a capillary in a 2(pi) - tilting device that allows object rotation into an optimal perspective. The application of the glass capillary instead of a standard slide has an additional influence on the imaging properties of the microscope. Therefore, another 2(pi) -tilting device was developed, using a glass fiber for object fixation and rotation. Such a fiber could be covered by standard cover glasses. To estimate the resolution of this setup, point spread functions were measured under different conditions using fluorescent microspheres of subwavelength dimensions. Results obtained from standard slide setups were compared to the glass fiber setup. These results showed that in practice rotation leads to an overall 3D-resolution improvement.

Optical-sectioning microscopy of protoporphyrin IX fluorescence in human gliomas: standardization and quantitative comparison with histology

NASA Astrophysics Data System (ADS)

Wei, Linpeng; Chen, Ye; Yin, Chengbo; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T. C.

2017-04-01

Systemic delivery of 5-aminolevulinic acid leads to enhanced fluorescence image contrast in many tumors due to the increased accumulation of protoporphyrin IX (PpIX), a fluorescent porphyrin that is associated with tumor burden and proliferation. The value of PpIX-guided resection of malignant gliomas has been demonstrated in prospective randomized clinical studies in which a twofold greater extent of resection and improved progression-free survival have been observed. In low-grade gliomas and at the diffuse infiltrative margins of all gliomas, PpIX fluorescence is often too weak to be detected with current low-resolution surgical microscopes that are used in operating rooms. However, it has been demonstrated that high-resolution optical-sectioning microscopes are capable of detecting the sparse and punctate accumulations of PpIX that are undetectable via conventional low-power surgical fluorescence microscopes. To standardize the performance of high-resolution optical-sectioning devices for future clinical use, we have developed an imaging phantom and methods to ensure that the imaging of PpIX-expressing brain tissues can be performed reproducibly. Ex vivo imaging studies with a dual-axis confocal microscope demonstrate that these methods enable the acquisition of images from unsectioned human brain tissues that quantitatively and consistently correlate with images of histologically processed tissue sections.

Confocal Microscopy Imaging with an Optical Transition Edge Sensor

NASA Astrophysics Data System (ADS)

Fukuda, D.; Niwa, K.; Hattori, K.; Inoue, S.; Kobayashi, R.; Numata, T.

2018-05-01

Fluorescence color imaging at an extremely low excitation intensity was performed using an optical transition edge sensor (TES) embedded in a confocal microscope for the first time. Optical TES has the ability to resolve incident single photon energy; therefore, the wavelength of each photon can be measured without spectroscopic elements such as diffraction gratings. As target objects, animal cells labeled with two fluorescent dyes were irradiated with an excitation laser at an intensity below 1 μW. In our confocal system, an optical fiber-coupled TES device is used to detect photons instead of the pinhole and photomultiplier tube used in typical confocal microscopes. Photons emitted from the dyes were collected by the objective lens, and sent to the optical TES via the fiber. The TES measures the wavelength of each photon arriving in an exposure time of 70 ms, and a fluorescent photon spectrum is constructed. This measurement is repeated by scanning the target sample, and finally a two-dimensional RGB-color image is obtained. The obtained image showed that the photons emitted from the dyes of mitochondria and cytoskeletons were clearly resolved at a detection intensity level of tens of photons. TES exhibits ideal performance as a photon detector with a low dark count rate (< 1 Hz) and wavelength resolving power. In the single-mode fiber-coupled system, the confocal microscope can be operated in the super-resolution mode. These features are very promising to realize high-sensitivity and high-resolution photon spectral imaging, and would help avoid cell damage and photobleaching of fluorescence dyes.

A laser scanning confocal imaging-surface plasmon resonance system application in real time detection of antibody-antigen interaction

NASA Astrophysics Data System (ADS)

Zhang, H. Y.; Yang, L. Q.; Liu, W. M.

2011-12-01

The laser scanning confocal microscope (LSCM) offers several advantages over conventional optical microscopy, but most LSCM work is qualitative analysis and it is very hard to achieve quantitative detection directly with the changing of the fluorescent intensity. A new real time sensor system for the antibody-antigen interaction detection was built integrating with a LSCM and a wavelength-dependent surface plasmon resonance (SPR) sensor. The system was applied to detect the bonding process of human IgG and fluorescent-labeled affinity purified antibody in real time. The fluorescence images changing is well with that of SPR wavelengths in real time, and the trend of the resonance wavelength shift with the concentrations of antibody is similar to that of the fluorescent intensity changing. The results show that SPR makes up the short of quantificational analysis with LSCM with the high spatial resolution. The sensor system shows the merits of the of the LSCM and SPR synergetic application, which are of great importance for practical application in biosensor and life science for interesting local interaction.

Compact divided-pupil line-scanning confocal microscope for investigation of human tissues

NASA Astrophysics Data System (ADS)

Glazowski, Christopher; Peterson, Gary; Rajadhyaksha, Milind

2013-03-01

Divided-pupil line-scanning confocal microscopy (DPLSCM) can provide a simple and low-cost approach for imaging of human tissues with pathology-like nuclear and cellular detail. Using results from a multidimensional numerical model of DPLSCM, we found optimal pupil configurations for improved axial sectioning, as well as control of speckle noise in the case of reflectance imaging. The modeling results guided the design and construction of a simple (10 component) microscope, packaged within the footprint of an iPhone, and capable of cellular resolution. We present the optical design with experimental video-images of in-vivo human tissues.

Dual-axis confocal microscope for high-resolution in vivo imaging

PubMed Central

Wang, Thomas D.; Mandella, Michael J.; Contag, Christopher H.; Kino, Gordon S.

2007-01-01

We describe a novel confocal microscope that uses separate low-numerical-aperture objectives with the illumination and collection axes crossed at angle θ from the midline. This architecture collects images in scattering media with high transverse and axial resolution, long working distance, large field of view, and reduced noise from scattered light. We measured transverse and axial (FWHM) resolution of 1.3 and 2.1 μm, respectively, in free space, and confirm subcellular resolution in excised esophageal mucosa. The optics may be scaled to millimeter dimensions and fiber coupled for collection of high-resolution images in vivo. PMID:12659264

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

PubMed

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.

Colonization of cashew plants by Lasiodiplodia theobromae: Microscopical features

USDA-ARS?s Scientific Manuscript database

Lasiodiplodia theobromae is a phytopathogenic fungus causing gummosis, a threatening disease for cashew plants in Brazil. In an attempt to investigate the ultrastructural features of the pathogen colonization and its response to immunofluorescence labeling, light, confocal and electron microscope st...

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

PubMed

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.

Fluorescence multi-scale endoscopy and its applications in the study and diagnosis of gastro-intestinal diseases: set-up design and software implementation

NASA Astrophysics Data System (ADS)

Gómez-García, Pablo Aurelio; Arranz, Alicia; Fresno, Manuel; Desco, Manuel; Mahmood, Umar; Vaquero, Juan José; Ripoll, Jorge

2015-06-01

Endoscopy is frequently used in the diagnosis of several gastro-intestinal pathologies as Crohn disease, ulcerative colitis or colorectal cancer. It has great potential as a non-invasive screening technique capable of detecting suspicious alterations in the intestinal mucosa, such as inflammatory processes. However, these early lesions usually cannot be detected with conventional endoscopes, due to lack of cellular detail and the absence of specific markers. Due to this lack of specificity, the development of new endoscopy technologies, which are able to show microscopic changes in the mucosa structure, are necessary. We here present a confocal endomicroscope, which in combination with a wide field fluorescence endoscope offers fast and specific macroscopic information through the use of activatable probes and a detailed analysis at cellular level of the possible altered tissue areas. This multi-modal and multi-scale imaging module, compatible with commercial endoscopes, combines near-infrared fluorescence (NIRF) measurements (enabling specific imaging of markers of disease and prognosis) and confocal endomicroscopy making use of a fiber bundle, providing a cellular level resolution. The system will be used in animal models exhibiting gastro-intestinal diseases in order to analyze the use of potential diagnostic markers in colorectal cancer. In this work, we present in detail the set-up design and the software implementation in order to obtain simultaneous RGB/NIRF measurements and short confocal scanning times.

Dimensional metrology of lab-on-a-chip internal structures: a comparison of optical coherence tomography with confocal fluorescence microscopy.

PubMed

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.

EVALUATION OF CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: APPLICATIONS FOR IMAGING MORPHOLOGY AND DEATH IN EMBRYOS AND REPRODUCTIVE TISSUE/ORGANS

EPA Science Inventory

The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. It is remarkable that procedures to test the performance of these machines are not done routinely by most investigators and thus many of the machines in the field are working at level...

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

EPA Science Inventory

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...

3D imaging of particle tracks in Solid State Nuclear Track Detectors

NASA Astrophysics Data System (ADS)

Wertheim, D.; Gillmore, G.; Brown, L.; Petford, N.

2009-04-01

Inhalation of radon gas (222Rn) and associated ionizing decay products is known to cause lung cancer in human. In the U.K., it has been suggested that 3 to 5 % of total lung cancer deaths can be linked to elevated radon concentrations in the home and/or workplace. Radon monitoring in buildings is therefore routinely undertaken in areas of known risk. Indeed, some organisations such as the Radon Council in the UK and the Environmental Protection Agency in the USA, advocate a ‘to test is best' policy. Radon gas occurs naturally, emanating from the decay of 238U in rock and soils. Its concentration can be measured using CR?39 plastic detectors which conventionally are assessed by 2D image analysis of the surface; however there can be some variation in outcomes / readings even in closely spaced detectors. A number of radon measurement methods are currently in use (for examples, activated carbon and electrets) but the most widely used are CR?39 solid state nuclear track?etch detectors (SSNTDs). In this technique, heavily ionizing alpha particles leave tracks in the form of radiation damage (via interaction between alpha particles and the atoms making up the CR?39 polymer). 3D imaging of the tracks has the potential to provide information relating to angle and energy of alpha particles but this could be time consuming. Here we describe a new method for rapid high resolution 3D imaging of SSNTDs. A ‘LEXT' OLS3100 confocal laser scanning microscope was used in confocal mode to successfully obtain 3D image data on four CR?39 plastic detectors. 3D visualisation and image analysis enabled characterisation of track features. This method may provide a means of rapid and detailed 3D analysis of SSNTDs. Keywords: Radon; SSNTDs; confocal laser scanning microscope; 3D imaging; LEXT

How the confocal laser scanning microscope entered biological research.

PubMed

Amos, W B; White, J G

2003-09-01

A history of the early development of the confocal laser scanning microscope in the MRC Laboratory of Molecular Biology in Cambridge is presented. The rapid uptake of this technology is explained by the wide use of fluorescence in the 80s. The key innovations were the scanning of the light beam over the specimen rather than vice-versa and a high magnification at the level of the detector, allowing the use of a macroscopic iris. These were followed by an achromatic all-reflective relay system, a non-confocal transmission detector and novel software for control and basic image processing. This design was commercialized successfully and has been produced and developed over 17 years, surviving challenges from alternative technologies, including solid-state scanning systems. Lessons are pointed out from the unusual nature of the original funding and research environment. Attention is drawn to the slow adoption of the instrument in diagnostic medicine, despite promising applications.

Dermoscopic and reflectance confocal microscopic features of exogenous ochronosis.

PubMed

Gil, Inmaculada; Segura, Sonia; Martínez-Escala, Estela; Lloreta, Josep; Puig, Susana; Vélez, Mariano; Pujol, Ramón M; Herrero-González, Josep E

2010-09-01

Exogenous ochronosis presents as an acquired asymptomatic hyperpigmentation on photoexposed areas, predominantly over bony prominences, and is caused by the topical application of several skin-lightening agents. We describe a 63-year-old Hispanic woman who developed exogenous ochronosis lesions on her face after using topical bleaching creams containing hydroquinone, 2% to 3%, and oxybenzone, 2%, for several years. Dermoscopy revealed irregular brown-gray globular, annular, and arciform structures that corresponded to focal deposition of ochronotic pigment on the dermis. These deposits correlated with multiple banana-shaped nonrefractile structures seen using reflectance confocal microscopy. Histopathologic sections revealed the deposition of a banana-shaped, yellow to brown material in the papillary and middle dermis. Ultrastructural examination revealed an amorphous electron-dense material mostly located in the core of elastic fibers and also in smaller amounts in the interstitium with prominent degenerative changes in the elastic fibers. A good correlation was observed between the results of both noninvasive techniques and the diagnostic histologic features of this condition. We characterized by means of dermoscopy, reflectance confocal microscopy, and electronic microscopy a case of exogenous ochronosis. To our knowledge, this is the first description of reflectance confocal microscopic findings in this condition. Dermoscopy and reflectance confocal microscopy are proved to be useful noninvasive techniques for the diagnosis of this pigmentary disorder.

Studies of porphyrin-containing specimens using an optical spectrometer connected to a confocal scanning laser microscope.

PubMed

Trepte, O; Rokahr, I; Andersson-Engels, S; Carlsson, K

1994-12-01

A spectrometer has been developed for use with a confocal scanning laser microscope. With this unit, spectral information from a single point or a user-defined region within the microscope specimen can be recorded. A glass prism is used to disperse the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit keeps the detector at 277 K, thereby allowing integration times of up to 60 s. The spectral resolving power, lambda/delta lambda, ranges from 350 at lambda = 400 nm to 100 at lambda = 700 nm. Since the entrance aperture of the spectrometer has the same size as the detector pinhole used during normal confocal scanning, the three-dimensional spatial resolution is equivalent to that of normal confocal scanning. Light from the specimen is deflected to the spectrometer by a solenoid controlled mirror, allowing fast and easy switching between normal confocal scanning and spectrometer readings. With this equipment, studies of rodent liver specimens containing porphyrins have been made. The subcellular localization is of interest for the mechanisms of photodynamic therapy (PDT) of malignant tumours. Spectroscopic detection is necessary to distinguish the porphyrin signal from other fluorescent components in the specimen. Two different substances were administered to the tissue, Photofrin, a haematoporphyrin derivative (HPD) and delta-amino levulinic acid (ALA), a precursor to protoporphyrin IX and haem in the haem cycle. Both are substances under clinical trials for PDT of malignant tumours. Following administration of these compounds to the tissue, the potent photosensitizer and fluorescent compound Photofrin, or protoporphyrin IX, respectively, is accumulated.(ABSTRACT TRUNCATED AT 250 WORDS)

Skin aging: in vivo microscopic assessment of epidermal and dermal changes by means of confocal microscopy.

PubMed

Longo, Caterina; Casari, Alice; Beretti, Francesca; Cesinaro, Anna Maria; Pellacani, Giovanni

2013-03-01

Skin aging is thought to be a complex biological process that is traditionally classified as intrinsic and extrinsic aging. Several clinical score and instrumental devices have been applied to obtain a precise assessment of skin aging. Among them, confocal microscopy has emerged as a new technique capable of assessing cytoarchitectural changes with a nearly histopathologic resolution. We sought to determine the microscopic skin changes occurring on the face in different age groups by means of confocal microscopy. The skin of the cheek in 63 volunteers belonging to distinct age groups was analyzed by confocal microscopy. In 4 cases, routine histopathology was performed on site-matched surplus areas from routine excisions for obtaining a better comparison with confocal findings. Young skin was characterized by regular polygonal keratinocytes and thin reticulated collagen fibers. With aging, more irregularly shaped keratinocytes and areas with unevenly distributed pigmentation and increased compactness of collagen fibers were observed. In the elderly, thinning of the epidermis, marked keratinocyte alterations, and huddles of collagen and curled fibers, corresponding to elastosis, were present. A side-by-side correlation between confocal descriptors and histopathologic aspects has been provided in a few cases. Reticular dermal changes cannot be assessed because of the limited depth laser penetration. Confocal microscopy was successfully applied to identify in vivo skin changes occurring in aged skin at both the epidermal and dermal levels at histopathologic resolution. This offers the possibility to test cosmetic product efficacy and to identify early signs of sun damage. Copyright © 2011 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.

Anti-translational research: from the bedside back to the bench for reflectance confocal microscopy

NASA Astrophysics Data System (ADS)

Gareau, Daniel

2014-03-01

The reflectance confocal microscope has made translational progress in dermatology. 0.5 micrometer lateral resolution, 0.75mm field-of-view and excellent temporal resolution at ~15 frames/second serve the VivaScope well in the clinic, but it may be overlooked in basic research. This work reviews high spatiotemporal confocal microscopy and presents images acquired of various samples: zebra fish embryo where melanocytes with excellent contrast overly the spinal column, chicken embryo, where myocardium is seen moving at 15 frames/ second, calcium spikes in dendrites (fluorescence mode) just beyond the temporal resolution, and human skin where blood cells race through the artereovenous microvasculature. For an introduction to confocal microscopy, see: http://dangareau.net.s69818.gridserver.com/science/confocal-microscopy

Dynamics of Unjammed Emulsions

NASA Astrophysics Data System (ADS)

Guerra, Rodrigo; Kodger, Thomas; Weitz, David

2014-03-01

Light scattering and NMR densitometry measurements of quiescent emulsions have shown that amorphous packings of soft, repulsive droplets unjam at osmotic pressures 105 times larger than the typical droplet thermal energy density: 3kB/T 4 πR3. This transition corresponds to the pressure at which the thermal fluctuations of individual droplet positions match the yield strain of the packing and drive the fluidization of the material. We use confocal microscopy to investigate the microscopic dynamics of this fluid-like phase and find them to be fundamentally different from those of conventional glass-forming liquids; cage-breaking dynamics are not evident from droplet mean squared displacements and the effective viscosity of the emulsion, though 105 larger than the background fluid, appears largely insensitive to the confining pressure.

Characterisation of a resolution enhancing image inversion interferometer.

PubMed

Wicker, Kai; Sindbert, Simon; Heintzmann, Rainer

2009-08-31

Image inversion interferometers have the potential to significantly enhance the lateral resolution and light efficiency of scanning fluorescence microscopes. Self-interference of a point source's coherent point spread function with its inverted copy leads to a reduction in the integrated signal for off-axis sources compared to sources on the inversion axis. This can be used to enhance the resolution in a confocal laser scanning microscope. We present a simple image inversion interferometer relying solely on reflections off planar surfaces. Measurements of the detection point spread function for several types of light sources confirm the predicted performance and suggest its usability for scanning confocal fluorescence microscopy.

4Pi-confocal microscopy of live cells

NASA Astrophysics Data System (ADS)

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.

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

PubMed Central

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.

Proposal for an in vivo histopathologic scoring system for skin aging by means of confocal microscopy.

PubMed

Longo, Caterina; Casari, Alice; De Pace, Barbara; Simonazzi, Silvia; Mazzaglia, Giovanna; Pellacani, Giovanni

2013-02-01

Many instrumental devices have been testing in analysing and quantifying the skin aging signs. However, histopathology still remains the only methods that allow a microscopic assessment of the skin. However, a skin biopsy is not feasible in aesthetically critical areas such as the face. Recently, confocal microscopy has been discovered as a noninvasive tool with a nearly histologic resolution. Distinct morphologic confocal aspects on facial skin have been described and correlated with the histopathologic counterparts. In our study we aim to develop an easy to use confocal aging score to quantify the skin aging related signs. A sample of facial skin of fifty volunteers has been subjected to confocal imaging. Combining the previously identified confocal features, three different semi-quantitative scores were calculated: - epidermal disarray score (irregular honeycombed pattern + epidermal thickness + furrow pattern); - epidermal hyperplasia score (mottled pigmentation + extent of polycyclic papillary + epidermal thickness; - collagen score (curled fibers, 2 for huddles of collagen, 1 for coarse collagen structures, and 0 for thin reticulated collagen) The epidermal disarray score showed a stable trend up to 65 years and a dramatic increase in the elderly subjects epidermal. Hyperplasia score was characterized by an ascending trend from younger subjects to middle age. The total collagen score showed a progressive trend with age with a different proportion of distinct collagen type. RCM is a powerful, noninvasive technique that could permit to microscopically quantify the aging signs and to test cosmetic efficacy. © 2012 John Wiley & Sons A/S.

Methods to calibrate and scale axial distances in confocal microscopy as a function of refractive index.

PubMed

Besseling, T H; Jose, J; Van Blaaderen, A

2015-02-01

Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive-index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high-end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily implemented. With these methods, we measured axial scaling factors as a function of refractive-index mismatch for high-aperture confocal microscopy imaging. We found that our scaling factors are almost completely linearly dependent on refractive index and that they were in good agreement with theoretical predictions that take the full vectorial properties of light into account. There was however a strong deviation with the theoretical predictions using (high-angle) geometrical optics, which predict much lower scaling factors. As an illustration, we measured the PSF of a correctly calibrated point-scanning confocal microscope and showed that a nearly index-matched, micron-sized spherical object is still significantly elongated due to this PSF, which signifies that care has to be taken when determining axial calibration or axial scaling using such particles. © 2014 The Authors Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.

Towards automated early cancer detection: Non-invasive, fluorescence-based approaches for quantitative assessment of cells and tissue to identify pre-cancers

NASA Astrophysics Data System (ADS)

Levitt, Jonathan Michael

Cancer is the second leading cause of death globally, second only to heart disease. As in many diseases, patient survival is directly related to how early lesions are detected. Using conventional screening methods, the early changes associated with cancer, which occur on the microscopic scale, can easily go overlooked. Due to the inherent drawbacks of conventional techniques we present non-invasive, optically based methods to acquire high resolution images from live samples and assess cellular function associated with the onset of disease. Specifically, we acquired fluorescence images from NADH and FAD to quantify morphology and metabolic activity. We first conducted studies to monitor monolayers of keratinocytes in response to apoptosis which has been shown to be disrupted during cancer progression. We found that as keratinocytes undergo apoptosis there are populations of mitochondria that exhibit a higher metabolic activity that become progressively confined to a gradually smaller perinuclear region. To further assess the changes associated with early cancer growth we developed automated methods to rapidly quantify fluorescence images and extract morphological and metabolic information from life tissue. In this study, we simultaneously quantified mitochondrial organization, metabolic activity, nuclear size distribution, and the localization of the structural protein keratin, to differentiate between normal and pre-cancerous engineered tissues. We found the degree mitochondrial organization, as determined from the fractal derived Hurst parameter, was well correlated to level of cellular differentiation. We also found that the metabolic activity in the pre-cancerous cells was greater and more consistent throughout tissue depths in comparison to normal tissue. Keratin localization, also quantified from the fluorescence images, we found it to be confined to the uppermost layers of normal tissue while it was more evenly distributed in the precancerous tissues. To allow for evaluation of the early cancerous changes in vivo, we developed video-rate confocal reflectance/multi-photon fluorescence microscope as a clinical prototype. This device was specifically designed to rapidly acquire and assess non-invasively acquire fluorescence images using the automated methods we have developed. We have demonstrated the ability of this microscope to simultaneously acquire fluorescence, confocal reflectance, and second-harmonic generation images as well as assess blood flow in vivo.

All-near-infrared multiphoton microscopy interrogates intact tissues at deeper imaging depths than conventional single- and two-photon near-infrared excitation microscopes

PubMed Central

Sarder, Pinaki; Yazdanfar, Siavash; Akers, Walter J.; Tang, Rui; Sudlow, Gail P.; Egbulefu, Christopher

2013-01-01

Abstract. The era of molecular medicine has ushered in the development of microscopic methods that can report molecular processes in thick tissues with high spatial resolution. A commonality in deep-tissue microscopy is the use of near-infrared (NIR) lasers with single- or multiphoton excitations. However, the relationship between different NIR excitation microscopic techniques and the imaging depths in tissue has not been established. We compared such depth limits for three NIR excitation techniques: NIR single-photon confocal microscopy (NIR SPCM), NIR multiphoton excitation with visible detection (NIR/VIS MPM), and all-NIR multiphoton excitation with NIR detection (NIR/NIR MPM). Homologous cyanine dyes provided the fluorescence. Intact kidneys were harvested after administration of kidney-clearing cyanine dyes in mice. NIR SPCM and NIR/VIS MPM achieved similar maximum imaging depth of ∼100  μm. The NIR/NIR MPM enabled greater than fivefold imaging depth (>500  μm) using the harvested kidneys. Although the NIR/NIR MPM used 1550-nm excitation where water absorption is relatively high, cell viability and histology studies demonstrate that the laser did not induce photothermal damage at the low laser powers used for the kidney imaging. This study provides guidance on the imaging depth capabilities of NIR excitation-based microscopic techniques and reveals the potential to multiplex information using these platforms. PMID:24150231

Super-resolved linear fluorescence localization microscopy using photostable fluorophores: A virtual microscopy study

NASA Astrophysics Data System (ADS)

Birk, Udo; Szczurek, Aleksander; Cremer, Christoph

2017-12-01

Current approaches to overcome the conventional limit of the resolution potential of light microscopy (of about 200 nm for visible light), often suffer from non-linear effects, which render the quantification of the image intensities in the reconstructions difficult, and also affect the quantification of the biological structure under investigation. As an attempt to face these difficulties, we discuss a particular method of localization microscopy which is based on photostable fluorescent dyes. The proposed method can potentially be implemented as a fast alternative for quantitative localization microscopy, circumventing the need for the acquisition of thousands of image frames and complex, highly dye-specific imaging buffers. Although the need for calibration remains in order to extract quantitative data (such as the number of emitters), multispectral approaches are largely facilitated due to the much less stringent requirements on imaging buffers. Furthermore, multispectral acquisitions can be readily obtained using commercial instrumentation such as e.g. the conventional confocal laser scanning microscope.

Reflectance confocal microscopy of oral epithelial tissue using an electrically tunable lens

NASA Astrophysics Data System (ADS)

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

2014-02-01

We present the use of a commercially available electrically tunable lens to achieve axial scanning in a reflectance confocal microscope. Over a 255 μm axial scan range, the lateral and axial resolutions varied from 1-2 μm and 4-14 μm, respectively, dependent on the variable focal length of the tunable lens. Confocal imaging was performed on normal human biopsies from the oral cavity ex vivo. Sub-cellular morphologic features were seen throughout the depth of the epithelium while axially scanning using the focus tunable lens.

Multimodal confocal mosaics enable high sensitivity and specificity in screening of in situ squamous cell carcinoma

NASA Astrophysics Data System (ADS)

Grados Luyando, Maria del Carmen; Bar, Anna; Snavely, Nicholas; Jacques, Steven; Gareau, Daniel S.

2014-02-01

Screening cancer in excision margins with confocal microscopy may potentially save time and cost over the gold standard histopathology (H and E). However, diagnostic accuracy requires sufficient contrast and resolution to reveal pathological traits in a growing set of tumor types. Reflectance mode images structural details due to microscopic refractive index variation. Nuclear contrast with acridine orange fluorescence provides enhanced diagnostic value, but fails for in situ squamous cell carcinoma (SCC), where the cytoplasm is important to visualize. Combination of three modes [eosin (Eo) fluorescence, reflectance (R) and acridine orange (AO) fluorescence] enable imaging of cytoplasm, collagen and nuclei respectively. Toward rapid intra-operative pathological margin assessment to guide staged cancer excisions, multimodal confocal mosaics can image wide surgical margins (~1cm) with sub-cellular resolution and mimic the appearance of conventional H and E. Absorption contrast is achieved by alternating the excitation wavelength: 488nm (AO fluorescence) and 532nm (Eo fluorescence). Superposition and false-coloring of these modes mimics H and E, enabling detection of the carcinoma in situ in the epidermal layer The sum mosaic Eo+R is false-colored pink to mimic eosins' appearance in H and E, while the AO mosaic is false-colored purple to mimic hematoxylins' appearance in H and E. In this study, mosaics of 10 Mohs surgical excisions containing SCC in situ and 5 containing only normal tissue were subdivided for digital presentation equivalent to 4X histology. Of the total 16 SCC in situ multimodal mosaics and 16 normal cases presented, two reviewers made 1 and 2 (respectively) type-2 errors (false positives) but otherwise scored perfectly when using the confocal images to screen for the presence of SCC in situ as compared to the gold standard histopathology. Limitations to precisely mimic H and E included occasional elastin staining by AO. These results suggest that confocal mosaics may effectively guide staged SCC excisions in skin and other tissues.

Rigid confocal endoscopy for in vivo imaging of experimental oral squamous intra-epithelial lesions.

PubMed

Farahati, Behnaz; Stachs, Oliver; Prall, Friedrich; Stave, Joachim; Guthoff, Rudolf; Pau, Hans Wilhelm; Just, Tino

2010-04-01

A rigid confocal endoscope has been developed to assess the oral squamous epithelium of mice and to determine sensitivity, specificity, and accuracy of this new technology. This endoscope is connected to the commercially available Heidelberg Retina Tomograph (HRT). HRT is a device with a 670-nm diode laser designed to acquire topographical measurements of the optic nerve head. Real-time rigid confocal endoscopy is demonstrated by imaging the epithelial lesions of a mice model. Six-week-old male C57Bl/6 mice were randomly divided into a non-treated group (n = 10) and into a 4-nitroquinoline 1-oxide (4-NQO)-treated group (n = 50). In the 4-NQO-treated group, the mice obtained 4-nitroquinoline 1-oxide in the drinking water (100 microg/ml) to induce tumourigenesis in the mouse tongue. The 4-NQO-solution was diluted in the drinking water for mice. After an 8-16-week carcinogen treatment with 4-NQO (ad libitum), mouse tongues were dissected within 3 h after CO(2) overdose. After confocal microscopy of all lesions of the tongue, conventional histopathological investigation was performed. The inter-rater reliability for the two observers of the confocal microscopic findings was found to be Kappa = 0.59 (P < 0.001). The penetration depth varied in the healthy tissue of the underside of the tongue throughout this study and was measured between 104 and 240 microm. In keratotic lesions, the penetration depths were diminished and varied between 80 and 140 microm. Strong keratinization inhibits the evaluation of the epithelium. For differentiation between low-grade and high-grade squamous intra-epithelial lesions, a sensitivity and specificity of 73% and 88% was reached. The animal experiment with this non-invasive new technology indicates that this imaging technology facilitates the detection of pre-cancerous lesions of the underside of the oropharynx. Human studies on oropharyngeal and laryngeal lesions are needed to prove the applicability of this method in the field of otorhinolaryngology.

3D image restoration for confocal microscopy: toward a wavelet deconvolution for the study of complex biological structures

NASA Astrophysics Data System (ADS)

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.

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

PubMed

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.

High-resolution resonant and nonresonant fiber-scanning confocal microscope.

PubMed

Hendriks, Benno H W; Bierhoff, Walter C J; Horikx, Jeroen J L; Desjardins, Adrien E; Hezemans, Cees A; 't Hooft, Gert W; Lucassen, Gerald W; Mihajlovic, Nenad

2011-02-01

We present a novel, hand-held microscope probe for acquiring confocal images of biological tissue. This probe generates images by scanning a fiber-lens combination with a miniature electromagnetic actuator, which allows it to be operated in resonant and nonresonant scanning modes. In the resonant scanning mode, a circular field of view with a diameter of 190 μm and an angular frequency of 127 Hz can be achieved. In the nonresonant scanning mode, a maximum field of view with a width of 69 μm can be achieved. The measured transverse and axial resolutions are 0.60 and 7.4 μm, respectively. Images of biological tissue acquired in the resonant mode are presented, which demonstrate its potential for real-time tissue differentiation. With an outer diameter of 3 mm, the microscope probe could be utilized to visualize cellular microstructures in vivo across a broad range of minimally-invasive procedures.

Characterization of a subwavelength-scale 3D void structure using the FDTD-based confocal laser scanning microscopic image mapping technique.

PubMed

Choi, Kyongsik; Chon, James W; Gu, Min; Lee, Byoungho

2007-08-20

In this paper, a simple confocal laser scanning microscopic (CLSM) image mapping technique based on the finite-difference time domain (FDTD) calculation has been proposed and evaluated for characterization of a subwavelength-scale three-dimensional (3D) void structure fabricated inside polymer matrix. The FDTD simulation method adopts a focused Gaussian beam incident wave, Berenger's perfectly matched layer absorbing boundary condition, and the angular spectrum analysis method. Through the well matched simulation and experimental results of the xz-scanned 3D void structure, we first characterize the exact position and the topological shape factor of the subwavelength-scale void structure, which was fabricated by a tightly focused ultrashort pulse laser. The proposed CLSM image mapping technique based on the FDTD can be widely applied from the 3D near-field microscopic imaging, optical trapping, and evanescent wave phenomenon to the state-of-the-art bio- and nanophotonics.

In vivo laser confocal microscopy findings of a cornea with osteogenesis imperfecta.

PubMed

Kobayashi, Akira; Higashide, Tomomi; Yokogawa, Hideaki; Yamazaki, Natsuko; Masaki, Toshinori; Sugiyama, Kazuhisa

2014-01-01

To report the in vivo laser confocal microscopy findings of a cornea with osteogenesis imperfecta (OI) with special attention to the abnormality of Bowman's layer and sub-Bowman's fibrous structures (K-structures). Two patients (67-year-old male and his 26-year-old son) with OI type I were included in this study. Slit lamp biomicroscopic and in vivo laser confocal microscopic examinations were performed for both patients. Central corneal thickness and central endothelial cell density were also measured. Although the corneas looked clear with normal endothelial density for both eyes in both patients, they were quite thin (386 μm oculus dexter (OD) (the right eye) and 384 μm oculus sinister (OS) (the left eye) in the father and 430 μm OD and 425 μm OS in the son). In both patients, slit lamp biomicroscopic and in vivo laser confocal microscopic examination showed similar results. Anterior corneal mosaics produced by rubbing the eyelid under fluorescein were completely absent in both eyes. In vivo laser confocal microscopy revealed an absent or atrophic Bowman's layer; a trace of a presumed Bowman's layer and/or basement membrane was barely visible with high intensity. Additionally, K-structures were completely absent in both eyes. The absence of K-structures and fluorescein anterior corneal mosaics strongly suggested an abnormality of Bowman's layer in these OI patients.

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

PubMed

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.

Application toward Confocal Full-Field Microscopic X-ray Absorption Near Edge Structure Spectroscopy.

PubMed

Tack, Pieter; Vekemans, Bart; Laforce, Brecht; Rudloff-Grund, Jennifer; Hernández, Willinton Y; Garrevoet, Jan; Falkenberg, Gerald; Brenker, Frank; Van Der Voort, Pascal; Vincze, Laszlo

2017-02-07

Using X-ray absorption near edge structure (XANES) spectroscopy, information on the local chemical structure and oxidation state of an element of interest can be acquired. Conventionally, this information can be obtained in a spatially resolved manner by scanning a sample through a focused X-ray beam. Recently, full-field methods have been developed to obtain direct 2D chemical state information by imaging a large sample area. These methods are usually in transmission mode, thus restricting the use to thin and transmitting samples. Here, a fluorescence method is displayed using an energy-dispersive pnCCD detector, the SLcam, characterized by measurement times far superior to what is generally applicable. Additionally, this method operates in confocal mode, thus providing direct 3D spatially resolved chemical state information from a selected subvolume of a sample, without the need of rotating a sample. The method is applied to two samples: a gold-supported magnesia catalyst (Au/MgO) and a natural diamond containing Fe-rich inclusions. Both samples provide XANES spectra that can be overlapped with reference XANES spectra, allowing this method to be used for fingerprinting and linear combination analysis of known XANES reference compounds.

Adaptive optics plug-and-play setup for high-resolution microscopes with multi-actuator adaptive lens

NASA Astrophysics Data System (ADS)

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.

Application of confocal Raman micro-spectroscopy for label-free monitoring of oxidative stress in living bronchial cells

NASA Astrophysics Data System (ADS)

Surmacki, Jakub M.; Quirós Gonzalez, Isabel; Bohndiek, Sarah E.

2018-02-01

Oxidative stress in cancer is implicated in tumor progression, being associated with increased therapy resistance and metastasis. Conventional approaches for monitoring oxidative stress in tissue such as high-performance liquid chromatography and immunohistochemistry are bulk measurements and destroy the sample, meaning that longitudinal monitoring of cancer cell heterogeneity remains elusive. Raman spectroscopy has the potential to overcome this challenge, providing a chemically specific, label free readout from single living cells. Here, we applied a standardized protocol for label-free confocal Raman micro-spectroscopy in living cells to monitor oxidative stress in bronchial cells. We used a quartz substrate in a commercial cell chamber contained within a microscope incubator providing culture media for cell maintenance. We studied the effect of a potent reactive oxygen species inducer, tert-butyl hydroperoxide (TBHP), and antioxidant, N-acetyl-L-cysteine (NAC) on living cells from a human bronchial epithelial cells (HBEC). We found that the Raman bands corresponding to nucleic acids, proteins and lipids were significantly different (p<0.05) for control, TBHP, and NAC. Encouragingly, partial least squares discriminant analysis applied to our data showed high sensitivity and specificity for identification of control (87.3%, 71.7%), NAC (92.3%, 85.1%) and TBHP (86.9%, 92.9%). These results suggest that confocal Raman micro-spectroscopy may be able to monitor the biological impact of oxidative and reductive processes in cells, hence enabling longitudinal studies of oxidative stress in therapy resistance and metastasis at the single cell level.

Assessment of fresh breast tissue specimens with confocal strip-mosaicking microscopy in an emulated pathology setting (Conference Presentation)

NASA Astrophysics Data System (ADS)

Abeytunge, Sanjeewa; Larson, Bjorg A.; Peterson, Gary; Rajadhyaksha, Milind; Murray, Melissa

2017-02-01

Confocal microscopy is in clinical use to diagnose skin cancers in the United States and in Europe. Potentially, this technology may provide bed-side pathology in breast cancer surgery during tumor removal. Initial studies have described major findings of invasive breast cancers as seen on fluorescence confocal microscopy. In many of these studies the region of interest (ROI) used in the analysis was user-selected and small (typically 15 square-mm). Although these important findings open exploration into rapid pathology, further development and implementation in a surgical setting will require examination of large specimens in a blinded fashion that will address the needs of typical surgical settings. In post surgery pathology viewing, pathologists inspect the entire pathology section with a low (2X) magnification objective lens initially and then zoomed in to ROIs with higher magnification lenses (10X to 40X) magnifications to further investigate suspected regions. In this study we explore the possibility of implementation in a typical surgical setting with a new microscope, termed confocal strip-mosaicking microscope (CSM microscope), which images an area of 400 square-mm (2 cm x 2 cm) of tissue with cellular level resolution in 10 minutes. CSM images of 34 human breast tissue specimens from 18 patients were blindly analyzed by a board-certified pathologist and correlated with the corresponding standard fixed histopathology. Invasive tumors and benign tissue were clearly identified in CSM images. Thirty specimens were concordant for images-to-histopathology correlation while four were discordant. Preliminary results from on-going work to molecularly target tumor margin will also be presented.

Fluorescence intensity and bright spot analyses using a confocal microscope for photodynamic diagnosis of brain tumors.

PubMed

Yoneyama, Takeshi; Watanabe, Tetsuyo; Kagawa, Hiroyuki; Hayashi, Yutaka; Nakada, Mitsutoshi

2017-03-01

In photodynamic diagnosis using 5-aminolevulinic acid (5-ALA), discrimination between the tumor and normal tissue is very important for a precise resection. However, it is difficult to distinguish between infiltrating tumor and normal regions in the boundary area. In this study, fluorescent intensity and bright spot analyses using a confocal microscope is proposed for the precise discrimination between infiltrating tumor and normal regions. From the 5-ALA-resected brain tumor tissue, the red fluorescent and marginal regions were sliced for observation under a confocal microscope. Hematoxylin and eosin (H&E) staining were performed on serial slices of the same tissue. According to the pathological inspection of the H&E slides, the tumor and infiltrating and normal regions on confocal microscopy images were investigated. From the fluorescent intensity of the image pixels, a histogram of pixel number with the same fluorescent intensity was obtained. The fluorescent bright spot sizes and total number were compared between the marginal and normal regions. The fluorescence intensity distribution and average intensity in the tumor were different from those in the normal region. The probability of a difference from the dark enhanced the difference between the tumor and the normal region. The bright spot size and number in the infiltrating tumor were different from those in the normal region. Fluorescence intensity analysis is useful to distinguish a tumor region, and a bright spot analysis is useful to distinguish between infiltrating tumor and normal regions. These methods will be important for the precise resection or photodynamic therapy of brain tumors. Copyright © 2016 Elsevier B.V. All rights reserved.

Fractal propagation method enables realistic optical microscopy simulations in biological tissues

PubMed Central

Glaser, Adam K.; Chen, Ye; Liu, Jonathan T.C.

2017-01-01

Current simulation methods for light transport in biological media have limited efficiency and realism when applied to three-dimensional microscopic light transport in biological tissues with refractive heterogeneities. We describe here a technique which combines a beam propagation method valid for modeling light transport in media with weak variations in refractive index, with a fractal model of refractive index turbulence. In contrast to standard simulation methods, this fractal propagation method (FPM) is able to accurately and efficiently simulate the diffraction effects of focused beams, as well as the microscopic heterogeneities present in tissue that result in scattering, refractive beam steering, and the aberration of beam foci. We validate the technique and the relationship between the FPM model parameters and conventional optical parameters used to describe tissues, and also demonstrate the method’s flexibility and robustness by examining the steering and distortion of Gaussian and Bessel beams in tissue with comparison to experimental data. We show that the FPM has utility for the accurate investigation and optimization of optical microscopy methods such as light-sheet, confocal, and nonlinear microscopy. PMID:28983499

A targeted illumination optical fiber probe for high resolution fluorescence imaging and optical switching

NASA Astrophysics Data System (ADS)

Shinde, Anant; Perinchery, Sandeep Menon; Murukeshan, Vadakke Matham

2017-04-01

An optical imaging probe with targeted multispectral and spatiotemporal illumination features has applications in many diagnostic biomedical studies. However, these systems are mostly adapted in conventional microscopes, limiting their use for in vitro applications. We present a variable resolution imaging probe using a digital micromirror device (DMD) with an achievable maximum lateral resolution of 2.7 μm and an axial resolution of 5.5 μm, along with precise shape selective targeted illumination ability. We have demonstrated switching of different wavelengths to image multiple regions in the field of view. Moreover, the targeted illumination feature allows enhanced image contrast by time averaged imaging of selected regions with different optical exposure. The region specific multidirectional scanning feature of this probe has facilitated high speed targeted confocal imaging.

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

PubMed

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.

Confocal Raman microscopy for identification of bacterial species in biofilms

NASA Astrophysics Data System (ADS)

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.

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

PubMed Central

Zhao, Ming; Li, Yu; Peng, Leilei

2014-01-01

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. PMID:24921725

Measuring skin penetration by confocal Raman microscopy (CRM): correlation to results from conventional experiments

NASA Astrophysics Data System (ADS)

Lunter, Dominique; Daniels, Rolf

2016-03-01

Confocal Raman microscopy has become an advancing technique in the characterization of drug transport into the skin. In this study the skin penetration of a local anesthetic from a semisolid preparation was investigated. Furthermore, the effect of the chemical enhancers propylene glycol and POE-23-lauryl ether on its penetration was investigated. The results show that confocal Raman microscopy may provide detailed information on the penetration of APIs into the skin and may elucidate their distribution within the skin with high resolution. The results of the CRM analysis are fully in line with those of conventional permeation and penetration experiments.

Three-dimensional scanning confocal laser microscope

DOEpatents

Anderson, R. Rox; Webb, Robert H.; Rajadhyaksha, Milind

1999-01-01

A confocal microscope for generating an image of a sample includes a first scanning element for scanning a light beam along a first axis, and a second scanning element for scanning the light beam at a predetermined amplitude along a second axis perpendicular to the first axis. A third scanning element scans the light beam at a predetermined amplitude along a third axis perpendicular to an imaging plane defined by the first and second axes. The second and third scanning element are synchronized to scan at the same frequency. The second and third predetermined amplitudes are percentages of their maximum amplitudes. A selector determines the second and third predetermined amplitudes such that the sum of the percentages is equal to one-hundred percent.

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

DOEpatents

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.

In vivo laser confocal microscopy findings of a cornea with osteogenesis imperfecta

PubMed Central

Kobayashi, Akira; Higashide, Tomomi; Yokogawa, Hideaki; Yamazaki, Natsuko; Masaki, Toshinori; Sugiyama, Kazuhisa

2014-01-01

Objective To report the in vivo laser confocal microscopy findings of a cornea with osteogenesis imperfecta (OI) with special attention to the abnormality of Bowman’s layer and sub-Bowman’s fibrous structures (K-structures). Patients and methods Two patients (67-year-old male and his 26-year-old son) with OI type I were included in this study. Slit lamp biomicroscopic and in vivo laser confocal microscopic examinations were performed for both patients. Central corneal thickness and central endothelial cell density were also measured. Results Although the corneas looked clear with normal endothelial density for both eyes in both patients, they were quite thin (386 μm oculus dexter (OD) (the right eye) and 384 μm oculus sinister (OS) (the left eye) in the father and 430 μm OD and 425 μm OS in the son). In both patients, slit lamp biomicroscopic and in vivo laser confocal microscopic examination showed similar results. Anterior corneal mosaics produced by rubbing the eyelid under fluorescein were completely absent in both eyes. In vivo laser confocal microscopy revealed an absent or atrophic Bowman’s layer; a trace of a presumed Bowman’s layer and/or basement membrane was barely visible with high intensity. Additionally, K-structures were completely absent in both eyes. Conclusion The absence of K-structures and fluorescein anterior corneal mosaics strongly suggested an abnormality of Bowman’s layer in these OI patients. PMID:24591812

Tracking protein dynamics with photoconvertible Dendra2 on spinning disk confocal systems.

PubMed

Woods, Elena; Courtney, Jane; Scholz, Dimitri; Hall, William W; Gautier, Virginie W

2014-12-01

Understanding the dynamic properties of cellular proteins in live cells and in real time is essential to delineate their function. In this context, we introduce the Fluorescence Recovery After Photobleaching-Photoactivation unit (Andor) combined with the Nikon Eclipse Ti E Spinning Disk (Andor) confocal microscope as an advantageous and robust platform to exploit the properties of the Dendra2 photoconvertible fluorescent protein (Evrogen) and analyse protein subcellular trafficking in living cells. A major advantage of the spinning disk confocal is the rapid acquisition speed, enabling high temporal resolution of cellular processes. Furthermore, photoconversion and imaging are less invasive on the spinning disk confocal as the cell exposition to illumination power is reduced, thereby minimizing photobleaching and increasing cell viability. We have tested this commercially available platform using experimental settings adapted to track the migration of fast trafficking proteins such as UBC9, Fibrillarin and have successfully characterized their differential motion between subnuclear structures. We describe here step-by-step procedures, with emphasis on cellular imaging parameters, to successfully perform the dynamic imaging and photoconversion of Dendra2-fused proteins at high spatial and temporal resolutions necessary to characterize the trafficking pathways of proteins. © 2014 The Authors. Journal of Microscopy published by John Wiley & Sons, Ltd on behalf of Royal Microscopical Society.

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

PubMed Central

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. PMID:25120924

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

PubMed

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.

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

PubMed

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.

Confocal reflectance quantitative phase microscope system for cellular membranes dynamics study (Conference Presentation)

NASA Astrophysics Data System (ADS)

Singh, Vijay Raj; Yaqoob, Zahid; So, Peter T. C.

2017-02-01

Quantitative phase microscopy (QPM) techniques developed so far primarily belongs to high speed transmitted light based systems that has enough sensitivity to resolve membrane fluctuations and dynamics, but has no depth resolution. Therefore, most biomechanics studies using QPM today is confined to simple cells, such as RBCs, without internal organelles. An important instrument that will greatly extend the biomedical applications of QPM is to develop next generation microscope with 3D capability and sufficient temporal resolution to study biomechanics of complex eukaryotic cells including the mechanics of their internal compartments. For eukaryotic cells, the depth sectioning capability is critical and should be sufficient to distinguish nucleic membrane fluctuations from plasma membrane fluctuations. Further, this microscope must provide high temporal resolution since typical eukaryotes membranes are substantially stiffer than RBCs. A confocal reflectance quantitative phase microscope is presented based on multi-pinhole scanning, with the capabilities of higher temporal resolution and sensitivity for nucleic and plasma membranes of eukaryotic cells. System hardware is developed based on an array of confocal pinhole generated by using the `ON' state of subset of micro-mirrors of digital micro-mirror device (DMD, from Texas Instruments) and high-speed raster scanning provides 14ms imaging speed in wide-field mode. A common path interferometer is integrated at the imaging arm for detection of specimens' quantitative phase information. Theoretical investigation of quantitative phase reconstructed from system is investigated and application of system is presented for dimensional fluctuations measurements of both cellular plasma and nucleic membranes of embryonic stem cells.

Cornea and ocular lens visualized with three-dimensional confocal microscopy

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1992-08-01

This paper demonstrates the advantages of three-dimensional reconstruction of the cornea and the ocular crystalline lens by confocal microscopy and volume rendering computer techniques. The advantages of noninvasive observation of ocular structures in living, unstained, unfixed tissue include the following: the tissue is in a natural living state without the artifacts of fixation, mechanical sectioning, and staining; the three-dimensional structure can be observed from any view point and quantitatively analyzed; the dynamics of morphological changes can be studied; and the use of confocal microscopic observation results in a reduction of the number of animals required for ocular morphometric studies. The main advantage is that the dynamic morphology of ocular structures can be investigated in living ocular tissue. A laser scanning confocal microscope was used in the reflected light mode to obtain the two- dimensional images from the cornea and the ocular lens of a freshly enucleated rabbit eye. The light source was an argon ion laser with 488 nm wavelength. The microscope objective was a Leitz 25X, NA 0.6 water immersion lens. The 400 micron thick cornea was optically sectioned into 133, three micron sections. The semi-transparent cornea and the in-situ ocular lens was visualized as high resolution, high contrast two-dimensional images. The under sampling resulted in a three-dimensional visualization rendering in which the corneal thickness (z-axis) is compressed. The structures observed in the cornea include: superficial epithelial cells and their nuclei, basal epithelial cells and their `beaded' cell borders, basal lamina, nerve plexus, nerve fibers, free nerve endings in the basal epithelial cells, nuclei of stromal keratocytes, and endothelial cells. The structures observed in the in-situ ocular lens include: lens capsule, lens epithelial cells, and individual lens fibers.

Passport examination by a confocal-type laser profile microscope.

PubMed

Sugawara, Shigeru

2008-06-10

The author proposes a nondestructive and highly precise method of measuring the thickness of a film pasted on a passport using a confocal-type laser profile microscope. The effectiveness of this method in passport examination is demonstrated. A confocal-type laser profile microscope is used to create profiles of the film surface and film-paper interface; these profiles are used to calculate the film thickness by employing an algorithm developed by the author. The film thicknesses of the passport samples--35 genuine and 80 counterfeit Japanese passports--are measured nondestructively. The intra-sample standard deviation of the film thicknesses of the genuine and counterfeit Japanese passports was of the order of 1 microm The intersample standard deviations of the film thicknesses of passports forged using the same tools and techniques are expected to be of the order of 1 microm. The thickness values of the films on the machine-readable genuine passports ranged between 31.95 microm and 36.95 microm. The likelihood ratio of this method in the authentication of machine-readable Japanese genuine passports is 11.7. Therefore, this method is effective for the authentification of genuine passports. Since the distribution of the film thickness of all forged passports was considerably larger than the accuracy of this method, this method is considered effective also for revealing the relation among the forged passports and acquiring proof of the crime.

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

PubMed

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.

Corneal confocal microscopy following conventional, transepithelial, and accelerated corneal collagen cross-linking procedures for keratoconus.

PubMed

Touboul, David; Efron, Nathan; Smadja, David; Praud, Delphine; Malet, Florence; Colin, Joseph

2012-11-01

To compare early corneal healing following conventional, transepithelial, and accelerated corneal collagen cross-linking (CXL) protocols. Twenty-four patients with progressive keratoconus were divided into three groups to receive conventional, transepithelial, or accelerated CXL. In vivo corneal confocal microscopy was performed on each patient preoperatively and at 1, 3, and 6 months postoperatively. Closure of the epithelial wound was complete 3 days following conventional and accelerated CXL. The subbasal nerve plexus was essentially obliterated immediately following conventional and accelerated CXL, and virtually no nerve fibers had regenerated by 6 months. The anterior stroma showed significant changes 1 month following conventional CXL; these changes were similar but more pronounced following accelerated CXL. Observed stromal changes included complete obliteration of keratocytes, increased tissue reflectivity, a honeycomb-like appearance, and circular lacunae. Some recovery of keratocyte density was noted after 6 months. These changes were less pronounced in the mid-stroma, and there were no apparent changes to the posterior stroma or endothelium. The cornea appeared to be unaltered following transepithelial CXL. In vivo corneal confocal microscopy analysis of the postoperative impact of CXL on the cornea revealed clear differences among conventional, accelerated, and transepithelial CXL protocols. Accelerated CXL had a greater impact than conventional CXL on the anterior cornea, whereas transepithelial CXL did not appear to alter corneal morphology. Copyright 2012, SLACK Incorporated.

Plasmon resonance and the imaging of metal-impregnated neurons with the laser scanning confocal microscope

PubMed Central

Thompson, Karen J; Harley, Cynthia M; Barthel, Grant M; Sanders, Mark A; Mesce, Karen A

2015-01-01

The staining of neurons with silver began in the 1800s, but until now the great resolving power of the laser scanning confocal microscope has not been utilized to capture the in-focus and three-dimensional cytoarchitecture of metal-impregnated cells. Here, we demonstrate how spectral confocal microscopy, typically reserved for fluorescent imaging, can be used to visualize metal-labeled tissues. This imaging does not involve the reflectance of metal particles, but rather the excitation of silver (or gold) nanoparticles and their putative surface plasmon resonance. To induce such resonance, silver or gold particles were excited with visible-wavelength laser lines (561 or 640 nm), and the maximal emission signal was collected at a shorter wavelength (i.e., higher energy state). Because the surface plasmon resonances of noble metal nanoparticles offer a superior optical signal and do not photobleach, our novel protocol holds enormous promise of a rebirth and further development of silver- and gold-based cell labeling protocols. DOI: http://dx.doi.org/10.7554/eLife.09388.001 PMID:26670545

Remote focusing in confocal microscopy by means of a modified Alvarez lens.

PubMed

Bawart, M; Jesacher, A; Bernet, S; Ritsch-Marte, M

2018-06-22

Alvarez lenses are actuated lens-pairs which allow one to tune the optical power by mechanical displacement of subelements. Here, we show that a recently realized modified Alvarez lens design which does not require mechanical actuation can be integrated into a confocal microscope. Instead of mechanically moving them, the sublenses are imaged onto each other in a 4f-configuration, where the lateral image shift leading to a change in optical power is created by a galvo-mirror. The avoidance of mechanical lens shifts leads to a large speed gain for axial (and hence also 3D) image scans compared to classical Alvarez lenses. We demonstrate that the suggested operation principle is compatible with confocal microscopy. In order to optimize the system, we have drawn advantage of the flexibility a liquid-crystal spatial light modulator offers for the implementation. For given specifications, dedicated diffractive optical elements or freeform elements can be used in combination with resonant galvo-scanners or acousto-optic beam deflectors, to achieve even faster z-scans than reported here, reaching video rate. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Use of portable devices and confocal Raman spectrometers at different wavelength to obtain the spectral information of the main organic components in tomato (Solanum lycopersicum) fruits.

PubMed

Trebolazabala, Josu; Maguregui, Maite; Morillas, Héctor; de Diego, Alberto; Madariaga, Juan Manuel

2013-03-15

Tomato (Solanum lycopersicum) fruit samples, in two ripening stages, ripe (red) and unripe (green), collected from a cultivar in the North of Spain (Barrika, Basque Country), were analyzed directly, without any sample pretreatment, with two different Raman instruments (portable spectrometer coupled to a micro-videocamera and a confocal Raman microscope), using two different laser excitation wavelengths (514 and 785 nm, only for the confocal microscope). The combined use of these laser excitation wavelengths allows obtaining, in a short period of time, the maximum spectral information about the main organic compounds present in this fruit. The major identified components of unripe tomatoes were cutin and cuticular waxes. On the other hand, the main components on ripe tomatoes were carotenes, polyphenoles and polysaccharides. Among the carotenes, it was possible to distinguish the presence of lycopene from β-carotene with the help of both excitation wavelengths, but specially using the 514 nm one, which revealed specific overtones and combination tones of this type of carotene. Copyright © 2012 Elsevier B.V. All rights reserved.

Use of portable devices and confocal Raman spectrometers at different wavelength to obtain the spectral information of the main organic components in tomato (Solanum lycopersicum) fruits

NASA Astrophysics Data System (ADS)

Trebolazabala, Josu; Maguregui, Maite; Morillas, Héctor; de Diego, Alberto; Madariaga, Juan Manuel

2013-03-01

Tomato (Solanum lycopersicum) fruit samples, in two ripening stages, ripe (red) and unripe (green), collected from a cultivar in the North of Spain (Barrika, Basque Country), were analyzed directly, without any sample pretreatment, with two different Raman instruments (portable spectrometer coupled to a micro-videocamera and a confocal Raman microscope), using two different laser excitation wavelengths (514 and 785 nm, only for the confocal microscope). The combined use of these laser excitation wavelengths allows obtaining, in a short period of time, the maximum spectral information about the main organic compounds present in this fruit. The major identified components of unripe tomatoes were cutin and cuticular waxes. On the other hand, the main components on ripe tomatoes were carotenes, polyphenoles and polysaccharides. Among the carotenes, it was possible to distinguish the presence of lycopene from β-carotene with the help of both excitation wavelengths, but specially using the 514 nm one, which revealed specific overtones and combination tones of this type of carotene.

Ex vivo confocal microscopy: an emerging technique in dermatology

PubMed Central

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

Direct observation of redox reactions in Candida parapsilosis ATCC 7330 by Confocal microscopic studies.

PubMed

Venkataraman, Sowmyalakshmi; Narayan, Shoba; Chadha, Anju

2016-10-14

Confocal microscopic studies with the resting cells of yeast, Candida parapsilosis ATCC 7330, a reportedly versatile biocatalyst for redox enzyme mediated preparation of optically pure secondary alcohols in high optical purities [enantiomeric excess (ee) up to >99%] and yields, revealed that the yeast cells had large vacuoles under the experimental conditions studied where the redox reaction takes place. A novel fluorescence method was developed using 1-(6-methoxynaphthalen-2-yl)ethanol to track the site of biotransformation within the cells. This alcohol, itself non-fluorescent, gets oxidized to produce a fluorescent ketone, 1-(6-methoxynaphthalen-2-yl)ethanone. Kinetic studies showed that the reaction occurs spontaneously and the products get released out of the cells in less time [5 mins]. The biotransformation was validated using HPLC.

Dual-slit confocal light sheet microscopy for in vivo whole-brain imaging of zebrafish

PubMed Central

Yang, Zhe; Mei, Li; Xia, Fei; Luo, Qingming; Fu, Ling; Gong, Hui

2015-01-01

In vivo functional imaging at single-neuron resolution is an important approach to visualize biological processes in neuroscience. Light sheet microscopy (LSM) is a cutting edge in vivo imaging technique that provides micron-scale spatial resolution at high frame rate. Due to the scattering and absorption of tissue, however, conventional LSM is inadequate to resolve cells because of the attenuated signal to noise ratio (SNR). Using dual-beam illumination and confocal dual-slit detection, here a dual-slit confocal LSM is demonstrated to obtain the SNR enhanced images with frame rate twice as high as line confocal LSM method. Through theoretical calculations and experiments, the correlation between the slit’s width and SNR was determined to optimize the image quality. In vivo whole brain structural imaging stacks and the functional imaging sequences of single slice were obtained for analysis of calcium activities at single-cell resolution. A two-fold increase in imaging speed of conventional confocal LSM makes it possible to capture the sequence of the neurons’ activities and help reveal the potential functional connections in the whole zebrafish’s brain. PMID:26137381

Imaging ion and molecular transport at subcellular resolution by secondary ion mass spectrometry

NASA Astrophysics Data System (ADS)

Chandra, Subhash; Morrison, George H.

1995-05-01

The transport of K+, Na+, and Ca2+ were imaged in individual cells with a Cameca IMS-3f ion microscope. Strict cryogenic frozen freeze-dry sample preparations were employed. Ion redistribution artifacts in conventional chemical preparations are discussed. Cryogenically prepared freeze-fractured freeze-dried cultured cells allowed the three-dimensional ion microscopic imaging of elements. As smaller structures in calcium images can be resolved with the 0.5 [mu]m spatial resolution, correlative techniques are needed to confirm their identity. The potentials of reflected light microscopy, scanning electron microscopy and laser scanning confocal microscopy are discussed for microfeature recognition in freeze-fractured freeze-dried cells. The feasibility of using frozen freeze-dried cells for imaging molecular transport at subcellular resolution was tested. Ion microscopy successfully imaged the transport of the isotopically tagged (13C, 15N) amino acid, -arginine. The labeled amino acid was imaged at mass 28 with a Cs+ primary ion beam as the 28(13C15N)- species. After a 4 h exposure of LLC-PK1 kidney cells to 4 mM labeled arginine, the amino acid was localized throughout the cell with a preferential incorporation into the nucleus and nucleolus. An example is also shown of the ion microscopic imaging of sodium borocaptate, an experimental therapeutic drug for brain tumors, in cryogenically prepared frozen freeze-dried Swiss 3T3 cells.

Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope

PubMed Central

Klauss, André; König, Marcelle; Hille, Carsten

2015-01-01

By overcoming the diffraction limit in light microscopy, super-resolution techniques, such as stimulated emission depletion (STED) microscopy, are experiencing an increasing impact on life sciences. High costs and technically demanding setups, however, may still hinder a wider distribution of this innovation in biomedical research laboratories. As far-field microscopy is the most widely employed microscopy modality in the life sciences, upgrading already existing systems seems to be an attractive option for achieving diffraction-unlimited fluorescence microscopy in a cost-effective manner. Here, we demonstrate the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout, previously proposed as “easy-STED”, achieving lateral resolution < λ/10 corresponding to a five-fold improvement over a confocal modality. For this purpose, both the excitation and depletion laser beams pass through a commercially available segmented phase plate that creates the STED-doughnut light distribution in the focal plane, while leaving the excitation beam unaltered when implemented into the joint beam path. Diffraction-unlimited imaging of 20 nm-sized fluorescent beads as reference were achieved with the wavelength combination of 635 nm excitation and 766 nm depletion. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and Abberior STAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. Here, we applied a recently proposed deconvolution approach and showed that images obtained from time-gated pulsed STED microscopy may benefit concerning the signal-to-background ratio, from the joint deconvolution of sub-images with different spatial information which were extracted from offline time gating. PMID:26091552

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

NASA Astrophysics Data System (ADS)

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.

Reflectance confocal microscope for imaging oral tissues in vivo, potentially with line scanning as a low-cost approach for clinical use

NASA Astrophysics Data System (ADS)

Peterson, Gary; Abeytunge, Sanjeewa; Eastman, Zachary; Rajadhyaksha, Milind

2012-02-01

Reflectance confocal microscopy with a line scanning approach potentially offers a smaller, simpler and less expensive approach than traditional methods of point scanning for imaging in living tissues. With one moving mechanical element (galvanometric scanner), a linear array detector and off-the-shelf optics, we designed a compact (102x102x76mm) line scanning confocal reflectance microscope (LSCRM) for imaging human tissues in vivo in a clinical setting. Custom-designed electronics, based on field programmable gate array (FPGA) logic has been developed. With 405 nm illumination and a custom objective lens of numerical aperture 0.5, lateral resolution was measured to be 0.8 um (calculated 0.64 um). The calculated optical sectioning is 3.2 um. Preliminary imaging shows nuclear and cellular detail in human skin and oral epithelium in vivo. Blood flow is also visualized in the deeper connective tissue (lamina propria) in oral mucosa. Since a line is confocal only in one dimension (parallel) but not in the other, the detection is more sensitive to multiply scattered out of focus background noise than in the traditional point scanning configuration. Based on the results of our translational studies thus far, a simpler, smaller and lower-cost approach based on a LSCRM appears to be promising for clinical imaging.

Development of a near-field/confocal polarization microscope for local measurements of anisotropy in organic films

NASA Astrophysics Data System (ADS)

Kosterin, Andrey Valentinovich

2000-10-01

Polarization microscopy is a powerful technique for imaging structure and stress distributions in many transparent materials, and has been particularly useful in morphology studies of polymer films. Recently the possibility of combining polarization imaging with near-field scanning optical microscopy (NSOM) has been demonstrated, offering new opportunities for studying molecular organization with better than 50 nm resolution. However, there are challenges associated with near-field polarization experiments on organic films: (1) the films are susceptible to damage by the near-field probe; (2) the phase shift or retardation (80) is small, often <0.1 rad; (3) interpretation of near-field images is complicated by topography and probe-sample coupling. To address these challenges, we have developed a new combined near-field/confocal polarization microscope and tested its sensitivity to linear birefringence in thin polymer films. For near-field imaging, the microscope employs a commercially available scanhead with cantilevered (bent) optical fiber probes. To study soft samples (point 1), we have modified the scanhead for tapping mode feedback, which eliminates probe-sample shear forces and prolongs the lifetime of the probe, while minimizing damage to the sample. To achieve sensitivity to small phase shifts (point 2), we have implemented the phase modulation (PM) technique in the optical path. Enhanced sensitivity relative to the standard crossed polarizers scheme is achieved because of the better signal-to-noise discrimination common to lock-in detection and because the detected first harmonic intensity, Io , is linearly proportional to deltatheta instead of (deltatheta) 2. To facilitate interpretation of near-field contrast (point 3), we have incorporated near-field and confocal channels in one instrument. This allows consecutive acquisition of both near-field and far-field images on the same sample area. Since the far-field images do not suffer from the same artifacts, they can be used as a source of independent information on sample optical properties. The combined near-field/confocal polarization microscope is discussed in this thesis as well as some of its applications. Specifically we consider the results of polyethylene oxide (PEO) and crosslinked polybutadiene (PB) thin film imaging.

High magnification bronchovideoscopy combined with narrow band imaging could detect capillary loops of angiogenic squamous dysplasia in heavy smokers at high risk for lung cancer.

PubMed

Shibuya, K; Hoshino, H; Chiyo, M; Iyoda, A; Yoshida, S; Sekine, Y; Iizasa, T; Saitoh, Y; Baba, M; Hiroshima, K; Ohwada, H; Fujisawa, T

2003-11-01

We investigated the use of high magnification bronchovideoscopy combined with narrow band imaging (NBI) for the detailed examination of angiogenic squamous dysplasia (ASD). This was carried out in relation to bronchial vascular patterns with abnormal mucosal fluorescence in heavy smokers at high risk for lung cancer. Forty eight patients with sputum cytology specimens suspicious or positive for malignancy were entered into the study. Conventional white light and fluorescence bronchoscopic examination was first performed. Observations by high magnification bronchovideoscopy with conventional white light were made primarily at sites of abnormal fluorescence, and then repeated with NBI light to examine microvascular networks in the bronchial mucosa. Spectral features on the RGB (Red/Green/Blue) sequential videoscope system were changed from the conventional RGB broadband filter to the new NBI filter. The wavelength ranges of the new NBI filter were B1: 400-430 nm, B2: 420-470 nm, and G: 560-590 nm. ASD tissues were also examined using a confocal laser scanning microscope equipped with argon-krypton (488 nm) and argon (514 nm) laser sources. The microvessels, vascular networks of various grades, and dotted vessels in ASD tissues were clearly observed in NBI-B1 images. Diameters of the dotted vessels visible on NBI-B1 images agreed with the diameters of ASD capillary blood vessels diagnosed by pathological examination. Capillary blood vessels were also clearly visualised by green fluorescence by confocal laser scanning microscopy. There was a significant association between the frequency of dotted vessels by NBI-B1 imaging and tissues confirmed as ASD pathologically (p=0.002). High magnification bronchovideoscopy combined with NBI was useful in the detection of capillary blood vessels in ASD lesions at sites of abnormal fluorescence. This may enable the discrimination between ASD and another pre-invasive bronchial lesion.

Differential high-speed digital micromirror device based fluorescence speckle confocal microscopy.

PubMed

Jiang, Shihong; Walker, John

2010-01-20

We report a differential fluorescence speckle confocal microscope that acquires an image in a fraction of a second by exploiting the very high frame rate of modern digital micromirror devices (DMDs). The DMD projects a sequence of predefined binary speckle patterns to the sample and modulates the intensity of the returning fluorescent light simultaneously. The fluorescent light reflecting from the DMD's "on" and "off" pixels is modulated by correlated speckle and anticorrelated speckle, respectively, to form two images on two CCD cameras in parallel. The sum of the two images recovers a widefield image, but their difference gives a near-confocal image in real time. Experimental results for both low and high numerical apertures are shown.

Confocal microscopy imaging of the biofilm matrix.

PubMed

Schlafer, Sebastian; Meyer, Rikke L

2017-07-01

The extracellular matrix is an integral part of microbial biofilms and an important field of research. Confocal laser scanning microscopy is a valuable tool for the study of biofilms, and in particular of the biofilm matrix, as it allows real-time visualization of fully hydrated, living specimens. Confocal microscopes are held by many research groups, and a number of methods for qualitative and quantitative imaging of the matrix have emerged in recent years. This review provides an overview and a critical discussion of techniques used to visualize different matrix compounds, to determine the concentration of solutes and the diffusive properties of the biofilm matrix. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed Central

Zehri, Aqib H.; Ramey, Wyatt; Georges, Joseph F.; Mooney, Michael A.; Martirosyan, Nikolay L.; Preul, Mark C.; Nakaji, Peter

2014-01-01

Background: The clinical application of fluorescent contrast agents (fluorescein, indocyanine green, and aminolevulinic acid) with intraoperative microscopy has led to advances in intraoperative brain tumor imaging. Their properties, mechanism of action, history of use, and safety are analyzed in this report along with a review of current laser scanning confocal endomicroscopy systems. Additional imaging modalities with potential neurosurgical utility are also analyzed. Methods: A comprehensive literature search was performed utilizing PubMed and key words: In vivo confocal microscopy, confocal endomicroscopy, fluorescence imaging, in vivo diagnostics/neoplasm, in vivo molecular imaging, and optical imaging. Articles were reviewed that discussed clinically available fluorophores in neurosurgery, confocal endomicroscopy instrumentation, confocal microscopy systems, and intraoperative cancer diagnostics. Results: Current clinically available fluorescent contrast agents have specific properties that provide microscopic delineation of tumors when imaged with laser scanning confocal endomicroscopes. Other imaging modalities such as coherent anti-Stokes Raman scattering (CARS) microscopy, confocal reflectance microscopy, fluorescent lifetime imaging (FLIM), two-photon microscopy, and second harmonic generation may also have potential in neurosurgical applications. Conclusion: In addition to guiding tumor resection, intraoperative fluorescence and microscopy have the potential to facilitate tumor identification and complement frozen section analysis during surgery by providing real-time histological assessment. Further research, including clinical trials, is necessary to test the efficacy of fluorescent contrast agents and optical imaging instrumentation in order to establish their role in neurosurgery. PMID:24872922

Intensity calibration of a laser scanning confocal microscope based on concentrated dyes.

PubMed

Model, Michael A; Blank, James L

2006-10-01

To find water-soluble fluorescent dyes with absorption in various regions of the spectrum and investigate their utility as standards for laser scanning confocal microscopy. Several dyes were found to have characteristics required for fluorescence microscopy standards. The intensity of biological fluorescent specimens was measured against the emission of concentrated dyes. Results using different optics and different microscopes were compared. Slides based on concentrated dyes can be prepared in a highly reproducible manner and are stable under laser scanning. Normalized fluorescence of biological specimens remains consistent with different objective lenses and is tolerant to some mismatch in optical filters or imperfect pinhole alignment. Careful choice of scanning parameters is necessary to ensure linearity of intensity measurements. Concentrated dyes provide a robust and inexpensive intensity standard that can be used in basic research or clinical studies.

Laser confocal microscope for analysis of 3013 inner container closure weld region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinez-Rodriguez, M. J.

As part of the protocol to investigate the corrosion in the inner container closure weld region (ICCWR) a laser confocal microscope (LCM) was used to perform close visual examination of the surface and measurements of corrosion features on the surface. However, initial analysis of selected destructively evaluated (DE) containers using the LCM revealed several challenges for acquiring, processing and interpreting the data. These challenges include topography of the ICCWR sample, surface features, and the amount of surface area for collecting data at high magnification conditions. In FY17, the LCM parameters were investigated to identify the appropriate parameter values for datamore » acquisition and identification of regions of interest. Using these parameter values, selected DE containers were analyzed to determine the extent of the ICCWR to be examined.« less

Confocal Microscopy

NASA Astrophysics Data System (ADS)

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.

Upright Imaging of Drosophila Egg Chambers

PubMed Central

Manning, Lathiena; Starz-Gaiano, Michelle

2015-01-01

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective. PMID:25867882

In Situ Observation of Kinetic Processes of Lath Bainite Nucleation and Growth by Laser Scanning Confocal Microscope in Reheated Weld Metals

NASA Astrophysics Data System (ADS)

Mao, Gaojun; Cao, Rui; Guo, Xili; Jiang, Yong; Chen, Jianhong

2017-12-01

The kinetic processes of nucleation and growth of bainite laths in reheated weld metals are observed and analyzed by a combination of a laser confocal scanning microscope and an electron backscattering diffraction with a field emission scanning electron microscope. The results indicate that the surface relief induced by phase transformation is able to reveal the real microstructural morphologies of bainite laths when viewed from various angles. Five nucleation modes and six types of growth behaviors of bainite laths are revealed. The bainite lath growth rates are measured to vary over a wide range, from 2 μm/s to higher than 2000 μm/s. The orientations of the bainite laths within a prior austenite grain are examined and denoted as different variants. On the basis of variant identification, the reason is analyzed for various growth rates which are demonstrated to be affected by (1) the density of the high-angle misorientation in it, (2) the included angle between habit planes of different variants, and (3) the direction of lath growth with respect to the free (polished) surface.

Parallel detecting super-resolution microscopy using correlation based image restoration

NASA Astrophysics Data System (ADS)

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

2017-12-01

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

SRRF: Universal live-cell super-resolution microscopy.

PubMed

Culley, Siân; Tosheva, Kalina L; Matos Pereira, Pedro; Henriques, Ricardo

2018-08-01

Super-resolution microscopy techniques break the diffraction limit of conventional optical microscopy to achieve resolutions approaching tens of nanometres. The major advantage of such techniques is that they provide resolutions close to those obtainable with electron microscopy while maintaining the benefits of light microscopy such as a wide palette of high specificity molecular labels, straightforward sample preparation and live-cell compatibility. Despite this, the application of super-resolution microscopy to dynamic, living samples has thus far been limited and often requires specialised, complex hardware. Here we demonstrate how a novel analytical approach, Super-Resolution Radial Fluctuations (SRRF), is able to make live-cell super-resolution microscopy accessible to a wider range of researchers. We show its applicability to live samples expressing GFP using commercial confocal as well as laser- and LED-based widefield microscopes, with the latter achieving long-term timelapse imaging with minimal photobleaching. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Microscopic and molecular identification of hemotropic mycoplasmas in South American coatis (Nasua nasua).

PubMed

Cubilla, Michelle P; Santos, Leonilda C; de Moraes, Wanderlei; Cubas, Zalmir S; Leutenegger, Christian M; Estrada, Marko; Lindsay, LeAnn L; Trindade, Edvaldo S; Franco, Célia Regina C; Vieira, Rafael F C; Biondo, Alexander W; Sykes, Jane E

2017-08-01

Hemoplasmas were detected in two apparently healthy captive South American coatis (Nasua nasua) from southern Brazil during an investigation for vector-borne pathogens. Blood was subjected to packed cell volume (PCV) determination, a commercial real-time PCR panel for the detection of Anaplasma spp., Babesia spp., Bartonella spp., Hepatozoon spp., Leishmania spp., Mycoplasma haemofelis, 'Candidatus Mycoplasma turicensis', 'Candidatus Mycoplasma haemominutum', Neorickettsia risticii, Rickettsia rickettsii and Leptospira spp., and a pan-hemoplasma conventional PCR assay. PCV was normal, but both coatis tested positive for hemoplasmas and negative for all the remaining pathogens tested. Using different techniques for microscopy (light, confocal or SEM), structures compatible with hemoplasmas were identified. Sequencing of the 16S rRNA gene identified an organism resembling Mycoplasma haemofelis and another hemotropic Mycoplasma sp., with a sequence identity of 96.8% to a Mycoplasma sp. previously detected in capybaras. Copyright © 2017 Elsevier Ltd. All rights reserved.

Application of confocal surface wave microscope to self-calibrated attenuation coefficient measurement by Goos-Hänchen phase shift modulation.

PubMed

Pechprasarn, Suejit; Chow, Terry W K; Somekh, Michael G

2018-06-04

In this paper, we present a direct method to measure surface wave attenuation arising from both ohmic and coupling losses using our recently developed phase spatial light modulator (phase-SLM) based confocal surface plasmon microscope. The measurement is carried out in the far-field using a phase-SLM to impose an artificial surface wave phase profile in the back focal plane (BFP) of a microscope objective. In other words, we effectively provide an artificially engineered backward surface wave by modulating the Goos Hänchen (GH) phase shift of the surface wave. Such waves with opposing phase and group velocities are well known in acoustics and electromagnetic metamaterials but usually require structured or layered surfaces, here the effective wave is produced externally in the microscope illumination path. Key features of the technique developed here are that it (i) is self-calibrating and (ii) can distinguish between attenuation arising from ohmic loss (k″ Ω ) and coupling (reradiation) loss (k″ c ). This latter feature has not been achieved with existing methods. In addition to providing a unique measurement the measurement occurs of over a localized region of a few microns. The results were then validated against the surface plasmons (SP) dip measurement in the BFP and a theoretical model based on a simplified Green's function.

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

NASA Astrophysics Data System (ADS)

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hödemann, S., E-mail: siim.hodemann@ut.ee; Möls, P.; Kiisk, V.

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 coefficientmore » is calculated and discussed. Our method opens up new possibilities for three-dimensional scattered light tomography of mechanical imaging in birefringent materials.« less

ConfocalGN: A minimalistic confocal image generator

NASA Astrophysics Data System (ADS)

Dmitrieff, Serge; Nédélec, François

Validating image analysis pipelines and training machine-learning segmentation algorithms require images with known features. Synthetic images can be used for this purpose, with the advantage that large reference sets can be produced easily. It is however essential to obtain images that are as realistic as possible in terms of noise and resolution, which is challenging in the field of microscopy. We describe ConfocalGN, a user-friendly software that can generate synthetic microscopy stacks from a ground truth (i.e. the observed object) specified as a 3D bitmap or a list of fluorophore coordinates. This software can analyze a real microscope image stack to set the noise parameters and directly generate new images of the object with noise characteristics similar to that of the sample image. With a minimal input from the user and a modular architecture, ConfocalGN is easily integrated with existing image analysis solutions.

Two-Photon Fluorescence Microscope for Microgravity Research

NASA Technical Reports Server (NTRS)

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 longer-wavelength excitation light and passes the shorter-wavelength fluorescence light. Also, the confocal pinhole has been removed to increase the signal strength. The laser beam is scanned by a twoperpendicular- axis pair of galvanometer mirrors through a pupil transfer lens into the side port of an inverted microscope. Finally, the beam is focused by a 63-magnification, 1.3-numerical- aperture oil-immersion objective lens onto a specimen. The pupil transfer lens serves to match the intermediate image planes of the scanning head and the microscope, and its location is critical. In order to maximize the quality of the image, (that is, the point spread function of the objective lens for all scan positions), the entire system was modeled in optical-design software, and the various free design parameters (the parameters of the spatial-filter components as well as the separations of all of the system components) were determined through an iterative optimization process. A modular design was chosen to facilitate access to the optical train for future fluorescence correlation spectroscopy and fluorescence-lifetime experiments.

[Revealing the chemical changes of tea cell wall induced by anthracnose with confocal Raman microscopy].

PubMed

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.

Towards modeling of cardiac micro-structure with catheter-based confocal microscopy: a novel approach for dye delivery and tissue characterization.

PubMed

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.

Dynamics of intracellular processes in live-cell systems unveiled by fluorescence correlation microscopy.

PubMed

González Bardeci, Nicolás; Angiolini, Juan Francisco; De Rossi, María Cecilia; Bruno, Luciana; Levi, Valeria

2017-01-01

Fluorescence fluctuation-based methods are non-invasive microscopy tools especially suited for the study of dynamical aspects of biological processes. These methods examine spontaneous intensity fluctuations produced by fluorescent molecules moving through the small, femtoliter-sized observation volume defined in confocal and multiphoton microscopes. The quantitative analysis of the intensity trace provides information on the processes producing the fluctuations that include diffusion, binding interactions, chemical reactions and photophysical phenomena. In this review, we present the basic principles of the most widespread fluctuation-based methods, discuss their implementation in standard confocal microscopes and briefly revise some examples of their applications to address relevant questions in living cells. The ultimate goal of these methods in the Cell Biology field is to observe biomolecules as they move, interact with targets and perform their biological action in the natural context. © 2016 IUBMB Life, 69(1):8-15, 2017. © 2016 International Union of Biochemistry and Molecular Biology.

Imaging Chromosome Separation in Mouse Oocytes by Responsive 3D Confocal Timelapse Microscopy.

PubMed

Lane, Simon I R; Crouch, Stephen; Jones, Keith T

2017-01-01

Accurate chromosome segregation is necessary so that genetic material is equally shared among daughter cells. However, maturing mammalian oocytes are particularly prone to chromosome segregation errors, making them a valuable tool for identifying the causes of mis-segregation. Factors such as aging, cohesion loss, DNA damage, and the roles of a plethora of kinetochore and cell cycle-related proteins are involved. To study chromosome segregation in oocytes in a live setting is an imaging challenge that requires advanced techniques. Here we describe a method for examining chromosomes in live oocytes in detail as they undergo maturation. Our method is based on tracking the "center of brightness" of fluorescently labeled chromosomes. Here we describe how to set up our software and run experiments on a Leica TCS SP8 confocal microscope, but the method would be transferable to other microscopes with computer-aided microscopy.

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

NASA Astrophysics Data System (ADS)

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.

Eyelid skin as a potential site for drug delivery to conjunctiva and ocular tissues.

PubMed

See, Gerard Lee; Sagesaka, Ayano; Sugasawa, Satoko; Todo, Hiroaki; Sugibayashi, Kenji

2017-11-25

The feasibility of topical application onto the (lower) eyelid skin to deliver hydrophilic and lipophilic compounds into the conjunctiva and ocular tissues was evaluated by comparing with conventional eye drop application. Skin permeation and the concentration of several model compounds, and skin impedance were determined utilizing eyelid skin from hairless rats, as well as abdominal skin in the same animals for comparison. In vitro static diffusion cells were used to assess the skin permeation in order to provide key insights into the relationship between the skin sites and drugs. The obtained results revealed that drug permeation through the eyelid skin was much higher than that through abdominal skin regardless of the drug lipophilicity. Specifically, diclofenac sodium salt and tranilast exhibited approximately 6-fold and 11-fold higher permeability coefficients, respectively, through eyelid skin compared with abdominal skin. Histomorphological evaluation and in vivo distribution of model fluorescent dyes were also examined in the conjunctiva and skin after eyelid administration by conventional microscope and confocal laser scanning microscope analyses. The result revealed that eyelid skin has a thinner stratum corneum, thereby showing lower impedance, which could be the reason for the higher drug permeation through eyelid skin. Comparative evaluation of lipophilic and hydrophilic model compounds administered via the eyelid skin over 8h revealed stronger fluorescence intensity in the skin and surrounding tissues compared with eye drop administration. These results suggested that the (lower) eyelid skin is valuable as a prospective site for ophthalmic medicines. Copyright © 2017 Elsevier B.V. All rights reserved.

Light sheet-based fluorescence microscopy (LSFM) reduces phototoxic effects and provides new means for the modern life sciences

NASA Astrophysics Data System (ADS)

Pampaloni, Francesco; Ansari, Nari; Girard, Philippe; Stelzer, Ernst H. K.

2011-07-01

Most optical technologies are applied to flat, basically two-dimensional cellular systems. However, physiological meaningful information relies on the morphology, the mechanical properties and the biochemistry of a cell's context. A cell requires the complex three-dimensional relationship to other cells. However, the observation of multi-cellular biological specimens remains a challenge. Specimens scatter and absorb light, thus, the delivery of the probing light and the collection of the signal light become inefficient; many endogenous biochemical compounds also absorb light and suffer degradation of some sort (photo-toxicity), which induces malfunction of a specimen. In conventional and confocal fluorescence microscopy, whenever a single plane, the entire specimen is illuminated. Recording stacks of images along the optical Z-axis thus illuminates the entire specimen once for each plane. Hence, cells are illuminated 10-20 and fish 100-300 times more often than they are observed. This can be avoided by changing the optical arrangement. The basic idea is to use light sheets, which are fed into the specimen from the side and overlap with the focal plane of a wide-field fluorescence microscope. In contrast to an epi-fluorescence arrangement, such an azimuthal fluorescence arrangement uses two independently operated lenses for illumination and detection. Optical sectioning and no photo-toxic damage or photo-bleaching outside a small volume close to the focal plane are intrinsic properties. Light sheet-based fluorescence microscopy (LSFM) takes advantage of modern camera technologies. LSFM can be operated with laser cutters and for fluorescence correlation spectroscopy. During the last few years, LSFM was used to record zebrafish development from the early 32-cell stage until late neurulation with sub-cellular resolution and short sampling periods (60-90 sec/stack). The recording speed was five 4-Megapixel large frames/sec with a dynamic range of 12-14 bit. We followed cell movements during gastrulation, revealed the development during cell migration processes and showed that an LSFM exposes an embryo to 200 times less energy than a conventional and 5,000 times less energy than a confocal fluorescence microscope. Most recently, we implemented incoherent structured illumination in our DSLM. The intensity modulated light sheets can be generated with dynamic frequencies and allow us to estimate the effect of the specimen on the image formation process at various depths in objects of different age.

Optically Sectioned Imaging of Microvasculature of In-Vivo and Ex-Vivo Thick Tissue Models with Speckle-illumination HiLo Microscopy and HiLo Image Processing Implementation in MATLAB Architecture

NASA Astrophysics Data System (ADS)

Suen, Ricky Wai

The work described in this thesis covers the conversion of HiLo image processing into MATLAB architecture and the use of speckle-illumination HiLo microscopy for use of ex-vivo and in-vivo imaging of thick tissue models. HiLo microscopy is a wide-field fluorescence imaging technique and has been demonstrated to produce optically sectioned images comparable to confocal in thin samples. The imaging technique was developed by Jerome Mertz and the Boston University Biomicroscopy Lab and has been implemented in our lab as a stand-alone optical setup and a modification to a conventional fluorescence microscope. Speckle-illumination HiLo microscopy combines two images taken under speckle-illumination and standard uniform-illumination to generate an optically sectioned image that reject out-of-focus fluorescence. The evaluated speckle contrast in the images is used as a weighting function where elements that move out-of-focus have a speckle contrast that decays to zero. The experiments shown here demonstrate the capability of our HiLo microscopes to produce optically-sectioned images of the microvasculature of ex-vivo and in-vivo thick tissue models. The HiLo microscope were used to image the microvasculature of ex-vivo mouse heart sections prepared for optical histology and the microvasculature of in-vivo rodent dorsal window chamber models. Studies in label-free surface profiling with HiLo microscopy is also presented.

Discrimination of liver cancer in cellular level based on backscatter micro-spectrum with PCA algorithm and BP neural network

NASA Astrophysics Data System (ADS)

Yang, Jing; Wang, Cheng; Cai, Gan; Dong, Xiaona

2016-10-01

The incidence and mortality rate of the primary liver cancer are very high and its postoperative metastasis and recurrence have become important factors to the prognosis of patients. Circulating tumor cells (CTC), as a new tumor marker, play important roles in the early diagnosis and individualized treatment. This paper presents an effective method to distinguish liver cancer based on the cellular scattering spectrum, which is a non-fluorescence technique based on the fiber confocal microscopic spectrometer. Combining the principal component analysis (PCA) with back propagation (BP) neural network were utilized to establish an automatic recognition model for backscatter spectrum of the liver cancer cells from blood cell. PCA was applied to reduce the dimension of the scattering spectral data which obtained by the fiber confocal microscopic spectrometer. After dimensionality reduction by PCA, a neural network pattern recognition model with 2 input layer nodes, 11 hidden layer nodes, 3 output nodes was established. We trained the network with 66 samples and also tested it. Results showed that the recognition rate of the three types of cells is more than 90%, the relative standard deviation is only 2.36%. The experimental results showed that the fiber confocal microscopic spectrometer combining with the algorithm of PCA and BP neural network can automatically identify the liver cancer cell from the blood cells. This will provide a better tool for investigating the metastasis of liver cancers in vivo, the biology metabolic characteristics of liver cancers and drug transportation. Additionally, it is obviously referential in practical application.

Odontoma: retrospective study and confocal laser scanning microscope analysis of 52 cases.

PubMed

Crincoli, V; Scivetti, M; Di Bisceglie, M B; Lucchese, A; Favia, G

2007-01-01

The aim of this study was to perform a retrospective analysis of 52 cases of odontoma treated at the Department of Dentistry and Surgery, University of Bari, in the period 1971-2005. The odontogenic tumors were diagnosed as complex or compound odontoma following histological analysis and clinical radiological examination, and applying the 2005 WHO classification. The data analysis was conducted by considering the following factors: gender, age, site of the lesion, association with impacted teeth, aplasia, presence of supernumerary teeth as well as preoperative diagnosis by panoramic and periapical radiographs. Biopsy tissue samples were conventionally processed for histopathologic paraffin embedding and then were observed by optical microscopy and subsequently by confocal laser scanning microscopy (CLSM) in autofluorescence. Thirty specimens (57.6%) were from females and 22 (42.3%) were from males patients. The patients' age ranged from 5 to 75 years. Fifty-one percent of the specimens were excised from the mandible. In the maxilla, the most common location for odontomas was the anterior region. Most odontomas were associated with impacted teeth and only in one case there was an odontoma instead of a permanent tooth. Odontomas are considered hamartomatous malformations whose diagnosis is generally formulated by routinary radiographic examination. The CLSM analysis could help in diagnosis and histopathological analysis showing well-defined follicular area entrapped in hard tissues and pointing out ghost cells, otherwise not identifiable by traditional microscopy.

Confocal ultrafast pump-probe spectroscopy: a new technique to explore nanoscale composites.

PubMed

Virgili, Tersilla; Grancini, Giulia; Molotokaite, Egle; Suarez-Lopez, Inma; Rajendran, Sai Kiran; Liscio, Andrea; Palermo, Vincenzo; Lanzani, Guglielmo; Polli, Dario; Cerullo, Giulio

2012-04-07

This article is devoted to the exploration of the benefits of a new ultrafast confocal pump-probe technique, able to study the photophysics of different structured materials with nanoscale resolution. This tool offers many advantages over standard stationary microscopy techniques because it directly interrogates excited state dynamics in molecules, providing access to both radiative and non-radiative deactivation processes at a local scale. In this paper we present a few different examples of its application to organic semiconductor systems. The first two are focussed on the study of the photophysics of phase-separated polymer blends: (i) a blue-emitting polyfluorene (PFO) in an inert matrix of PMMA and (ii) an electron donor polythiophene (P3HT) mixed with an electron acceptor fullerene derivative (PCBM). The experimental results on these samples demonstrate the capability of the technique to unveil peculiar interfacial dynamics at the border region between phase-segregated domains, which would be otherwise averaged out using conventional pump-probe spectroscopy. The third example is the study of the photophysics of isolated mesoscopic crystals of the PCBM molecule. Our ultrafast microscope could evidence the presence of two distinctive regions within the crystals. In particular, we could pinpoint for the first time areas within the crystals showing photobleaching/stimulated emission signals from a charge-transfer state. This journal is © The Royal Society of Chemistry 2012

Slide-free histology via MUSE: UV surface excitation microscopy for imaging unsectioned tissue (Conference Presentation)

NASA Astrophysics Data System (ADS)

Levenson, Richard M.; Harmany, Zachary; Demos, Stavros G.; Fereidouni, Farzad

2016-03-01

Widely used methods for preparing and viewing tissue specimens at microscopic resolution have not changed for over a century. They provide high-quality images but can involve time-frames of hours or even weeks, depending on logistics. There is increasing interest in slide-free methods for rapid tissue analysis that can both decrease turn-around times and reduce costs. One new approach is MUSE (microscopy with UV surface excitation), which exploits the shallow penetration of UV light to excite fluorescent signals from only the most superficial tissue elements. The method is non-destructive, and eliminates requirement for conventional histology processing, formalin fixation, paraffin embedding, or thin sectioning. It requires no lasers, confocal, multiphoton or optical coherence tomography optics. MUSE generates diagnostic-quality histological images that can be rendered to resemble conventional hematoxylin- and eosin-stained samples, with enhanced topographical information, from fresh or fixed, but unsectioned tissue, rapidly, with high resolution, simply and inexpensively. We anticipate that there could be widespread adoption in research facilities, hospital-based and stand-alone clinical settings, in local or regional pathology labs, as well as in low-resource environments.

Control algorithms and applications of the wavefront sensorless adaptive optics

NASA Astrophysics Data System (ADS)

Ma, Liang; Wang, Bin; Zhou, Yuanshen; Yang, Huizhen

2017-10-01

Compared with the conventional adaptive optics (AO) system, the wavefront sensorless (WFSless) AO system need not to measure the wavefront and reconstruct it. It is simpler than the conventional AO in system architecture and can be applied to the complex conditions. Based on the analysis of principle and system model of the WFSless AO system, wavefront correction methods of the WFSless AO system were divided into two categories: model-free-based and model-based control algorithms. The WFSless AO system based on model-free-based control algorithms commonly considers the performance metric as a function of the control parameters and then uses certain control algorithm to improve the performance metric. The model-based control algorithms include modal control algorithms, nonlinear control algorithms and control algorithms based on geometrical optics. Based on the brief description of above typical control algorithms, hybrid methods combining the model-free-based control algorithm with the model-based control algorithm were generalized. Additionally, characteristics of various control algorithms were compared and analyzed. We also discussed the extensive applications of WFSless AO system in free space optical communication (FSO), retinal imaging in the human eye, confocal microscope, coherent beam combination (CBC) techniques and extended objects.

Dye-enhanced reflectance and fluorescence confocal microscopy as an optical pathology tool

NASA Astrophysics Data System (ADS)

Yaroslavsky, Anna N.; Salomatina, Elena; Novak, John; Amat-Roldan, Ivan; Castano, Ana; Hamblin, Michael

2006-02-01

Early detection and precise excision of neoplasms are imperative requirements for successful cancer treatment. In this study we evaluated the use of dye-enhanced confocal microscopy as an optical pathology tool in the ex vivo trial with fresh thick non-melanoma skin cancer excisions and in vivo trial with B16F10 melanoma cancer in mice. For the experiments the tumors were rapidly stained using aqueous solutions of either toluidine blue or methylene blue and imaged using multimodal confocal microscope. Reflectance images were acquired at the wavelengths of 630nm and 650 nm. Fluorescence was excited at 630 nm and 650 nm. Fluorescence emission was registered in the range between 680 nm and 710 nm. The images were compared to the corresponding en face frozen H&E sections. The results of the study indicate confocal images of stained cancerous tissue closely resemble corresponding H&E sections both in vivo and in vitro. This remarkable similarity enables interpretation of confocal images in a manner similar to that of histopathology. The developed technique may provide an efficient real-time optical tool for detecting skin pathology.

Potential effect of cationic liposomes on interactions with oral bacterial cells and biofilms.

PubMed

Sugano, Marika; Morisaki, Hirobumi; Negishi, Yoichi; Endo-Takahashi, Yoko; Kuwata, Hirotaka; Miyazaki, Takashi; Yamamoto, Matsuo

2016-01-01

Although oral infectious diseases have been attributed to bacteria, drug treatments remain ineffective because bacteria and their products exist as biofilms. Cationic liposomes have been suggested to electrostatically interact with the negative charge on the bacterial surface, thereby improving the effects of conventional drug therapies. However, the electrostatic interaction between oral bacteria and cationic liposomes has not yet been examined in detail. The aim of the present study was to examine the behavior of cationic liposomes and Streptococcus mutans in planktonic cells and biofilms. Liposomes with or without cationic lipid were prepared using a reverse-phase evaporation method. The zeta potentials of conventional liposomes (without cationic lipid) and cationic liposomes were -13 and 8 mV, respectively, and both had a mean particle size of approximately 180 nm. We first assessed the interaction between liposomes and planktonic bacterial cells with a flow cytometer. We then used a surface plasmon resonance method to examine the binding of liposomes to biofilms. We confirmed the binding behavior of liposomes with biofilms using confocal laser scanning microscopy. The interactions between cationic liposomes and S. mutans cells and biofilms were stronger than those of conventional liposomes. Microscopic observations revealed that many cationic liposomes interacted with the bacterial mass and penetrated the deep layers of biofilms. In this study, we demonstrated that cationic liposomes had higher affinity not only to oral bacterial cells, but also biofilms than conventional liposomes. This electrostatic interaction may be useful as a potential drug delivery system to biofilms.

Lattice relations and solidification of the complex regular eutectic (Cr,Fe)-(Cr,Fe)23C6

NASA Astrophysics Data System (ADS)

Lai, Hsuan-Han; Hsieh, Chih-Chun; Lin, Chi-Ming; Wu, Weite

2017-05-01

The eutectic (Cr,Fe)-(Cr,Fe)23C6 showed a triaxial fishbone structure and could be categorized as a "complex regular structure". In this study, the lattice relations of the fishbone (Cr,Fe)23C6 were examined and the solidification process was observed using a transmission electron microscope and a confocal laser scanning microscope. For one of the three fish bones in a eutectic cell, parallel (Cr,Fe)23C6 lamellas at one side of the spine had the same lattice direction, as did those in the (Cr,Fe) phase. The lattices of neighboring (Cr,Fe)23C6 and (Cr,Fe) phases were not coherent. Lamellar (Cr,Fe)23C6 on opposite sides of a spine had different lattice directions, and their lattice boundary was in the spine. By using the confocal laser scanning microscope, the solidification of lamellar eutectic structure could be observed. At the low cooling rate of 5 o C·min-1, parallel lamellas would grow thick blocks instead of thin plates. To obtain a thin lamellar eutectic structure, the cooling rate should be higher, like the rate in welding.

Flexible conformable hydrophobized surfaces for turbulent flow drag reduction

NASA Astrophysics Data System (ADS)

Brennan, Joseph C.; Geraldi, Nicasio R.; Morris, Robert H.; Fairhurst, David J.; McHale, Glen; Newton, Michael I.

2015-05-01

In recent years extensive work has been focused onto using superhydrophobic surfaces for drag reduction applications. Superhydrophobic surfaces retain a gas layer, called a plastron, when submerged underwater in the Cassie-Baxter state with water in contact with the tops of surface roughness features. In this state the plastron allows slip to occur across the surface which results in a drag reduction. In this work we report flexible and relatively large area superhydrophobic surfaces produced using two different methods: Large roughness features were created by electrodeposition on copper meshes; Small roughness features were created by embedding carbon nanoparticles (soot) into Polydimethylsiloxane (PDMS). Both samples were made into cylinders with a diameter under 12 mm. To characterize the samples, scanning electron microscope (SEM) images and confocal microscope images were taken. The confocal microscope images were taken with each sample submerged in water to show the extent of the plastron. The hydrophobized electrodeposited copper mesh cylinders showed drag reductions of up to 32% when comparing the superhydrophobic state with a wetted out state. The soot covered cylinders achieved a 30% drag reduction when comparing the superhydrophobic state to a plain cylinder. These results were obtained for turbulent flows with Reynolds numbers 10,000 to 32,500.

Quantitative and structural analyses of the in vitro and ex vivo biofilm-forming ability of dermatophytes.

PubMed

Brilhante, Raimunda Sâmia Nogueira; Correia, Edmilson Emanuel Monteiro; Guedes, Glaucia Morgana de Melo; Pereira, Vandbergue Santos; Oliveira, Jonathas Sales de; Bandeira, Silviane Praciano; Alencar, Lucas Pereira de; Andrade, Ana Raquel Colares de; Castelo-Branco, Débora de Souza Collares Maia; Cordeiro, Rossana de Aguiar; Pinheiro, Adriana de Queiroz; Chaves, Lúcio Jackson Queiroz; Pereira Neto, Waldemiro de Aquino; Sidrim, José Júlio Costa; Rocha, Marcos Fábio Gadelha

2017-07-01

The aim of this study was to evaluate the in vitro and ex vivo biofilm-forming ability of dermatophytes on a nail fragment. Initially, four isolates of Trichophyton rubrum, six of Trichophyton tonsurans, three of Trichophyton mentagrophytes, ten of Microsporum canis and three of Microsporum gypseum were tested for production biomass by crystal violet assay. Then, one strain per species presenting the best biofilm production was chosen for further studies by optical microscopy (Congo red staining), confocal laser scanning (LIVE/DEAD staining) and scanning electron (secondary electron) microscopy. Biomass quantification by crystal violet assay, optical microscope images of Congo red staining, confocal microscope and scanning electron microscope images revealed that all species studied are able to form biofilms both in vitro and ex vivo, with variable density and architecture. M. gypseum, T. rubrum and T. tonsurans produced robust biofilms, with abundant matrix and biomass, while M. canis produced the weakest biofilms compared to other species. This study sheds light on biofilms of different dermatophyte species, which will contribute to a better understanding of the pathophysiology of dermatophytosis. Further studies of this type are necessary to investigate the processes involved in the formation and composition of dermatophyte biofilms.

Imaging fluorescence detected linear dichroism of plant cell walls in laser scanning confocal microscope.

PubMed

Steinbach, Gábor; Pomozi, István; Zsiros, Ottó; Páy, Anikó; Horváth, Gábor V; Garab, Gyozo

2008-03-01

Anisotropy carries important information on the molecular organization of biological samples. Its determination requires a combination of microscopy and polarization spectroscopy tools. The authors constructed differential polarization (DP) attachments to a laser scanning microscope in order to determine physical quantities related to the anisotropic distribution of molecules in microscopic samples; here the authors focus on fluorescence-detected linear dichroism (FDLD). By modulating the linear polarization of the laser beam between two orthogonally polarized states and by using a demodulation circuit, the authors determine the associated transmitted and fluorescence intensity-difference signals, which serve the basis for LD (linear dichroism) and FDLD, respectively. The authors demonstrate on sections of Convallaria majalis root tissue stained with Acridin Orange that while (nonconfocal) LD images remain smeared and weak, FDLD images recorded in confocal mode reveal strong anisotropy of the cell wall. FDLD imaging is suitable for mapping the anisotropic distribution of transition dipoles in 3 dimensions. A mathematical model is proposed to account for the fiber-laminate ultrastructure of the cell wall and for the intercalation of the dye molecules in complex, highly anisotropic architecture. Copyright 2007 International Society for Analytical Cytology.

Site-specific accumulation and dynamic change of flavonoids in Apocyni Veneti Folium.

PubMed

Chen, Cui-Hua; Xu, Hu; Liu, Xun-Hong; Zou, Li-Si; Wang, Mei; Liu, Zi-Xiu; Fu, Xing-Sheng; Zhao, Hui; Yan, Ying

2017-12-01

Site-specific accumulation of flavonoids in Apocyni Veneti Folium was determined by laser scanning confocal microscope (LSCM) and the localization of catechins also was observed via vanillin-HCl staining under the conventional optical microscope. The contents of five flavonoids in Apocyni Veneti Folium from different harvest times and growth parts were measured using HPLC method. LSCM observation showed that flavonoids are accumulated in cuticle of epidermal cells and vessel walls, especially in protoplasts and nucleolus of the collenchyma cells and the epidermal cells. Catechins are localized in the palisade parenchyma cells and vessel walls, particularly in the laticifers found in the phloem. On the basis of the difference of the maximal emission wavelength between quercetin and kaempferol derivatives which have fluorescence behavior by appropriate treatment, kaempferol and its derivatives are localized exclusively in the cuticle. Results showed that the content of astragalin in Apocyni Veneti Folium from different parts revealed the decreasing trend, while hyperin and isoquercitrin were higher in June and July analyzed by HPLC. In summary, the site-specific accumulation of flavonoids in Apocyni Veneti Folium can be determined by LSCM and vanillin-HCl staining. The contents of flavonoids in Apocyni Veneti Folium are correlated with harvest times and growth parts. © 2017 Wiley Periodicals, Inc.

Facile fabrication of bowl-shaped microparticles for oral curcumin delivery to ulcerative colitis tissue.

PubMed

Chen, Qiubing; Gou, Shuangquan; Huang, Yamei; Zhou, Xin; Li, Qian; Han, Moon Kwon; Kang, Yuejun; Xiao, Bo

2018-05-05

Oral microparticles (MPs) have been considered as promising drug carriers in the treatment of ulcerative colitis (UC). Here, a facile strategy based on a conventional emulsion-solvent evaporation technique was used to fabricate bowl-shaped MPs (BMPs), and these MPs loaded with anti-inflammatory drug (curcumin, CUR) during the fabrication process. The physicochemical properties of the resultant BMPs were characterized by dynamic light scattering, scanning electron microscope, confocal laser scanning microscope and X-ray diffraction as well as contact angle goniometer. Results indicated that BMPs had a desirable hydrodynamic diameter (1.84 ± 0.20 μm), a negative zeta potential (-26.5 ± 1.13 mV), smooth surface morphology, high CUR encapsulation efficiency and controlled drug release profile. It was found that CUR molecules were dispersed in an amorphous state within the polymeric matrixes. In addition, BMPs showed excellent hydrophilicity due to the presence of Pluronic F127 and poly(vinyl alcohol) on their surface. More importantly, orally administered BMPs could efficiently alleviate UC based on a dextran sulfate sodium-induced mouse model. These results collectively suggest that BMP can be exploited as a readily scalable oral drug delivery system for UC therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Quantification of whey in fluid milk using confocal Raman microscopy and artificial neural network.

PubMed

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.

Novel approach to real-time flash photolysis and confocal [Ca2+] imaging

PubMed Central

Sobie, Eric A.; Kao, Joseph P.Y.; Lederer, W. J.

2008-01-01

Flash photolysis of “caged” compounds using ultraviolet light is a powerful experimental technique for producing rapid changes in concentrations of bioactive signaling molecules. Studies that employ this technique have used diverse strategies for controlling the spatial and temporal application of light to the specimen. Here we describe a new system for flash photolysis that delivers light from a pulsed, adjustable intensity laser through an optical fiber coupled into the epifluorescence port of a commercial confocal microscope. Photolysis is achieved with extremely brief (5 ns) pulses of ultraviolet light (355 nm) that can be synchronized with respect to confocal laser scanning. The system described also localizes the UV intensity spatially so that uncaging only occurs in defined sub-cellular regions; moreover, since the microscope optics are used in localization, the photolysis volume can be easily adjusted. Experiments performed on rat ventricular myocytes loaded with the Ca2+ indicator fluo-3 and the Ca2+ cage NP-EGTA demonstrate the system's capabilities. Localized intracellular increases in [Ca2+] can trigger sarcoplasmic reticular Ca2+ release events such as Ca2+ sparks and, under certain conditions, regenerative Ca2+ waves. This relatively simple and inexpensive system is therefore a useful tool for examining local signaling in heart and other tissues. PMID:17323075

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.

PubMed

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.

Epiphany sealer penetration into dentinal tubules: Confocal laser scanning microscopic study.

PubMed

Ravi, S V; Nageswar, Rao; Swapna, Honwad; Sreekant, Puthalath; Ranjith, Madhavan; Mahidhar, Surabhi

2014-03-01

The aim of the following study was to evaluate the percentage and average depth of epiphany sealer penetration into dentinal tubules among the coronal, middle and apical thirds of the root using the confocal laser scanning microscopy (CLSM). A total of 10 maxillary central incisors were prepared and obturated with Resilon-Epiphany system. Sealer was mixed with fluorescent rhodamine B isothiyocyanate dye for visibility under confocal microscope. Teeth were cross-sectioned into coronal, middle and apical sections-2 mm thick. Sections were observed under CLSM. Images were analyzed for percentage and average depth of sealer penetration into dentinal tubules using the lasso tool in Adobe Photoshop CS3 (Adobe systems incorporated, San jose, CA) and laser scanning microscopy (LSM 5) image analyzer. One-way analysis of variance with Student Neuman Keuls post hoc tests, Kruskal-Wallis test and Wilcoxon signed-rank post hoc tests. The results showed that a higher percentage of sealer penetration in coronal section-89.23%, followed by middle section-84.19% and the apical section-64.9%. Average depth of sealer penetration for coronal section was 526.02 μm, middle-385.26 μm and apical-193.49 μm. Study concluded that there was higher epiphany sealer penetration seen in coronal followed by middle and least at apical third of the roots.

Investigating the impact of blood pressure increase to the brain using high resolution serial histology and image processing

NASA Astrophysics Data System (ADS)

Lesage, F.; Castonguay, A.; Tardif, P. L.; Lefebvre, J.; Li, B.

2015-09-01

A combined serial OCT/confocal scanner was designed to image large sections of biological tissues at microscopic resolution. Serial imaging of organs embedded in agarose blocks is performed by cutting through tissue using a vibratome which sequentially cuts slices in order to reveal new tissue to image, overcoming limited light penetration encountered in microscopy. Two linear stages allow moving the tissue with respect to the microscope objective, acquiring a 2D grid of volumes (1x1x0.3 mm) with OCT and a 2D grid of images (1x1mm) with the confocal arm. This process is repeated automatically, until the entire sample is imaged. Raw data is then post-processed to re-stitch each individual acquisition and obtain a reconstructed volume of the imaged tissue. This design is being used to investigate correlations between white matter and microvasculature changes with aging and with increase in pulse pressure following transaortic constriction in mice. The dual imaging capability of the system allowed to reveal different contrast information: OCT imaging reveals changes in refractive indices giving contrast between white and grey matter in the mouse brain, while transcardial perfusion of FITC or pre-sacrifice injection of Evans Blue shows microsvasculature properties in the brain with confocal imaging.

Fluorescence fibre-optic confocal microscopy of skin in vivo: microscope and fluorophores.

PubMed

Suihko, Christian; Swindle, Lucinda D; Thomas, Steven G; Serup, Jørgen

2005-11-01

Fibre-optic confocal imaging in vivo is a new approach in the assessment of human skin. The objective is to describe a novel instrument and its operation and use in combination with fluorophores. The Stratum is a fibre-optic fluorescence confocal microscope especially developed for the study of skin and mucous membranes. The system is flexible and any body site can be studied with a hand-held scanner. The light source is a 488 nm argon ion laser. Horizontal (en face) images of the epidermis and outer dermis are produced with cellular resolution. Magnification is approximately 1000 x . Fluorescein sodium is routinely used as fluorophore (intradermal injection or application to the skin surface). This fluorophore is safe for human use in vivo, but other substances (rhodamine B, Acridine Orange, green fluorescent protein, curcumin) have also been studied. The instrument produces sharp images of epidermal cell layers from the epidermal surface to the sub-papillary dermis, with sub-cellular resolution. The scanner is flexible in use. The technique of intradermal fluorophore injection requires some skill. We consider this fibre-optic instrument a potentially important tool in skin research for non-invasive optical biopsy of primarily the epidermis. Present use is focussed on research applications, where the fluorophore distribution in the skin may illustrate morphological changes in the epidermis.

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

PubMed

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.

Handheld tunable focus confocal microscope utilizing a double-clad fiber coupler for in vivo imaging of oral epithelium

NASA Astrophysics Data System (ADS)

Olsovsky, Cory; Hinsdale, Taylor; Cuenca, Rodrigo; Cheng, Yi-Shing Lisa; Wright, John M.; Rees, Terry D.; Jo, Javier A.; Maitland, Kristen C.

2017-05-01

A reflectance confocal endomicroscope with double-clad fiber coupler and electrically tunable focus lens is applied to imaging of the oral mucosa. The instrument is designed to be lightweight and robust for clinical use. The tunable lens allows axial scanning through >250 μm in the epithelium when the probe tip is placed in contact with tissue. Images are acquired at 6.6 frames per second with a field of view diameter up to 850 μm. In vivo imaging of a wide range of normal sites in the oral cavity demonstrates the accessibility of the handheld probe. In vivo imaging of clinical lesions diagnosed as inflammation and dysplasia illustrates the ability of reflectance confocal endomicroscopy to image cellular changes associated with pathology.

Mapping owl's eye cells of patients with cytomegalovirus corneal endotheliitis using in vivo laser confocal microscopy.

PubMed

Yokogawa, Hideaki; Kobayashi, Akira; Sugiyama, Kazuhisa

2013-01-01

To produce a two-dimensional reconstruction map of owl's eye cells using in vivo laser confocal microscopy in patients with cytomegalovirus (CMV) corneal endotheliitis, and to demonstrate any association between owl's eye cells and coin-shaped lesions observed with slit-lamp biomicroscopy. Two patients (75- and 77-year-old men) with polymerase chain reaction-proven CMV corneal endotheliitis were evaluated in this study. Slit-lamp biomicroscopy and in vivo laser confocal microscopy were performed. Images of owl's eye cells in the endothelial cell layer were arranged and mapped into subconfluent montages. Montage images of owl's eye cells were then superimposed on a slit-lamp photo of the corresponding coin-shaped lesion. Degree of concordance between the confocal microscopic images and slit-lamp photos was evaluated. In both eyes, a two-dimensional reconstruction map of the owl's eye cells was created by computer software using acquired confocal images; the maps showed circular patterns. Superimposing montage images of owl's eye cells onto the photos of a coin-shaped lesion showed good concordance in the two eyes. This study suggests that there is an association between owl's eye cells observed by confocal microscopy and coin-shaped lesions observed by slit-lamp biomicroscopy in patients with CMV corneal endotheliitis. The use of in vivo laser confocal microscopy may provide clues as to the underlying causes of CMV corneal endotheliitis.

Confocal Endomicroscopy: Instrumentation and Medical Applications

PubMed Central

Jabbour, Joey M.; Saldua, Meagan A.; Bixler, Joel N.; Maitland, Kristen C.

2013-01-01

Advances in fiber optic technology and miniaturized optics and mechanics have propelled confocal endomicroscopy into the clinical realm. This high resolution, non-invasive imaging technology provides the ability to microscopically evaluate cellular and sub-cellular features in tissue in vivo by optical sectioning. Because many cancers originate in epithelial tissues accessible by endoscopes, confocal endomicroscopy has been explored to detect regions of possible neoplasia at an earlier stage by imaging morphological features in vivo that are significant in histopathologic evaluation. This technique allows real-time assessment of tissue which may improve diagnostic yield by guiding biopsy. Research and development continues to reduce the overall size of the imaging probe, increase the image acquisition speed, and improve resolution and field of view of confocal endomicroscopes. Technical advances will continue to enable application to less accessible organs and more complex systems in the body. Lateral and axial resolutions down to 0.5 μm and 3 μm, respectively, field of view as large as 800×450 μm, and objective lens and total probe outer diameters down to 350 μm and 1.25 mm, respectively, have been achieved. We provide a review of the historical developments of confocal imaging in vivo, the evolution of endomicroscope instrumentation, and the medical applications of confocal endomicroscopy. PMID:21994069

Portable oral cancer detection using a miniature confocal imaging probe with a large field of view

NASA Astrophysics Data System (ADS)

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.

Photon event distribution sampling: an image formation technique for scanning microscopes that permits tracking of sub-diffraction particles with high spatial and temporal resolutions.

PubMed

Larkin, J D; Publicover, N G; Sutko, J L

2011-01-01

In photon event distribution sampling, an image formation technique for scanning microscopes, the maximum likelihood position of origin of each detected photon is acquired as a data set rather than binning photons in pixels. Subsequently, an intensity-related probability density function describing the uncertainty associated with the photon position measurement is applied to each position and individual photon intensity distributions are summed to form an image. Compared to pixel-based images, photon event distribution sampling images exhibit increased signal-to-noise and comparable spatial resolution. Photon event distribution sampling is superior to pixel-based image formation in recognizing the presence of structured (non-random) photon distributions at low photon counts and permits use of non-raster scanning patterns. A photon event distribution sampling based method for localizing single particles derived from a multi-variate normal distribution is more precise than statistical (Gaussian) fitting to pixel-based images. Using the multi-variate normal distribution method, non-raster scanning and a typical confocal microscope, localizations with 8 nm precision were achieved at 10 ms sampling rates with acquisition of ~200 photons per frame. Single nanometre precision was obtained with a greater number of photons per frame. In summary, photon event distribution sampling provides an efficient way to form images when low numbers of photons are involved and permits particle tracking with confocal point-scanning microscopes with nanometre precision deep within specimens. © 2010 The Authors Journal of Microscopy © 2010 The Royal Microscopical Society.

Confocal Raman microscopy and multivariate statistical analysis for determination of different penetration abilities of caffeine and propylene glycol applied simultaneously in a mixture on porcine skin ex vivo.

PubMed

Mujica Ascencio, Saul; Choe, ChunSik; Meinke, Martina C; Müller, Rainer H; Maksimov, George V; Wigger-Alberti, Walter; Lademann, Juergen; Darvin, Maxim E

2016-07-01

Propylene glycol is one of the known substances added in cosmetic formulations as a penetration enhancer. Recently, nanocrystals have been employed also to increase the skin penetration of active components. Caffeine is a component with many applications and its penetration into the epidermis is controversially discussed in the literature. In the present study, the penetration ability of two components - caffeine nanocrystals and propylene glycol, applied topically on porcine ear skin in the form of a gel, was investigated ex vivo using two confocal Raman microscopes operated at different excitation wavelengths (785nm and 633nm). Several depth profiles were acquired in the fingerprint region and different spectral ranges, i.e., 526-600cm(-1) and 810-880cm(-1) were chosen for independent analysis of caffeine and propylene glycol penetration into the skin, respectively. Multivariate statistical methods such as principal component analysis (PCA) and linear discriminant analysis (LDA) combined with Student's t-test were employed to calculate the maximum penetration depths of each substance (caffeine and propylene glycol). The results show that propylene glycol penetrates significantly deeper than caffeine (20.7-22.0μm versus 12.3-13.0μm) without any penetration enhancement effect on caffeine. The results confirm that different substances, even if applied onto the skin as a mixture, can penetrate differently. The penetration depths of caffeine and propylene glycol obtained using two different confocal Raman microscopes are comparable showing that both types of microscopes are well suited for such investigations and that multivariate statistical PCA-LDA methods combined with Student's t-test are very useful for analyzing the penetration of different substances into the skin. Copyright © 2016 Elsevier B.V. All rights reserved.

The Light Microscopy Module: An On-Orbit Multi-User Microscope Facility

NASA Technical Reports Server (NTRS)

Motil, Susan M.; Snead, John H.

2002-01-01

The Light Microscopy Module (LMM) is planned as a remotely controllable on-orbit microscope subrack facility, allowing flexible scheduling and operation of fluids and biology experiments within the Fluids and Combustion Facility (FCF) Fluids Integrated Rack (FIR) on the International Space Station (ISS). The LMM will be the first integrated payload with the FIR to conduct four fluid physics experiments. A description of the LMM diagnostic capabilities, including video microscopy, interferometry, laser tweezers, confocal, and spectrophotometry, will be provided.

Shear-driven dynamic clusters in a colloidal glass

NASA Astrophysics Data System (ADS)

Eisenmann, Christoph; Kim, Chanjoong; Mattsson, Johan; Weitz, David

2007-03-01

We investigate the effect of shear applied to a colloidal glass on a microscopic level using a shear device that can be mounted on top of a confocal microscope. We find that the glass yields at a critical strain of about 10%, independently of the shear rate. Surprisingly, the yielding is accompanied by an increase of cooperative particle movements and a formation of dynamic clusters which is in contrast to the normal glass transition where one typically finds heterogeneity increasing whilst moving towards the glass transition.

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

NASA Astrophysics Data System (ADS)

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

2012-03-01

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

Programmable Illumination and High-Speed, Multi-Wavelength, Confocal Microscopy Using a Digital Micromirror

PubMed Central

Martial, Franck P.; Hartell, Nicholas A.

2012-01-01

Confocal microscopy is routinely used for high-resolution fluorescence imaging of biological specimens. Most standard confocal systems scan a laser across a specimen and collect emitted light passing through a single pinhole to produce an optical section of the sample. Sequential scanning on a point-by-point basis limits the speed of image acquisition and even the fastest commercial instruments struggle to resolve the temporal dynamics of rapid cellular events such as calcium signals. Various approaches have been introduced that increase the speed of confocal imaging. Nipkov disk microscopes, for example, use arrays of pinholes or slits on a spinning disk to achieve parallel scanning which significantly increases the speed of acquisition. Here we report the development of a microscope module that utilises a digital micromirror device as a spatial light modulator to provide programmable confocal optical sectioning with a single camera, at high spatial and axial resolution at speeds limited by the frame rate of the camera. The digital micromirror acts as a solid state Nipkov disk but with the added ability to change the pinholes size and separation and to control the light intensity on a mirror-by-mirror basis. The use of an arrangement of concave and convex mirrors in the emission pathway instead of lenses overcomes the astigmatism inherent with DMD devices, increases light collection efficiency and ensures image collection is achromatic so that images are perfectly aligned at different wavelengths. Combined with non-laser light sources, this allows low cost, high-speed, multi-wavelength image acquisition without the need for complex wavelength-dependent image alignment. The micromirror can also be used for programmable illumination allowing spatially defined photoactivation of fluorescent proteins. We demonstrate the use of this system for high-speed calcium imaging using both a single wavelength calcium indicator and a genetically encoded, ratiometric, calcium sensor. PMID:22937130

Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror.

PubMed

Martial, Franck P; Hartell, Nicholas A

2012-01-01

Confocal microscopy is routinely used for high-resolution fluorescence imaging of biological specimens. Most standard confocal systems scan a laser across a specimen and collect emitted light passing through a single pinhole to produce an optical section of the sample. Sequential scanning on a point-by-point basis limits the speed of image acquisition and even the fastest commercial instruments struggle to resolve the temporal dynamics of rapid cellular events such as calcium signals. Various approaches have been introduced that increase the speed of confocal imaging. Nipkov disk microscopes, for example, use arrays of pinholes or slits on a spinning disk to achieve parallel scanning which significantly increases the speed of acquisition. Here we report the development of a microscope module that utilises a digital micromirror device as a spatial light modulator to provide programmable confocal optical sectioning with a single camera, at high spatial and axial resolution at speeds limited by the frame rate of the camera. The digital micromirror acts as a solid state Nipkov disk but with the added ability to change the pinholes size and separation and to control the light intensity on a mirror-by-mirror basis. The use of an arrangement of concave and convex mirrors in the emission pathway instead of lenses overcomes the astigmatism inherent with DMD devices, increases light collection efficiency and ensures image collection is achromatic so that images are perfectly aligned at different wavelengths. Combined with non-laser light sources, this allows low cost, high-speed, multi-wavelength image acquisition without the need for complex wavelength-dependent image alignment. The micromirror can also be used for programmable illumination allowing spatially defined photoactivation of fluorescent proteins. We demonstrate the use of this system for high-speed calcium imaging using both a single wavelength calcium indicator and a genetically encoded, ratiometric, calcium sensor.

The Relationship between Neurite Density Measured with Confocal Microscopy in a Cleared Mouse Brain and Metrics Obtained from Diffusion Tensor and Diffusion Kurtosis Imaging

PubMed Central

Irie, Ryusuke; Kamagata, Koji; Kerever, Aurelien; Ueda, Ryo; Yokosawa, Suguru; Otake, Yosuke; Ochi, Hisaaki; Yoshizawa, Hidekazu; Hayashi, Ayato; Tagawa, Kazuhiko; Okazawa, Hitoshi; Takahashi, Kohske; Sato, Kanako; Hori, Masaaki; Arikawa-Hirasawa, Eri; Aoki, Shigeki

2018-01-01

Purpose: Diffusional kurtosis imaging (DKI) enables sensitive measurement of tissue microstructure by quantifying the non-Gaussian diffusion of water. Although DKI is widely applied in many situations, histological correlation with DKI analysis is lacking. The purpose of this study was to determine the relationship between DKI metrics and neurite density measured using confocal microscopy of a cleared mouse brain. Methods: One thy-1 yellow fluorescent protein 16 mouse was deeply anesthetized and perfusion fixation was performed. The brain was carefully dissected out and whole-brain MRI was performed using a 7T animal MRI system. DKI and diffusion tensor imaging (DTI) data were obtained. After the MRI scan, brain sections were prepared and then cleared using aminoalcohols (CUBIC). Confocal microscopy was performed using a two-photon confocal microscope with a laser. Forty-eight ROIs were set on the caudate putamen, seven ROIs on the anterior commissure, and seven ROIs on the ventral hippocampal commissure on the confocal microscopic image and a corresponding MR image. In each ROI, histological neurite density and the metrics of DKI and DTI were calculated. The correlations between diffusion metrics and neurite density were analyzed using Pearson correlation coefficient analysis. Results: Mean kurtosis (MK) (P = 5.2 × 10−9, r = 0.73) and radial kurtosis (P = 2.3 × 10−9, r = 0.74) strongly correlated with neurite density in the caudate putamen. The correlation between fractional anisotropy (FA) and neurite density was moderate (P = 0.0030, r = 0.42). In the anterior commissure and the ventral hippocampal commissure, neurite density and FA are very strongly correlated (P = 1.3 × 10−5, r = 0.90). MK in these areas were very high value and showed no significant correlation (P = 0.48). Conclusion: DKI accurately reflected neurite density in the area with crossing fibers, potentially allowing evaluation of complex microstructures. PMID:29213008

The Relationship between Neurite Density Measured with Confocal Microscopy in a Cleared Mouse Brain and Metrics Obtained from Diffusion Tensor and Diffusion Kurtosis Imaging.

PubMed

Irie, Ryusuke; Kamagata, Koji; Kerever, Aurelien; Ueda, Ryo; Yokosawa, Suguru; Otake, Yosuke; Ochi, Hisaaki; Yoshizawa, Hidekazu; Hayashi, Ayato; Tagawa, Kazuhiko; Okazawa, Hitoshi; Takahashi, Kohske; Sato, Kanako; Hori, Masaaki; Arikawa-Hirasawa, Eri; Aoki, Shigeki

2018-04-10

Diffusional kurtosis imaging (DKI) enables sensitive measurement of tissue microstructure by quantifying the non-Gaussian diffusion of water. Although DKI is widely applied in many situations, histological correlation with DKI analysis is lacking. The purpose of this study was to determine the relationship between DKI metrics and neurite density measured using confocal microscopy of a cleared mouse brain. One thy-1 yellow fluorescent protein 16 mouse was deeply anesthetized and perfusion fixation was performed. The brain was carefully dissected out and whole-brain MRI was performed using a 7T animal MRI system. DKI and diffusion tensor imaging (DTI) data were obtained. After the MRI scan, brain sections were prepared and then cleared using aminoalcohols (CUBIC). Confocal microscopy was performed using a two-photon confocal microscope with a laser. Forty-eight ROIs were set on the caudate putamen, seven ROIs on the anterior commissure, and seven ROIs on the ventral hippocampal commissure on the confocal microscopic image and a corresponding MR image. In each ROI, histological neurite density and the metrics of DKI and DTI were calculated. The correlations between diffusion metrics and neurite density were analyzed using Pearson correlation coefficient analysis. Mean kurtosis (MK) (P = 5.2 × 10 -9 , r = 0.73) and radial kurtosis (P = 2.3 × 10 -9 , r = 0.74) strongly correlated with neurite density in the caudate putamen. The correlation between fractional anisotropy (FA) and neurite density was moderate (P = 0.0030, r = 0.42). In the anterior commissure and the ventral hippocampal commissure, neurite density and FA are very strongly correlated (P = 1.3 × 10 -5 , r = 0.90). MK in these areas were very high value and showed no significant correlation (P = 0.48). DKI accurately reflected neurite density in the area with crossing fibers, potentially allowing evaluation of complex microstructures.

Clinical evaluation of a confocal microendoscope system for imaging the ovary

NASA Astrophysics Data System (ADS)

Tanbakuchi, Anthony A.; Rouse, Andrew R.; Hatch, Kenneth D.; Sampliner, Richard E.; Udovich, Josh A.; Gmitro, Arthur F.

2008-02-01

We have developed a mobile confocal microendoscope system that provides live cellular imaging during surgery to aid in diagnosing microscopic abnormalities including cancer. We present initial clinical trial results using the device to image ovaries in-vivo using fluorescein and ex-vivo results using acridine orange. The imaging catheter has improved depth control and localized dye delivery mechanisms than previously presented. A manual control now provides a simple way for the surgeon to adjust and optimize imaging depth during the procedure while a tiny piezo valve in the imaging catheter controls the dye delivery.

In vitro confocal imaging of the rabbit cornea.

PubMed

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.

Handheld confocal Raman microspectrometer for in-vivo skin cancer measurement

NASA Astrophysics Data System (ADS)

Lieber, Chad A.; Ellis, Darrel L.; Billheimer, D. D.; Mahadevan-Jansen, Anita

2004-07-01

Several studies have demonstrated Raman spectroscopy to be capable of tissue diagnosis with accuracy rivaling that of histopathologic analysis. This technique obtains biochemical-specific information noninvasively, and can eliminate the pain, time, and cost associated with biopsy and pathological analysis. Furthermore, when used in a confocal arrangement, Raman spectra can be obtained from localized regions of the tissue. Skin cancers are an ideal candidate for this emerging technology, due to their obvious accessibility and presentation at specific depths. However, most commercially available confocal Raman microspectrometers are large, rigid systems ill-suited for clinical application. We developed a bench-top confocal Raman microspectrometer using a portable external-cavity diode laser excitation source. This system was used to study several skin lesions in vitro. Results show the depth-resolved Raman spectra can diagnose in vitro skin lesions with 96% sensitivity, 88% specificity, and 86% pathological classification accuracy. Based on the success of this study, a portable Raman system with a handheld confocal microscope was developed for clinical application. Preliminary in vivo data show several distinct spectral differences between skin pathologies. Diagnostic algorithms are planned for this continuing study to assess the capability of Raman spectroscopy for clinical skin cancer diagnosis.

Remineralization Potential of Three Different Dentifrices using Raman Spectroscopy and Confocal Laser Scanning Microscope.

PubMed

Job, Tisson V; Narayana, Girish T; Venkappa, Kishan K; Nathan, K Binu; Ahsan, Shameem; Harikaran, Jayakkodi

2018-04-01

Aim: The aim of this study was to compare the remineralization potential of three different dentifrices using Raman spectroscopy and confocal laser scanning microscopy (CLSM). Materials and methods: Totally, 30 extracted intact impacted third molar teeth were selected and the crown of each tooth in a group was separated from the root and longitudinally sectioned into four parts with each section under a subgroup, of which one section was an untreated section, the second and the third sections were demineralized in a demineralizing solution, and the third section was remineralized after demineralization. The teeth in the three groups were demineralized for 4 days and then treated with 0.21% sodium fluoride dentifrice with trical-cium phosphate, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), and NovaMin for 14 days, following which the teeth surfaces were studied using Raman spec-troscopy and CLSM to assess the remineralization potential of the three dentifrices. The data were recorded and analyzed statistically. Results: Raman spectroscopic analysis revealed better remin-eralization with CPP-ACP, which was statistically significant from the groups treated with the NovaMin dentifrice and the fluoride-containing dentifrice.Confocal laser scanning microscopic examination also revealed significant differences between the three groups with the NovaMin-containing dentifrice demonstrating a greater remineralization of the surface when compared with the CPP-ACP dentifrice. The teeth samples treated with fluoride-containing dentifrice demonstrated the least reminer-alization among the three groups. Conclusion: It can be concluded that the demineralized samples of teeth treated with CPP-ACP showed the highest concentration of phosphate ions when analyzed using Raman spectroscopy, and the microscopic examination using confocal laser revealed a better surface remineralization of the demin-eralized samples when treated with the NovaMin technology. Clinical significance: There is a great need to find ways to enhance the remineralization process and transfer such knowledge into clinical therapy to alter caries balance for the better, especially in individuals with a high cariogenic bacterial challenge. Keywords: Casein phosphopeptide-amorphous calcium phosphate, Fluoride, NovaMin, Remineralization, Tricalcium phosphate.

Diffraction-limited IR Microspectroscopy with IRENI

Treesearch

J. Sedlmair; B. Illman; M. Unger; C. Hirschmugl

2012-01-01

In a unique way, IRENI (Infrared environmental Imaging), operated at the Synchrotron Radiation Center in Madison, combines IR spectroscopy and IR imaging, revealing the chemical morphology of a sample. Most storage ring based IR confocal microscopes have to overcome a trade-off between spatial resolution versus...

Digital Hilbert transformation for separation measurement of thicknesses and refractive indices of layered objects by use of a wavelength-scanning heterodyne interference confocal microscope.

PubMed

Watanabe, Yuuki; Yamaguchi, Ichirou

2002-08-01

A wavelength-scanning heterodyne interference confocal microscope quickly accomplishes the simultaneous measurement of the thickness and the refractive index of a sample by detection of the amplitude and the phase of the interference signal during a sample scan. However, the measurement range of the optical path difference (OPD) that is obtained from the phase changes is limited by the time response of the phase-locked loop circuit in the FM demodulator. To overcome this limitation and to improve the accuracy of the separation measurement, we propose an OPD detection using digital signal processing with a Hilbert transform. The measurement range is extended approximately five times, and the resolution of the OPD is improved to 5.5 from 9 microm without the electrical noise of the FM demodulator circuit. By applying this method for simultaneous measurement of thickness and the refractive index, we can measure samples 20-30-microm thick with refractive indices between 1 and 1.5.

Digital Hilbert transformation for separation measurement of thicknesses and refractive indices of layered objects by use of a wavelength-scanning heterodyne interference confocal microscope

NASA Astrophysics Data System (ADS)

Watanabe, Yuuki; Yamaguchi, Ichirou

2002-08-01

A wavelength-scanning heterodyne interference confocal microscope quickly accomplishes the simultaneous measurement of the thickness and the refractive index of a sample by detection of the amplitude and the phase of the interference signal during a sample scan. However, the measurement range of the optical path difference (OPD) that is obtained from the phase changes is limited by the time response of the phase-locked loop circuit in the FM demodulator. To overcome this limitation and to improve the accuracy of the separation measurement, we propose an OPD detection using digital signal processing with a Hilbert transform. The measurement range is extended approximately five times, and the resolution of the OPD is improved to 5.5 from 9 mum without the electrical noise of the FM demodulator circuit. By applying this method for simultaneous measurement of thickness and the refractive index, we can measure samples 20-30-mum thick with refractive indices between 1 and 1.5.

Spectral ophthalmoscopy based on supercontinuum

NASA Astrophysics Data System (ADS)

Cheng, Yueh-Hung; Yu, Jiun-Yann; Wu, Han-Hsuan; Huang, Bo-Jyun; Chu, Shi-Wei

2010-02-01

Confocal scanning laser ophthalmoscope (CSLO) has been established to be an important diagnostic tool for retinopathies like age-related macular degeneration, glaucoma and diabetes. Compared to a confocal laser scanning microscope, CSLO is also capable of providing optical sectioning on retina with the aid of a pinhole, but the microscope objective is replaced by the optics of eye. Since optical spectrum is the fingerprint of local chemical composition, it is attractive to incorporate spectral acquisition into CSLO. However, due to the limitation of laser bandwidth and chromatic/geometric aberration, the scanning systems in current CSLO are not compatible with spectral imaging. Here we demonstrate a spectral CSLO by combining a diffraction-limited broadband scanning system and a supercontinuum laser source. Both optical sectioning capability and sub-cellular resolution are demonstrated on zebrafish's retina. To our knowledge, it is also the first time that CSLO is applied onto the study of fish vision. The versatile spectral CSLO system will be useful to retinopathy diagnosis and neuroscience research.

Light-sheet microscopy by confocal line scanning of dual-Bessel beams

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Pengfei; Phipps, Mary Elizabeth; Goodwin, Peter Marvin

Here, we have developed a light-sheet microscope that uses confocal scanning of dual-Bessel beams for illumination. A digital micromirror device (DMD) is placed in the intermediate image plane of the objective used to collect fluorescence and is programmed with two lines of pixels in the “on” state such that the DMD functions as a spatial filter to reject the out-of-focus background generated by the side-lobes of the Bessel beams. The optical sectioning and out-of-focus background rejection capabilities of this microscope were demonstrated by imaging of fluorescently stained actin in human A431 cells. The dual-Bessel beam system enables twice as manymore » photons to be detected per imaging scan, which is useful for low light applications (e.g., single-molecule localization) or imaging at high speed with a superior signal to noise. While demonstrated for two Bessel beams, this approach is scalable to a larger number of beams.« less

Light-sheet microscopy by confocal line scanning of dual-Bessel beams

DOE PAGES

Zhang, Pengfei; Phipps, Mary Elizabeth; Goodwin, Peter Marvin; ...

2016-10-25

Here, we have developed a light-sheet microscope that uses confocal scanning of dual-Bessel beams for illumination. A digital micromirror device (DMD) is placed in the intermediate image plane of the objective used to collect fluorescence and is programmed with two lines of pixels in the “on” state such that the DMD functions as a spatial filter to reject the out-of-focus background generated by the side-lobes of the Bessel beams. The optical sectioning and out-of-focus background rejection capabilities of this microscope were demonstrated by imaging of fluorescently stained actin in human A431 cells. The dual-Bessel beam system enables twice as manymore » photons to be detected per imaging scan, which is useful for low light applications (e.g., single-molecule localization) or imaging at high speed with a superior signal to noise. While demonstrated for two Bessel beams, this approach is scalable to a larger number of beams.« less

MEMS-based handheld confocal microscope for in-vivo skin imaging

PubMed Central

Arrasmith, Christopher L.; Dickensheets, David L.; Mahadevan-Jansen, Anita

2010-01-01

This paper describes a handheld laser scanning confocal microscope for skin microscopy. Beam scanning is accomplished with an electromagnetic MEMS bi-axial micromirror developed for pico projector applications, providing 800x600 (SVGA) resolution at 56 frames per second. The design uses commercial objective lenses with an optional hemisphere front lens, operating with a range of numerical aperture from NA=0.35 to NA=1.1 and corresponding diagonal field of view ranging from 653 μm to 216 μm. Using NA=1.1 and a laser wavelength of 830 nm we measured the axial response to be 1.14 μm full width at half maximum, with a corresponding 10%-90% lateral edge response of 0.39 μm. Image examples showing both epidermal and dermal features including capillary blood flow are provided. These images represent the highest resolution and frame rate yet achieved for tissue imaging with a MEMS bi-axial scan mirror. PMID:20389391

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cabrera, Humberto, E-mail: hcabrera@ictp.it; Centro Multidisciplinartio de Ciencias, Instituto Venezolano de Investigaciones Científicas; Korte, Dorota

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 techniquemore » 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.« less

Demonstration of bacterial biofilms in culture-negative silicone stent and jones tube.

PubMed

Parsa, Kami; Schaudinn, Christoph; Gorur, Amita; Sedghizadeh, Parish P; Johnson, Thomas; Tse, David T; Costerton, John W

2010-01-01

To demonstrate the presence of bacterial biofilms on a dacryocystorhinostomy silicone stent and a Jones tube. One dacryocystorhinostomy silicone stent and one Jones tube were removed from 2 patients who presented with an infection of their respective nasolacrimal system. Cultures were obtained, and the implants were processed for scanning electron microscopy and confocal laser scanning microscopy, advanced microscopic methods that are applicable for detection of uncultivable biofilm organisms. Routine bacterial cultures revealed no growth, but bacterial biofilms on outer and inner surfaces of both implants were confirmed by advanced microscopic techniques. To the authors' knowledge, this is the first article that documents the presence of biofilms on a Crawford stent or a Jones tube on patients who presented with infections involving the nasolacrimal system. Although initial cultures revealed absence of any bacterial growth, confocal laser scanning microscopy and scanning electron microscopy documented bacterial colonization. Clinicians should consider the role of biofilms and the limitation of our standard culturing techniques while treating patients with device- or implant-related infections.

[Preparation and characterization of nanoemulsion].

PubMed

Sun, Yu-Jing; Wu, Dao-Cheng; Cao, Yun-Xin; Sui, Yan-Fang

2005-01-01

To prepare nanoemulsion-encapsulated BSA-FITC (NEBSA-FITC), study its characteristics, and measure its uptake by dendritic cells (DCs) and peritoneal macrophages. NEBSA-FITC was prepared by a method of interfacial polymerization.The encapsulation rate, drug-carrying capacity and stability of the nanoemulsion were determined by Sephadex-G100 chromatography. The shape and size of NEBSA-FITC were observed under electron microscope. The uptake of NEBSA-FITC by DCs and macrophage cells was detected by FACS and laser confocal microscopy. The mean size of NEBSA-FITC was (25+/-10) nm. The encapsulation rate was 91%, the drug-carrying capacity was 0.091 g/L and NEBSA-FITC had a good stability. The FACS analysis showed that DCs and macrophage cells could take in more NEBSA-FITC than free BSA. The observation under laser confocal microscope found that NEBSA-FITC was located in the cytoplasm of DCs. Nanoemulsion can be efficiently taken by DCs and macrophage cells, and therefore may be promising efficient carrier of APCs-targeted antitumor vaccine.

Assessment of nerve ultrastructure by fibre-optic confocal microscopy.

PubMed

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.

Handheld tunable focus confocal microscope utilizing a double-clad fiber coupler for in vivo imaging of oral epithelium

PubMed Central

Olsovsky, Cory; Hinsdale, Taylor; Cuenca, Rodrigo; Cheng, Yi-Shing Lisa; Wright, John M.; Rees, Terry D.; Jo, Javier A.; Maitland, Kristen C.

2017-01-01

Abstract. A reflectance confocal endomicroscope with double-clad fiber coupler and electrically tunable focus lens is applied to imaging of the oral mucosa. The instrument is designed to be lightweight and robust for clinical use. The tunable lens allows axial scanning through >250  μm in the epithelium when the probe tip is placed in contact with tissue. Images are acquired at 6.6 frames per second with a field of view diameter up to 850  μm. In vivo imaging of a wide range of normal sites in the oral cavity demonstrates the accessibility of the handheld probe. In vivo imaging of clinical lesions diagnosed as inflammation and dysplasia illustrates the ability of reflectance confocal endomicroscopy to image cellular changes associated with pathology. PMID:28541447

Confocal microscopy with strip mosaicing for rapid imaging over large areas of excised tissue

NASA Astrophysics Data System (ADS)

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

2013-06-01

Confocal mosaicing microscopy is a developing technology platform for imaging tumor margins directly in freshly excised tissue, without the processing required for conventional pathology. Previously, mosaicing on 12-×-12 mm2 of excised skin tissue from Mohs surgery and detection of basal cell carcinoma margins was demonstrated in 9 min. Last year, we reported the feasibility of a faster approach called "strip mosaicing," which was demonstrated on a 10-×-10 mm2 of tissue in 3 min. Here we describe further advances in instrumentation, software, and speed. A mechanism was also developed to flatten tissue in order to enable consistent and repeatable acquisition of images over large areas. We demonstrate mosaicing on 10-×-10 mm2 of skin tissue with 1-μm lateral resolution in 90 s. A 2.5-×-3.5 cm2 piece of breast tissue was scanned with 0.8-μm lateral resolution in 13 min. Rapid mosaicing of confocal images on large areas of fresh tissue potentially offers a means to perform pathology at the bedside. Imaging of tumor margins with strip mosaicing confocal microscopy may serve as an adjunct to conventional (frozen or fixed) pathology for guiding surgery.

Multidepth imaging by chromatic dispersion confocal microscopy

NASA Astrophysics Data System (ADS)

Olsovsky, Cory A.; Shelton, Ryan L.; Saldua, Meagan A.; Carrasco-Zevallos, Oscar; Applegate, Brian E.; Maitland, Kristen C.

2012-03-01

Confocal microscopy has shown potential as an imaging technique to detect precancer. Imaging cellular features throughout the depth of epithelial tissue may provide useful information for diagnosis. However, the current in vivo axial scanning techniques for confocal microscopy are cumbersome, time-consuming, and restrictive when attempting to reconstruct volumetric images acquired in breathing patients. Chromatic dispersion confocal microscopy (CDCM) exploits severe longitudinal chromatic aberration in the system to axially disperse light from a broadband source and, ultimately, spectrally encode high resolution images along the depth of the object. Hyperchromat lenses are designed to have severe and linear longitudinal chromatic aberration, but have not yet been used in confocal microscopy. We use a hyperchromat lens in a stage scanning confocal microscope to demonstrate the capability to simultaneously capture information at multiple depths without mechanical scanning. A photonic crystal fiber pumped with a 830nm wavelength Ti:Sapphire laser was used as a supercontinuum source, and a spectrometer was used as the detector. The chromatic aberration and magnification in the system give a focal shift of 140μm after the objective lens and an axial resolution of 5.2-7.6μm over the wavelength range from 585nm to 830nm. A 400x400x140μm3 volume of pig cheek epithelium was imaged in a single X-Y scan. Nuclei can be seen at several depths within the epithelium. The capability of this technique to achieve simultaneous high resolution confocal imaging at multiple depths may reduce imaging time and motion artifacts and enable volumetric reconstruction of in vivo confocal images of the epithelium.

Confocal mosaicing microscopy of basal-cell carcinomas ex vivo: progress in digital staining to simulate histology-like appearance

NASA Astrophysics Data System (ADS)

Bini, Jason; Spain, James; Nehal, Kishwer; Hazelwood, Vikki; DiMarzio, Charles; Rajadhyaksha, Milind

2011-03-01

Confocal mosaicing microscopy enables rapid imaging of large areas of fresh tissue, without the processing that is necessary for conventional histology. Using acridine orange (1 milliMolar, 20 seconds) to stain nuclei, basal cell carcinomas were detected in fluorescence confocal mosaics of Mohs surgical excisions with sensitivity of 96.6% and specificity of 89.2%. A possible barrier toward clinical acceptance is that confocal mosaics are based on a single mode of contrast and appear in grayscale, whereas histology is based on two (hematoxylin for nuclei, eosin for cellular cytoplasm and dermis) and appears purple-and-pink. Toward addressing this barrier, we report progress in developing a multispectral analytical model for digital staining: fluorescence confocal mosaics, which show only nuclei, are digitally stained purple and overlaid on reflectance confocal mosaics, which show only cellular cytoplasm and dermis, and digitally stained pink, to mimic the appearance of histology. Comparison of digitally stained confocal mosaics by our Mohs surgeon to the corresponding Mohs histology shows good correlation for normal and tumor detail. Digitally stained confocal mosaicing microscopy may allow direct examination of freshly excised tissue and serve as an adjunct for rapid pathology at-the-bedside.

Growth and Brilliant Photo-Emission of Crystalline Hexagonal Column of Alq3 Microwires

PubMed Central

Kim, Seokho; Kim, Do Hyoung; Choi, Jinho; Lee, Hojin; Kim, Sun-Young; Park, Jung Woon; Park, Dong Hyuk

2018-01-01

We report the growth and nanoscale luminescence characteristics of 8-hydroxyquinolinato aluminum (Alq3) with a crystalline hexagonal column morphology. Pristine Alq3 nanoparticles (NPs) were prepared using a conventional reprecipitation method. Crystal hexagonal columns of Alq3 were grown by using a surfactant-assisted self-assembly technique as an adjunct to the aforementioned reprecipitation method. The formation and structural properties of the crystalline and non-crystalline Alq3 NPs were analyzed with scanning electron microscopy and X-ray diffraction. The nanoscale photoluminescence (PL) characteristics and the luminescence color of the Alq3 single NPs and their crystal microwires (MWs) were evaluated from color charge-coupled device images acquired using a high-resolution laser confocal microscope. In comparison with the Alq3 NPs, the crystalline MWs exhibited a very bright and sharp emission. This enhanced and sharp emission from the crystalline Alq3 single MWs originated from effective π-π stacking of the Alq3 molecules due to strong interactions in the crystalline structure. PMID:29565306

Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations

PubMed Central

Gustafsson, Nils; Culley, Siân; Ashdown, George; Owen, Dylan M.; Pereira, Pedro Matos; Henriques, Ricardo

2016-01-01

Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challenging data sets for super-resolution, such as those obtained in low-illumination live-cell imaging with GFP, we show that SRRF is generally capable of achieving resolutions better than 150 nm. Meanwhile, for data sets similar to those obtained in PALM or STORM imaging, SRRF achieves resolutions approaching those of standard single-molecule localization analysis. The broad applicability of SRRF and its performance at low signal-to-noise ratios allows super-resolution using modern widefield, confocal or TIRF microscopes with illumination orders of magnitude lower than methods such as PALM, STORM or STED. We demonstrate this by super-resolution live-cell imaging over timescales ranging from minutes to hours. PMID:27514992

In vivo correlation mapping microscopy

NASA Astrophysics Data System (ADS)

McGrath, James; Alexandrov, Sergey; Owens, Peter; Subhash, Hrebesh; Leahy, Martin

2016-04-01

To facilitate regular assessment of the microcirculation in vivo, noninvasive imaging techniques such as nailfold capillaroscopy are required in clinics. Recently, a correlation mapping technique has been applied to optical coherence tomography (OCT), which extends the capabilities of OCT to microcirculation morphology imaging. This technique, known as correlation mapping optical coherence tomography, has been shown to extract parameters, such as capillary density and vessel diameter, and key clinical markers associated with early changes in microvascular diseases. However, OCT has limited spatial resolution in both the transverse and depth directions. Here, we extend this correlation mapping technique to other microscopy modalities, including confocal microscopy, and take advantage of the higher spatial resolution offered by these modalities. The technique is achieved as a processing step on microscopy images and does not require any modification to the microscope hardware. Results are presented which show that this correlation mapping microscopy technique can extend the capabilities of conventional microscopy to enable mapping of vascular networks in vivo with high spatial resolution in both the transverse and depth directions.

Far-field nanoscopy on a semiconductor quantum dot via a rapid-adiabatic-passage-based switch

NASA Astrophysics Data System (ADS)

Kaldewey, Timo; Kuhlmann, Andreas V.; Valentin, Sascha R.; Ludwig, Arne; Wieck, Andreas D.; Warburton, Richard J.

2018-02-01

The diffraction limit prevents a conventional optical microscope from imaging at the nanoscale. However, nanoscale imaging of molecules is possible by exploiting an intensity-dependent molecular switch1-3. This switch is translated into a microscopy scheme, stimulated emission depletion microscopy4-7. Variants on this scheme exist3,8-13, yet all exploit an incoherent response to the lasers. We present a scheme that relies on a coherent response to a laser. Quantum control of a two-level system proceeds via rapid adiabatic passage, an ideal molecular switch. We implement this scheme on an ensemble of quantum dots. Each quantum dot results in a bright spot in the image with extent down to 30 nm (λ/31). There is no significant loss of intensity with respect to confocal microscopy, resulting in a factor of 10 improvement in emitter position determination. The experiments establish rapid adiabatic passage as a versatile tool in the super-resolution toolbox.

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.

PubMed

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.

[Tripartite motif-containing protein 34 (TRIM34) colocalized with micronuclei chromosome and hampers its movement to equatorial plate during the metaphase stage of mitosis].

PubMed

Sun, Dakang; An, Xinye; Ji, Bing; Cheng, Yanli; Gao, Honglian; Tian, Mingming

2016-06-01

Objective To examine whether tripartite motif-containing protein 34 (TRIM34) is colocalized with micronuclei and investigate the influence on the movement of micronuclei chromosome in mitosis. Methods The eukaryotic expression vector TRIM34-pEGFP-N3 was constructed, identified and then transfected into HEK293T cells. With 4', 6-diamidino-2-phenylindole 2HCI (DAPI) staining, the colocalization between TRIM34 and micronuclei was observed under a fluorescence microscope. Moreover, MitoTracker(R)Deep Red was used to identify the colocalization between the complex of TRIM34-micronulei and mitochondria under a confocal microscope. Finally, the effect of TRIM34 on the movement of micronuclei chromosome in mitosis was examined. Results DNA sequencing confirmed that the vector TRIM34-pEGFP-N3 was constructed successfully. A fluorescence microscope revealed that TRIM34 could be colocalized with micronuclei in HEK293T cells transfected with TRIM34-pEGFP-N3. In the same manner, a confocal microscope distinctly showed that TRIM34 was colocalized with micronuclei similarly in appearance. However, there was no distinguished colocalization relationship between the complex of TRIM34-micronulei and mitochondria. Interestingly, the micronuclei chromosome conjugated with TRIM34 was hardly transferred to equatorial plate during the metaphase stage of mitosis. Conclusion TRIM34 is colocalized with micronuclei chromosome and hampers its movement to equatorial plate in mitosis.

Fluorescence correlation spectroscopy, Raster image correlation spectroscopy and Number & Brightness on a commercial confocal laser scanning microscope with analog detectors (Nikon C1)

PubMed Central

Moens, Pierre D.J.; Gratton, Enrico; Salvemini, Iyrri L.

2010-01-01

Fluorescence correlation spectroscopy (FCS) was developed in 1972 by Magde, Elson and Webb (Magde et al., 1972). Photon counting detectors and avalanche photodiodes have become standards in FCS to the point that there is a widespread belief that these detectors are essential to perform FCS experiments, despite the fact that FCS was developed using analog detectors. Spatial and temporal intensity fluctuation correlations using analog detection on a commercial Olympus Fluoview 300 microscope has been reported by Brown et al. (2008). However, each analog instrument has its own idiosyncrasies that need to be understood before using the instrument for FCS. In this work we explore the capabilities of the Nikon C1, a low cost confocal microscope, to obtain single point FCS, Raster-scan Image Correlation Spectroscopy (RICS) and Number & Brightness data both in solution and incorporated into the membrane of Giant Unilamellar Vesicles (GUVs). We show that it is possible to obtain dynamic information about fluorescent molecules from single point FCS, RICS and Number & Brightness using the Nikon C1. We highlighted the fact that care should be taken in selecting the acquisition parameters in order to avoid possible artifacts due to the detector noise. However, due to relatively large errors in determining the distribution of digital levels for a given microscope setting, the system is probably only adequate for determining relative brightness within the same image. PMID:20734406

Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning.

PubMed

Lee, Dong-Ryoung; Kim, Young-Duk; Gweon, Dae-Gab; Yoo, Hongki

2013-07-29

We propose a new method for high-speed, three-dimensional (3-D) fluorescence imaging, which we refer to as dual-detection confocal fluorescence microscopy (DDCFM). In contrast to conventional beam-scanning confocal fluorescence microscopy, where the focal spot must be scanned either optically or mechanically over a sample volume to reconstruct a 3-D image, DDCFM can obtain the depth of a fluorescent emitter without depth scanning. DDCFM comprises two photodetectors, each with a pinhole of different size, in the confocal detection system. Axial information on fluorescent emitters can be measured by the axial response curve through the ratio of intensity signals. DDCFM can rapidly acquire a 3-D fluorescent image from a single two-dimensional scan with less phototoxicity and photobleaching than confocal fluorescence microscopy because no mechanical depth scans are needed. We demonstrated the feasibility of the proposed method by phantom studies.

Analysis of replication factories in human cells by super-resolution light microscopy

PubMed Central

2009-01-01

Background DNA replication in human cells is performed in discrete sub-nuclear locations known as replication foci or factories. These factories form in the nucleus during S phase and are sites of DNA synthesis and high local concentrations of enzymes required for chromatin replication. Why these structures are required, and how they are organised internally has yet to be identified. It has been difficult to analyse the structure of these factories as they are small in size and thus below the resolution limit of the standard confocal microscope. We have used stimulated emission depletion (STED) microscopy, which improves on the resolving power of the confocal microscope, to probe the structure of these factories at sub-diffraction limit resolution. Results Using immunofluorescent imaging of PCNA (proliferating cell nuclear antigen) and RPA (replication protein A) we show that factories are smaller in size (approximately 150 nm diameter), and greater in number (up to 1400 in an early S- phase nucleus), than is determined by confocal imaging. The replication inhibitor hydroxyurea caused an approximately 40% reduction in number and a 30% increase in diameter of replication factories, changes that were not clearly identified by standard confocal imaging. Conclusions These measurements for replication factory size now approach the dimensions suggested by electron microscopy. This agreement between these two methods, that use very different sample preparation and imaging conditions, suggests that we have arrived at a true measurement for the size of these structures. The number of individual factories present in a single nucleus that we measure using this system is greater than has been previously reported. This analysis therefore suggests that each replication factory contains fewer active replication forks than previously envisaged. PMID:20015367

Confocal reflectance microscopy of basal cell cancers ex vivo: progress toward enhancing contrast and detectability of nuclei relative to dermis

NASA Astrophysics Data System (ADS)

Patel, Yogesh G.; Nehal, Kishwer S.; Halpern, Allan C.; Rajadhyaksha, Milind

2005-04-01

Mohs surgery is a staged procedure for microscopically excising basal cell carcinomas (BCCs) while preserving the surrounding normal skin. Serial excisions are performed with each excision being guided by examination of the frozen histology. Mohs surgery is a meticulous and time-consuming (15-45 minutes per excision) procedure requiring several (2-20) excisions and frozen histology prepared for each excision. Real-time confocal reflectance microscopy may make Mohs surgery more efficient by enabling rapid detection of BCCs directly in fresh, unprocessed excisions, and thereby possibly avoiding frozen histology. As previously reported, we are developing an acetowhitening-and-cross polarized method to detect BCCs with a confocal reflectance microscope. Acetowhitening compacts the chromatin within the nucleus, increasing nuclear backscatter, and brightening the nuclei in the confocal images of the tissue. Our experiments to optimize acetowhitening, using acetic acid concentrations from 1% to 30% and treatment times from 30 seconds to 5 minutes, show that a minimum concentration of 2% with minimum washing time of 2 minutes is required for enhancing nuclear morphology. Increased depolarization is observed within the compacted chromatin relative to the surrounding collagen, and imaging in brightfield or crossed polarization brightens or darkens the cellular cytoplasm and birefringent dermis; thus, we may potentially vary nuclear/cytoplasm and nuclear/dermis contrast. Images are collected, oriented, and tiled to create mosaics and sub-mosaics to view large excisions at variable 2X - 10X magnifications. To create and display mosaics, adequate pixelation relative to resolution must be maintained and precise mechanical fixturing is necessary to control tilt, sag, flattening and stability of the excised tissue specimen.

k-Space Image Correlation Spectroscopy: A Method for Accurate Transport Measurements Independent of Fluorophore Photophysics

PubMed Central

Kolin, David L.; Ronis, David; Wiseman, Paul W.

2006-01-01

We present the theory and application of reciprocal space image correlation spectroscopy (kICS). This technique measures the number density, diffusion coefficient, and velocity of fluorescently labeled macromolecules in a cell membrane imaged on a confocal, two-photon, or total internal reflection fluorescence microscope. In contrast to r-space correlation techniques, we show kICS can recover accurate dynamics even in the presence of complex fluorophore photobleaching and/or “blinking”. Furthermore, these quantities can be calculated without nonlinear curve fitting, or any knowledge of the beam radius of the exciting laser. The number densities calculated by kICS are less sensitive to spatial inhomogeneity of the fluorophore distribution than densities measured using image correlation spectroscopy. We use simulations as a proof-of-principle to show that number densities and transport coefficients can be extracted using this technique. We present calibration measurements with fluorescent microspheres imaged on a confocal microscope, which recover Stokes-Einstein diffusion coefficients, and flow velocities that agree with single particle tracking measurements. We also show the application of kICS to measurements of the transport dynamics of α5-integrin/enhanced green fluorescent protein constructs in a transfected CHO cell imaged on a total internal reflection fluorescence microscope using charge-coupled device area detection. PMID:16861272

Real-time restoration of white-light confocal microscope optical sections

PubMed Central

Balasubramanian, Madhusudhanan; Iyengar, S. Sitharama; Beuerman, Roger W.; Reynaud, Juan; Wolenski, Peter

2009-01-01

Confocal microscopes (CM) are routinely used for building 3-D images of microscopic structures. Nonideal imaging conditions in a white-light CM introduce additive noise and blur. The optical section images need to be restored prior to quantitative analysis. We present an adaptive noise filtering technique using Karhunen–Loéve expansion (KLE) by the method of snapshots and a ringing metric to quantify the ringing artifacts introduced in the images restored at various iterations of iterative Lucy–Richardson deconvolution algorithm. The KLE provides a set of basis functions that comprise the optimal linear basis for an ensemble of empirical observations. We show that most of the noise in the scene can be removed by reconstructing the images using the KLE basis vector with the largest eigenvalue. The prefiltering scheme presented is faster and does not require prior knowledge about image noise. Optical sections processed using the KLE prefilter can be restored using a simple inverse restoration algorithm; thus, the methodology is suitable for real-time image restoration applications. The KLE image prefilter outperforms the temporal-average prefilter in restoring CM optical sections. The ringing metric developed uses simple binary morphological operations to quantify the ringing artifacts and confirms with the visual observation of ringing artifacts in the restored images. PMID:20186290

Flexible conformable hydrophobized surfaces for turbulent flow drag reduction

PubMed Central

Brennan, Joseph C; Geraldi, Nicasio R; Morris, Robert H; Fairhurst, David J; McHale, Glen; Newton, Michael I

2015-01-01

In recent years extensive work has been focused onto using superhydrophobic surfaces for drag reduction applications. Superhydrophobic surfaces retain a gas layer, called a plastron, when submerged underwater in the Cassie-Baxter state with water in contact with the tops of surface roughness features. In this state the plastron allows slip to occur across the surface which results in a drag reduction. In this work we report flexible and relatively large area superhydrophobic surfaces produced using two different methods: Large roughness features were created by electrodeposition on copper meshes; Small roughness features were created by embedding carbon nanoparticles (soot) into Polydimethylsiloxane (PDMS). Both samples were made into cylinders with a diameter under 12 mm. To characterize the samples, scanning electron microscope (SEM) images and confocal microscope images were taken. The confocal microscope images were taken with each sample submerged in water to show the extent of the plastron. The hydrophobized electrodeposited copper mesh cylinders showed drag reductions of up to 32% when comparing the superhydrophobic state with a wetted out state. The soot covered cylinders achieved a 30% drag reduction when comparing the superhydrophobic state to a plain cylinder. These results were obtained for turbulent flows with Reynolds numbers 10,000 to 32,500. PMID:25975704

Corneal Structural Changes in Nonneoplastic and Neoplastic Monoclonal Gammopathies.

PubMed

Aragona, Pasquale; Allegra, Alessandro; Postorino, Elisa Imelde; Rania, Laura; Innao, Vanessa; Wylegala, Edward; Nowinska, Anna; Ieni, Antonio; Pisani, Antonina; Musolino, Caterina; Puzzolo, Domenico; Micali, Antonio

2016-05-01

To investigate corneal confocal microscopic changes in nonneoplastic and neoplastic monoclonal gammopathies. Three groups of subjects were considered: group 1, twenty normal subjects; group 2, fifteen patients with monoclonal gammopathy of undetermined significance (MGUS); group 3, eight patients with smoldering multiple myeloma and eight patients with untreated multiple myeloma. After hematologic diagnosis, patients underwent ophthalmologic exam and in vivo confocal microscopic study. The statistical analysis was performed using ANOVA and Student-Newman-Keuls tests and receiver operating characteristic (ROC) curve analysis. Epithelial cells of gammopathic patients showed significantly higher reflectivity than controls, demonstrated by optical density (P < 0.001). Subbasal nerve density, branching, and beading were significantly altered in gammopathic patients (P = 0.01, P = 0.02, P = 0.02, respectively). The number of keratocytes was significantly reduced in neoplastic patients (P < 0.001 versus both normal and MGUS) in the anterior, medium, and posterior stroma. The ROC curve analysis showed good sensitivity and specificity for this parameter. Group 2 and 3 keratocytes showed higher nuclear and cytoplasmatic reflectivity in the medium and posterior stroma. Endothelial cells were not affected. Patients with neoplastic gammopathies showed peculiar alterations of the keratocyte number, which appeared significantly reduced. A follow-up with corneal confocal microscopy of patients with MGUS is suggested as a useful tool to identify peripheral tissue alterations linked to possible neoplastic disease development.

Imaging the microscopic structure of shear thinning and thickening colloidal suspensions.

PubMed

Cheng, Xiang; McCoy, Jonathan H; Israelachvili, Jacob N; Cohen, Itai

2011-09-02

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.

Physics and engineering aspects of cell and tissue imaging systems: microscopic devices and computer assisted diagnosis.

PubMed

Chen, Xiaodong; Ren, Liqiang; Zheng, Bin; Liu, Hong

2013-01-01

The conventional optical microscopes have been used widely in scientific research and in clinical practice. The modern digital microscopic devices combine the power of optical imaging and computerized analysis, archiving and communication techniques. It has a great potential in pathological examinations for improving the efficiency and accuracy of clinical diagnosis. This chapter reviews the basic optical principles of conventional microscopes, fluorescence microscopes and electron microscopes. The recent developments and future clinical applications of advanced digital microscopic imaging methods and computer assisted diagnosis schemes are also discussed.

Use of stereo vision and 24-bit false-color imagery to enhance visualization of multimodal confocal images

NASA Astrophysics Data System (ADS)

Beltrame, Francesco; Diaspro, Alberto; Fato, Marco; Martin, I.; Ramoino, Paola; Sobel, Irwin E.

1995-03-01

Confocal microscopy systems can be linked to 3D data oriented devices for the interactive navigation of the operator through a 3D object space. Sometimes, such environments are named `virtual reality' or `augmented reality' systems. We consider optical confocal laser scanning microscopy images, in fluorescence with various excitations and emissions, and versus time The aim of our study has been the quantitative spatial analysis of confocal data using the false-color composition technique. Starting from three 2D confocal fluorescent images at the same slice location in a given biological specimen, a new single image representation of all three parameters has been generated by the false-color technique on a HP 9000/735 workstation, connected to the confocal microscope. The color composite result of the mapping of the three parameters is displayed using a resolution of 24 bits per pixel. The operator may independently vary the mix of each of the three components in the false-color composite via three (R, G, B) mixing sliders. Furthermore, by using the pixel data in the three fluorescent component images, a 3D space containing the density distribution of these three parameters has been constructed. The histogram has been displayed in stereo: it can be used for clustering purposes from the operator, through an original thresholding algorithm.

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging.

PubMed

Nyström, Sofie; Bäck, Marcus; Nilsson, K Peter R; Hammarström, Per

2017-10-20

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer's and Parkinson's disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive β-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy.

PubMed

Fuchs, Felix M; Raguse, Marina; Fiebrandt, Marcel; Madela, Kazimierz; Awakowicz, Peter; Laue, Michael; Stapelmann, Katharina; Moeller, Ralf

2017-11-30

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Large-scale imaging of cortical network activity with calcium indicators.

PubMed

Ikegaya, Yuji; Le Bon-Jego, Morgane; Yuste, Rafael

2005-06-01

Bulk loading of calcium indicators has provided a unique opportunity to reconstruct the activity of cortical networks with single-cell resolution. Here we describe the detailed methods of bulk loading of AM dyes we developed and have been improving for imaging with a spinning disk confocal microscope.

In vivo laser confocal microscopic analysis of murine cornea and lens microstructures.

PubMed

Yuasa, Masashi; Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2008-01-01

The purpose of the current study is to investigate in vivo microstructures of anterior segments of normal murine eyes by new-generation in vivo laser confocal microscopy. Twenty-six corneas and lenses from 13 mice were analyzed by in vivo laser confocal microscopy. Murine corneal superficial cells formed a polygonal cell pattern, with a mean cell density of 577 +/- 115 cells/mm2 (mean +/- standard deviation). Corneal basal epithelial cells had dark cytoplasm and were closely organized (9,312 +/- 1,777 cells/mm2). Sub-basal nerve fiber bundles were arranged in a whorl pattern, with both clockwise and counter-clockwise patterns. In the stroma, keratocytes were observed as numerous reflective stellate structures. The endothelial cells were organized in a honeycomb pattern (2,463 +/- 292 cells/mm2). Deeper inside the eye, murine lens epithelial cells were organized in a regular pattern (4,168 +/- 636 cells/mm2) and numerous lens fibers were observed. In vivo laser confocal microscopy can provide high-resolution images of all corneal layers and lens structures of mice without sacrificing animals or tissue preparation.

Combining total internal reflection sum frequency spectroscopy spectral imaging and confocal fluorescence microscopy.

PubMed

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.

Multiple microscopic approaches demonstrate linkage between chromoplast architecture and carotenoid composition in diverse Capsicum annuum fruit.

PubMed

Kilcrease, James; Collins, Aaron M; Richins, Richard D; Timlin, Jerilyn A; O'Connell, Mary A

2013-12-01

Increased accumulation of specific carotenoids in plastids through plant breeding or genetic engineering requires an understanding of the limitations that storage sites for these compounds may impose on that accumulation. Here, using Capsicum annuum L. fruit, we demonstrate directly the unique sub-organellar accumulation sites of specific carotenoids using live cell hyperspectral confocal Raman microscopy. Further, we show that chromoplasts from specific cultivars vary in shape and size, and these structural variations are associated with carotenoid compositional differences. Live-cell imaging utilizing laser scanning confocal (LSCM) and confocal Raman microscopy, as well as fixed tissue imaging by scanning and transmission electron microscopy (SEM and TEM), all demonstrated morphological differences with high concordance for the measurements across the multiple imaging modalities. These results reveal additional opportunities for genetic controls on fruit color and carotenoid-based phenotypes. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Inverted follicular keratosis: dermoscopic and reflectance confocal microscopic features.

PubMed

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.

Comparison of LED and Conventional Fluorescence Microscopy for Detection of Acid Fast Bacilli in a Low-Incidence Setting

PubMed Central

Minion, Jessica; Pai, Madhukar; Ramsay, Andrew; Menzies, Dick; Greenaway, Christina

2011-01-01

Introduction Light emitting diode fluorescence microscopes have many practical advantages over conventional mercury vapour fluorescence microscopes, which would make them the preferred choice for laboratories in both low- and high-resource settings, provided performance is equivalent. Methods In a nested case-control study, we compared diagnostic accuracy and time required to read slides with the Zeiss PrimoStar iLED, LW Scientific Lumin, and a conventional fluorescence microscope (Leica DMLS). Mycobacterial culture was used as the reference standard, and subgroup analysis by specimen source and organism isolated were performed. Results There was no difference in sensitivity or specificity between the three microscopes, and agreement was high for all comparisons and subgroups. The Lumin and the conventional fluorescence microscope were equivalent with respect to time required to read smears, but the Zeiss iLED was significantly time saving compared to both. Conclusions Light emitting diode microscopy should be considered by all tuberculosis diagnostic laboratories, including those in high income countries, as a replacement for conventional fluorescence microscopes. Our findings provide support to the recent World Health Organization policy recommending that conventional fluorescence microscopy be replaced by light emitting diode microscopy using auramine staining in all settings where fluorescence microscopy is currently used. PMID:21811622

Optical sectioning microscopes with no moving parts using a micro-stripe array light emitting diode.

PubMed

Poher, V; Zhang, H X; Kennedy, G T; Griffin, C; Oddos, S; Gu, E; Elson, D S; Girkin, M; French, P M W; Dawson, M D; Neil, M A

2007-09-03

We describe an optical sectioning microscopy system with no moving parts based on a micro-structured stripe-array light emitting diode (LED). By projecting arbitrary line or grid patterns onto the object, we are able to implement a variety of optical sectioning microscopy techniques such as grid-projection structured illumination and line scanning confocal microscopy, switching from one imaging technique to another without modifying the microscope setup. The micro-structured LED and driver are detailed and depth discrimination capabilities are measured and calculated.

Factors Essential for Prostate Cancer Metastasis Revealed Through a Novel 3D Microtissue Assay

DTIC Science & Technology

2016-06-01

with Ob-niche spheroid and then conducted confocal microscopic analysis with frozen sections for HRE -mediated GFP expression. The results...activity in response to CoCl2. (B) The microscopic images demonstrate the HRE -dependent expression of GFP in a spheroid-specific manner. (C) The...io n C om bi ne d A375 MB231 Fr oz en s ec . 582 µm B C D SecNLuc Puro 4x HRE 0 5 10 15 None 100 A375 0 1 2 3 4 5 6 None 100

Light Microscopy Microscope Experiment

NASA Image and Video Library

2016-02-04

Ground testing for the first confocal Light Microscopy Microscope (LMM) Experiment. Procter and Gamble is working with NASA Glenn scientists to prepare for a study that examines product stabilizers in a microgravity environment. The particles in the tube glow orange because they have been fluorescently tagged with a dye that reacts to green laser lights to allow construction of a 3D image point by point. The experiment, which will be sent to the ISS later this year, will help P&G develop improved product stabilizers to extend shelf life and develop more environmentally friendly packaging.

Application of laser scanning confocal microscopy in the soft tissue exquisite structure for 3D scan

PubMed Central

Zhang, Zhaoqiang; Ibrahim, Mohamed; Fu, Yang; Wu, Xujia; Ren, Fei; Chen, Lei

2018-01-01

Three-dimensional (3D) printing is a new developing technology for printing individualized materials swiftly and precisely in the field of biological medicine (especially tissue-engineered materials). Prior to printing, it is necessary to scan the structure of the natural biological tissue, then construct the 3D printing digital model through optimizing the scanned data. By searching the literatures, magazines at home and abroad, this article reviewed the current status, main processes and matters needing attention of confocal laser scanning microscope (LSCM) in the application of soft tissue fine structure 3D scanning, empathizing the significance of LSCM in this field. PMID:29755838

Quantitative detection of caffeine in human skin by confocal Raman spectroscopy--A systematic in vitro validation study.

PubMed

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.

Dye-enhanced multimodal confocal imaging as a novel approach to intraoperative diagnosis of brain tumors.

PubMed

Snuderl, Matija; Wirth, Dennis; Sheth, Sameer A; Bourne, Sarah K; Kwon, Churl-Su; Ancukiewicz, Marek; Curry, William T; Frosch, Matthew P; Yaroslavsky, Anna N

2013-01-01

Intraoperative diagnosis plays an important role in accurate sampling of brain tumors, limiting the number of biopsies required and improving the distinction between brain and tumor. The goal of this study was to evaluate dye-enhanced multimodal confocal imaging for discriminating gliomas from nonglial brain tumors and from normal brain tissue for diagnostic use. We investigated a total of 37 samples including glioma (13), meningioma (7), metastatic tumors (9) and normal brain removed for nontumoral indications (8). Tissue was stained in 0.05 mg/mL aqueous solution of methylene blue (MB) for 2-5 minutes and multimodal confocal images were acquired using a custom-built microscope. After imaging, tissue was formalin fixed and paraffin embedded for standard neuropathologic evaluation. Thirteen pathologists provided diagnoses based on the multimodal confocal images. The investigated tumor types exhibited distinctive and complimentary characteristics in both the reflectance and fluorescence responses. Images showed distinct morphological features similar to standard histology. Pathologists were able to distinguish gliomas from normal brain tissue and nonglial brain tumors, and to render diagnoses from the images in a manner comparable to haematoxylin and eosin (H&E) slides. These results confirm the feasibility of multimodal confocal imaging for intravital intraoperative diagnosis. © 2012 The Authors; Brain Pathology © 2012 International Society of Neuropathology.

Quantitative Live-Cell Confocal Imaging of 3D Spheroids in a High-Throughput Format.

PubMed

Leary, Elizabeth; Rhee, Claire; Wilks, Benjamin T; Morgan, Jeffrey R

2018-06-01

Accurately predicting the human response to new compounds is critical to a wide variety of industries. Standard screening pipelines (including both in vitro and in vivo models) often lack predictive power. Three-dimensional (3D) culture systems of human cells, a more physiologically relevant platform, could provide a high-throughput, automated means to test the efficacy and/or toxicity of novel substances. However, the challenge of obtaining high-magnification, confocal z stacks of 3D spheroids and understanding their respective quantitative limitations must be overcome first. To address this challenge, we developed a method to form spheroids of reproducible size at precise spatial locations across a 96-well plate. Spheroids of variable radii were labeled with four different fluorescent dyes and imaged with a high-throughput confocal microscope. 3D renderings of the spheroid had a complex bowl-like appearance. We systematically analyzed these confocal z stacks to determine the depth of imaging and the effect of spheroid size and dyes on quantitation. Furthermore, we have shown that this loss of fluorescence can be addressed through the use of ratio imaging. Overall, understanding both the limitations of confocal imaging and the tools to correct for these limits is critical for developing accurate quantitative assays using 3D spheroids.

Confocal microscopy with strip mosaicing for rapid imaging over large areas of excised tissue

PubMed Central

Li, Yongbiao; Larson, Bjorg; Peterson, Gary; Seltzer, Emily; Toledo-Crow, Ricardo; Rajadhyaksha, Milind

2013-01-01

Abstract. Confocal mosaicing microscopy is a developing technology platform for imaging tumor margins directly in freshly excised tissue, without the processing required for conventional pathology. Previously, mosaicing on 12-×-12  mm2 of excised skin tissue from Mohs surgery and detection of basal cell carcinoma margins was demonstrated in 9 min. Last year, we reported the feasibility of a faster approach called “strip mosaicing,” which was demonstrated on a 10-×-10  mm2 of tissue in 3 min. Here we describe further advances in instrumentation, software, and speed. A mechanism was also developed to flatten tissue in order to enable consistent and repeatable acquisition of images over large areas. We demonstrate mosaicing on 10-×-10  mm2 of skin tissue with 1-μm lateral resolution in 90 s. A 2.5-×-3.5  cm2 piece of breast tissue was scanned with 0.8-μm lateral resolution in 13 min. Rapid mosaicing of confocal images on large areas of fresh tissue potentially offers a means to perform pathology at the bedside. Imaging of tumor margins with strip mosaicing confocal microscopy may serve as an adjunct to conventional (frozen or fixed) pathology for guiding surgery. PMID:23389736

Nanoscale Spatiotemporal Diffusion Modes Measured by Simultaneous Confocal and Stimulated Emission Depletion Nanoscopy Imaging.

PubMed

Schneider, Falk; Waithe, Dominic; Galiani, Silvia; Bernardino de la Serna, Jorge; Sezgin, Erdinc; Eggeling, Christian

2018-06-19

The diffusion dynamics in the cellular plasma membrane provide crucial insights into molecular interactions, organization, and bioactivity. Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (scanning STED-FCS) measures such dynamics with high spatial and temporal resolution. It reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially for each pixel along the scanned line. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED-FCS measurement method, line interleaved excitation scanning STED-FCS (LIESS-FCS), that discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS-FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS-FCS to investigate the spatiotemporal organization of glycosylphosphatidylinositol-anchored proteins in the plasma membrane of live cells, which, interestingly, show multiple diffusion modes at different spatial positions.

Resolution enhancement in a double-helix phase engineered scanning microscope (RESCH microscope) (Presentation Recording)

NASA Astrophysics Data System (ADS)

Jesacher, Alexander; Ritsch-Marte, Monika; Piestun, Rafael

2015-08-01

Recently we introduced RESCH microscopy [1] - a scanning microscope that allows slightly refocusing the sample after the acquisition has been performed, solely by performing appropriate data post-processing. The microscope features a double-helix phase-engineered emission point spread function in combination with camera-based detection. Based on the principle of transverse resolution enhancement in Image Scanning Microscopy [2,3], we demonstrate similar resolution improvement in RESCH. Furthermore, we outline a pathway for how the collected 3D sample information can be used to construct sharper optical sections. [1] A. Jesacher, M. Ritsch-Marte and R. Piestun, accepted for Optica. [2] C.J.R. Sheppard, "Super-resolution in Confocal imaging," Optik, 80, 53-54 (1988). [3] C.B. Müller and J. Enderlein "Image Scanning Microscopy," Phys. Rev. Lett. 104, 198101 (2010).

Crystallization Behavior of Perovskite in the Synthesized High-Titanium-Bearing Blast Furnace Slag Using Confocal Scanning Laser Microscope

NASA Astrophysics Data System (ADS)

Hu, Meilong; Liu, Lu; Lv, Xuewei; Bai, Chenguang; Zhang, Shengfu

2014-01-01

The isothermal phase composition of high-titanium-bearing slag (23 mass pct TiO2) under an argon atmosphere during cooling process from 1723 K (1450 °C) was calculated by FactSage.6.3 (CRCT-ThermFact Inc., Montréal, Canada). Three main phases, which were perovskite, titania spinel, and clinopyroxene, could form during the cooling process and they precipitated at 1713 K, 1603 K, and 1498 K (1440 °C, 1330 °C, and 1225 °C), respectively. The nonisothermal crystallization process of perovskite in synthesized high-titanium-bearing slag was studied in situ by a confocal scanning laser microscope (CSLM) with cooling rate of 30 K/min. The results showed that the primary phase was perovskite that precipitated at 1703 K (1430 °C). The whole precipitation and growth process of perovskite was obtained, whereas other phases formed as glass under the current experimental conditions. Perovskite grew along a specific growth track and finally appeared with snowflake morphology. The growing kinetics of perovskite formation from molten slag were also mentioned.

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.

PubMed

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.

Surface topographical and structural analysis of Ag+-implanted polymethylmethacrylate

NASA Astrophysics Data System (ADS)

Arif, Shafaq; Rafique, M. Shahid; Saleemi, Farhat; Naab, Fabian; Toader, Ovidiu; Sagheer, Riffat; Bashir, Shazia; Zia, Rehana; Siraj, Khurram; Iqbal, Saman

2016-08-01

Specimens of polymethylmethacrylate (PMMA) were implanted with 400-keV Ag+ ions at different ion fluences ranging from 1 × 1014 to 5 × 1015 ions/cm2 using a 400-kV NEC ion implanter. The surface topographical features of the implanted PMMA were investigated by a confocal microscope. Modifications in the structural properties of the implanted specimens were analyzed in comparison with pristine PMMA by X-ray diffraction (XRD) and Raman spectroscopy. UV-Visible spectroscopy was applied to determine the effects of ion implantation on optical transmittance of the implanted PMMA. The confocal microscopic images revealed the formation of hillock-like microstructures along the ion track on the implanted PMMA surface. The increase in ion fluence led to more nucleation of hillocks. The XRD pattern confirmed the amorphous nature of pristine and implanted PMMA, while the Raman studies justified the transformation of Ag+-implanted PMMA into amorphous carbon at the ion fluence of ⩾5 × 1014 ions/cm2. Moreover, the decrease in optical transmittance of PMMA is associated with the formation of hillocks and ion-induced structural modifications after implantation.

LabVIEW control software for scanning micro-beam X-ray fluorescence spectrometer.

PubMed

Wrobel, Pawel; Czyzycki, Mateusz; Furman, Leszek; Kolasinski, Krzysztof; Lankosz, Marek; Mrenca, Alina; Samek, Lucyna; Wegrzynek, Dariusz

2012-05-15

Confocal micro-beam X-ray fluorescence microscope was constructed. The system was assembled from commercially available components - a low power X-ray tube source, polycapillary X-ray optics and silicon drift detector - controlled by an in-house developed LabVIEW software. A video camera coupled to optical microscope was utilized to display the area excited by X-ray beam. The camera image calibration and scan area definition software were also based entirely on LabVIEW code. Presently, the main area of application of the newly constructed spectrometer is 2-dimensional mapping of element distribution in environmental, biological and geological samples with micrometer spatial resolution. The hardware and the developed software can already handle volumetric 3-D confocal scans. In this work, a front panel graphical user interface as well as communication protocols between hardware components were described. Two applications of the spectrometer, to homogeneity testing of titanium layers and to imaging of various types of grains in air particulate matter collected on membrane filters, were presented. Copyright © 2012 Elsevier B.V. All rights reserved.

MultiMap: A Tool to Automatically Extract and Analyse Spatial Microscopic Data From Large Stacks of Confocal Microscopy Images

PubMed Central

Varando, Gherardo; Benavides-Piccione, Ruth; Muñoz, Alberto; Kastanauskaite, Asta; Bielza, Concha; Larrañaga, Pedro; DeFelipe, Javier

2018-01-01

The development of 3D visualization and reconstruction methods to analyse microscopic structures at different levels of resolutions is of great importance to define brain microorganization and connectivity. MultiMap is a new tool that allows the visualization, 3D segmentation and quantification of fluorescent structures selectively in the neuropil from large stacks of confocal microscopy images. The major contribution of this tool is the posibility to easily navigate and create regions of interest of any shape and size within a large brain area that will be automatically 3D segmented and quantified to determine the density of puncta in the neuropil. As a proof of concept, we focused on the analysis of glutamatergic and GABAergic presynaptic axon terminals in the mouse hippocampal region to demonstrate its use as a tool to provide putative excitatory and inhibitory synaptic maps. The segmentation and quantification method has been validated over expert labeled images of the mouse hippocampus and over two benchmark datasets, obtaining comparable results to the expert detections. PMID:29875639

MultiMap: A Tool to Automatically Extract and Analyse Spatial Microscopic Data From Large Stacks of Confocal Microscopy Images.

PubMed

Varando, Gherardo; Benavides-Piccione, Ruth; Muñoz, Alberto; Kastanauskaite, Asta; Bielza, Concha; Larrañaga, Pedro; DeFelipe, Javier

2018-01-01

The development of 3D visualization and reconstruction methods to analyse microscopic structures at different levels of resolutions is of great importance to define brain microorganization and connectivity. MultiMap is a new tool that allows the visualization, 3D segmentation and quantification of fluorescent structures selectively in the neuropil from large stacks of confocal microscopy images. The major contribution of this tool is the posibility to easily navigate and create regions of interest of any shape and size within a large brain area that will be automatically 3D segmented and quantified to determine the density of puncta in the neuropil. As a proof of concept, we focused on the analysis of glutamatergic and GABAergic presynaptic axon terminals in the mouse hippocampal region to demonstrate its use as a tool to provide putative excitatory and inhibitory synaptic maps. The segmentation and quantification method has been validated over expert labeled images of the mouse hippocampus and over two benchmark datasets, obtaining comparable results to the expert detections.

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions.

PubMed

Zheng, Haocheng; Goldner, Lori S; Leuba, Sanford H

2007-03-01

Many technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing the background signal. A second major innovation was the advent of high quantum-yield, low noise, single-photon counting detectors. The superior background rejection of SCFM combined with low-noise, high-yield detectors makes it possible to detect the fluorescence from single-dye molecules. By labeling a DNA molecule or a DNA/protein complex with a donor/acceptor dye pair, fluorescence resonance energy transfer (FRET) can be used to track conformational changes in the molecule/complex itself, on a single molecule/complex basis. In this methods paper, we describe the core concepts of SCFM in the context of a study that uses FRET to reveal conformational fluctuations in individual Holliday junction DNA molecules and nucleosomal particles. We also discuss data processing methods for SCFM.

Large area fabrication of plasmonic nanoparticle grating structure by conventional scanning electron microscope

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sudheer,, E-mail: sudheer@rrcat.gov.in; Tiwari, P.; Rai, V. N.

Plasmonic nanoparticle grating (PNG) structure of different periods has been fabricated by electron beam lithography using silver halide based transmission electron microscope film as a substrate. Conventional scanning electron microscope is used as a fabrication tool for electron beam lithography. Optical microscope and energy dispersive spectroscopy (EDS) have been used for its morphological and elemental characterization. Optical characterization is performed by UV-Vis absorption spectroscopic technique.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sadetaporn, D; The University of Texas MD Anderson Cancer Center, Houston, TX; Flint, D

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 hmore » 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.« less

In vivo confocal microscopic analysis of normal human anterior limbal stroma

PubMed Central

Mathews, Saumi; Chidambaram, Jaya Devi; Lanjewar, Shruti; Mascarenhas, Jeena; Prajna, Namperumalsamy Venkatesh; Muthukkaruppan, Veerappan; Chidambaranathan, Gowri Priya

2015-01-01

Purpose To characterize the microarchitecture of the anterior limbal stroma in healthy individuals using in vivo confocal microscopy (IVCM) and to correlate it with mesenchymal stem cells (MSCs), a component of the limbal-niche. Methods The corneal side of the superior limbus was scanned in 30 eyes of 17 normal subjects beyond the basal epithelium, deep into the stroma using a HRT III laser scanning microscope. The IVCM findings were correlated with the immunohistochemical features of MSCs in the anterior limbal stroma. Results Clusters of hyperreflective structures were observed in the anterior limbal stroma, subjacent to the basal epithelium (depth: 50.2±8.7 - 98±12.8 μm), but not in the corneal stroma. The structures showed unique morphology compared to epithelial cells, keratocytes, neurons and dendritic cells. In parallel, confocal analysis of immunostained sections showed clusters of cells, double positive for MSC specific markers (CD90 and CD105) in the anterior limbal stroma at a depth of 55.3±12.7 μm to 72±37.6 μm. The organization and distribution of the MSC clusters locates them within the hyperreflective region in the anterior limbal stroma. Conclusions The hyperreflective structures, demonstrated for the first time in the human anterior limbal stroma, probably represent an important component of the limbal-niche. Our approach of in vivo imaging may pave the way for assessing the limbal stromal health. PMID:25742388

Improving axial resolution in confocal microscopy with new high refractive index mounting media.

PubMed

Fouquet, Coralie; Gilles, Jean-François; Heck, Nicolas; Dos Santos, Marc; Schwartzmann, Richard; Cannaya, Vidjeacoumary; Morel, Marie-Pierre; Davidson, Robert Stephen; Trembleau, Alain; Bolte, Susanne

2015-01-01

Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required.

Acquisition of multiple image stacks with a confocal laser scanning microscope

NASA Astrophysics Data System (ADS)

Zuschratter, Werner; Steffen, Thomas; Braun, Katharina; Herzog, Andreas; Michaelis, Bernd; Scheich, Henning

1998-06-01

Image acquisition at high magnification is inevitably correlated with a limited view over the entire tissue section. To overcome this limitation we designed software for multiple image-stack acquisition (3D-MISA) in confocal laser scanning microscopy (CLSM). The system consists of a 4 channel Leica CLSM equipped with a high resolution z- scanning stage mounted on a xy-monitorized stage. The 3D- MISA software is implemented into the microscope scanning software and uses the microscope settings for the movements of the xy-stage. It allows storage and recall of 70 xyz- positions and the automatic 3D-scanning of image arrays between selected xyz-coordinates. The number of images within one array is limited only by the amount of disk space or memory available. Although for most applications the accuracy of the xy-scanning stage is sufficient for a precise alignment of tiled views, the software provides the possibility of an adjustable overlap between two image stacks by shifting the moving steps of the xy-scanning stage. After scanning a tiled image gallery of the extended focus-images of each channel will be displayed on a graphic monitor. In addition, a tiled image gallery of individual focal planes can be created. In summary, the 3D-MISA allows 3D-image acquisition of coherent regions in combination with high resolution of single images.

Bessel light sheet structured illumination microscopy

NASA Astrophysics Data System (ADS)

Noshirvani Allahabadi, Golchehr

Biomedical study researchers using animals to model disease and treatment need fast, deep, noninvasive, and inexpensive multi-channel imaging methods. Traditional fluorescence microscopy meets those criteria to an extent. Specifically, two-photon and confocal microscopy, the two most commonly used methods, are limited in penetration depth, cost, resolution, and field of view. In addition, two-photon microscopy has limited ability in multi-channel imaging. Light sheet microscopy, a fast developing 3D fluorescence imaging method, offers attractive advantages over traditional two-photon and confocal microscopy. Light sheet microscopy is much more applicable for in vivo 3D time-lapsed imaging, owing to its selective illumination of tissue layer, superior speed, low light exposure, high penetration depth, and low levels of photobleaching. However, standard light sheet microscopy using Gaussian beam excitation has two main disadvantages: 1) the field of view (FOV) of light sheet microscopy is limited by the depth of focus of the Gaussian beam. 2) Light-sheet images can be degraded by scattering, which limits the penetration of the excitation beam and blurs emission images in deep tissue layers. While two-sided sheet illumination, which doubles the field of view by illuminating the sample from opposite sides, offers a potential solution, the technique adds complexity and cost to the imaging system. We investigate a new technique to address these limitations: Bessel light sheet microscopy in combination with incoherent nonlinear Structured Illumination Microscopy (SIM). Results demonstrate that, at visible wavelengths, Bessel excitation penetrates up to 250 microns deep in the scattering media with single-side illumination. Bessel light sheet microscope achieves confocal level resolution at a lateral resolution of 0.3 micron and an axial resolution of 1 micron. Incoherent nonlinear SIM further reduces the diffused background in Bessel light sheet images, resulting in confocal quality images in thick tissue. The technique was applied to live transgenic zebra fish tg(kdrl:GFP), and the sub-cellular structure of fish vasculature genetically labeled with GFP was captured in 3D. The superior speed of the microscope enables us to acquire signal from 200 layers of a thick sample in 4 minutes. The compact microscope uses exclusively off-the-shelf components and offers a low-cost imaging solution for studying small animal models or tissue samples.

Hyper-spectral imaging in scanning-confocal-fluorescence microscopy using a novel broadband diffractive optic

NASA Astrophysics Data System (ADS)

Wang, Peng; Ebeling, Carl G.; Gerton, Jordan; Menon, Rajesh

In this paper, we demonstrate hyper-spectral imaging of fluorescent microspheres in a scanning-confocal-fluorescence microscope by spatially dispersing the spectra using a novel broadband diffractive optic, and applying a nonlinear optimization technique to extract the full-incident spectra. This broadband diffractive optic has a designed optical efficiency of over 90% across the entire visible spectrum. We used this technique to create two-color images of two fluorophores and also extracted their emission spectra with good fidelity. This method can be extended to image both spatially and spectrally overlapping fluorescent samples. Full control in the number of emission spectra and the feasibility of enhanced imaging speed are demonstrated as well.

Group refractive index reconstruction with broadband interferometric confocal microscopy

PubMed Central

Marks, Daniel L.; Schlachter, Simon C.; Zysk, Adam M.; Boppart, Stephen A.

2010-01-01

We propose a novel method of measuring the group refractive index of biological tissues at the micrometer scale. The technique utilizes a broadband confocal microscope embedded into a Mach–Zehnder interferometer, with which spectral interferograms are measured as the sample is translated through the focus of the beam. The method does not require phase unwrapping and is insensitive to vibrations in the sample and reference arms. High measurement stability is achieved because a single spectral interferogram contains all the information necessary to compute the optical path delay of the beam transmitted through the sample. Included are a physical framework defining the forward problem, linear solutions to the inverse problem, and simulated images of biologically relevant phantoms. PMID:18451922

In vivo integrated photoacoustic and confocal microscopy of hemoglobin oxygen saturation and oxygen partial pressure.

PubMed

Wang, Yu; Hu, Song; Maslov, Konstantin; Zhang, Yu; Xia, Younan; Wang, Lihong V

2011-04-01

We developed dual-modality microscope integrating photoacoustic microscopy (PAM) and fluorescence confocal microscopy (FCM) to noninvasively image hemoglobin oxygen saturation (sO₂) and oxygen partial pressure (pO₂) in vivo in single blood vessels with high spatial resolution. While PAM measures sO₂ by imaging hemoglobin optical absorption at two wavelengths, FCM quantifies pO₂ using phosphorescence quenching. The variations of sO₂ and pO₂ values in multiple orders of vessel branches under hyperoxic (100% oxygen) and normoxic (21% oxygen) conditions correlate well with the oxygen-hemoglobin dissociation curve. In addition, the total concentration of hemoglobin is imaged by PAM at an isosbestic wavelength.

Ultrahigh resolution multicolor colocalization of single fluorescent probes

DOEpatents

Weiss, Shimon; Michalet, Xavier; Lacoste, Thilo D.

2005-01-18

A novel optical ruler based on ultrahigh-resolution colocalization of single fluorescent probes is described. Two unique families of fluorophores are used, namely energy-transfer fluorescent beads and semiconductor nanocrystal (NC) quantum dots, that can be excited by a single laser wavelength but emit at different wavelengths. A novel multicolor sample-scanning confocal microscope was constructed which allows one to image each fluorescent light emitter, free of chromatic aberrations, by scanning the sample with nanometer scale steps using a piezo-scanner. The resulting spots are accurately localized by fitting them to the known shape of the excitation point-spread-function of the microscope.

Optimal model-based sensorless adaptive optics for epifluorescence microscopy.

PubMed

Pozzi, Paolo; Soloviev, Oleg; Wilding, Dean; Vdovin, Gleb; Verhaegen, Michel

2018-01-01

We report on a universal sample-independent sensorless adaptive optics method, based on modal optimization of the second moment of the fluorescence emission from a point-like excitation. Our method employs a sample-independent precalibration, performed only once for the particular system, to establish the direct relation between the image quality and the aberration. The method is potentially applicable to any form of microscopy with epifluorescence detection, including the practically important case of incoherent fluorescence emission from a three dimensional object, through minor hardware modifications. We have applied the technique successfully to a widefield epifluorescence microscope and to a multiaperture confocal microscope.

Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging

PubMed Central

Shimozawa, Togo; Yamagata, Kazuo; Kondo, Takefumi; Hayashi, Shigeo; Shitamukai, Atsunori; Konno, Daijiro; Matsuzaki, Fumio; Takayama, Jun; Onami, Shuichi; Nakayama, Hiroshi; Kosugi, Yasuhito; Watanabe, Tomonobu M.; Fujita, Katsumasa; Mimori-Kiyosue, Yuko

2013-01-01

A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to “pinhole cross-talk,” which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging. PMID:23401517

Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging.

PubMed

Shimozawa, Togo; Yamagata, Kazuo; Kondo, Takefumi; Hayashi, Shigeo; Shitamukai, Atsunori; Konno, Daijiro; Matsuzaki, Fumio; Takayama, Jun; Onami, Shuichi; Nakayama, Hiroshi; Kosugi, Yasuhito; Watanabe, Tomonobu M; Fujita, Katsumasa; Mimori-Kiyosue, Yuko

2013-02-26

A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to "pinhole cross-talk," which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging.

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

NASA Astrophysics Data System (ADS)

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.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

PubMed Central

Bertrand, Vincent; Lenne, Pierre-François

2014-01-01

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time. PMID:24836407

Virtual reality microscope versus conventional microscope regarding time to diagnosis: an experimental study.

PubMed

Randell, Rebecca; Ruddle, Roy A; Mello-Thoms, Claudia; Thomas, Rhys G; Quirke, Phil; Treanor, Darren

2013-01-01

  To create and evaluate a virtual reality (VR) microscope that is as efficient as the conventional microscope, seeking to support the introduction of digital slides into routine practice.   A VR microscope was designed and implemented by combining ultra-high-resolution displays with VR technology, techniques for fast interaction, and high usability. It was evaluated using a mixed factorial experimental design with technology and task as within-participant variables and grade of histopathologist as a between-participant variable. Time to diagnosis was similar for the conventional and VR microscopes. However, there was a significant difference in the mean magnification used between the two technologies, with participants working at a higher level of magnification on the VR microscope.   The results suggest that, with the right technology, efficient use of digital pathology for routine practice is a realistic possibility. Further work is required to explore what magnification is required on the VR microscope for histopathologists to identify diagnostic features, and the effect on this of the digital slide production process. © 2012 Blackwell Publishing Limited.

Two-Photon Fluorescence Microscopy Developed for Microgravity Fluid Physics

NASA Technical Reports Server (NTRS)

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

2004-01-01

Recent research efforts within the Microgravity Fluid Physics Branch of the NASA Glenn Research Center have necessitated the development of a microscope capable of high-resolution, three-dimensional imaging of intracellular structure and tissue morphology. Standard optical microscopy works well for thin samples, but it does not allow the imaging of thick samples because of severe degradation caused by out-of-focus object structure. Confocal microscopy, which is a laser-based scanning microscopy, provides improved three-dimensional imaging and true optical sectioning by excluding the out-of-focus light. However, in confocal microscopy, out-of-focus object structure is still illuminated by the incoming beam, which can lead to substantial photo-bleaching. In addition, confocal microscopy is plagued by limited penetration depth, signal loss due to the presence of a confocal pinhole, and the possibility of live-cell damage. Two-photon microscopy is a novel form of laser-based scanning microscopy that allows three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon microscopy, it utilizes the nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption because of the nonlinear (i.e., quadratic) electric field dependence, so an ultrafast pulsed laser source must typically be employed. On the other hand, this stringent energy density requirement effectively localizes fluorophore excitation to the focal volume. Consequently, two-photon microscopy provides optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction in photo-damage because of the longer excitation wavelength, a reduction in background fluorescence, and a 4 increase in penetration depth over confocal methods because of the reduction in Rayleigh scattering.

In vivo laser confocal microscopy findings in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy.

PubMed

Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2012-01-01

The purpose of this study was to investigate pathological changes of the corneal cell layer in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy by in vivo laser corneal confocal microscopy. Two patients were evaluated using a cornea-specific in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph 2 Rostock Cornea Module, HRT 2-RCM). The affected corneal areas of both patients were examined. Image analysis was performed to identify corneal epithelial and stromal deposits correlated with this dystrophy. Variously shaped (linear, multilaminar, curvilinear, ring-shape, geographic) highly reflective materials were observed in the "map" area, mainly in the basal epithelial cell layer. In "fingerprint" lesions, multiple linear and curvilinear hyporeflective lines were observed. Additionally, in the affected corneas, infiltration of possible Langerhans cells and other inflammatory cells was observed as highly reflective Langerhans cell-like or dot images. Finally, needle-shaped materials were observed in one patient. HRT 2-RCM laser confocal microscopy is capable of identifying corneal microstructural changes related to map-dot-fingerprint corneal dystrophy in vivo. The technique may be useful in elucidating the pathogenesis and natural course of map-dot-fingerprint corneal dystrophy and other similar basement membrane abnormalities.

Correlative Single-Molecule Localization Microscopy and Confocal Microscopy.

PubMed

Soeller, Christian; Hou, Yufeng; Jayasinghe, Isuru D; Baddeley, David; Crossman, David

2017-01-01

Single-molecule localization microscopy allows the ability to image fluorescence labeled molecular targets at nanoscale resolution. However, for many biological questions the ability to provide tissue and cellular context in addition to these high resolution data is eminently informative. Here, we describe a procedure to achieve this aim by correlatively imaging human cardiac tissue first at the nanoscale with direct stochastic optical reconstruction microscopy (dSTORM) and then at the diffraction limit with conventional confocal microscopy.

A new diagnostic technique for tinea incognito: in vivo reflectance confocal microscopy. Report of five cases.

PubMed

Turan, Enver; Erdemir, Asli Turgut; Gurel, Mehmet Salih; Yurt, Nurdan

2013-02-01

In vivo confocal laser scanning microscopy (CLSM) is a modern non-invasive method for investigation of the skin that allows real-time visualization of individual cells and subcellular structures with the highest resolution imaging comparable to the routine histopathology. Our aim was to demonstrate the potential of CLSM for non-invasive diagnosis of difficult tinea incognito cases. Clinically atypical lesions in five cases of tinea incognito due to dermatophyte spp. were demonstrated using reflectance confocal laser scanning microscopy (RCM), parallel to KOH preparation and fungal culture of skin scrapings performed in the same patients. The morphological features characteristic for tinea incognito, namely linear branched hyphae in the intercellular area of the stratum corneum, were readily detectable by means of CLSM. In vivo tissue imaging were performed at three different wavelengths (785, 658, 445 nm) and the best images of fungal elements were obtained at 445 nm. All of our five cases had similar reflectance confocal microscopical findings. Our findings suggest the potential of CLSM as a non-invasive tool for the diagnosis of tinea incognito having atypical clinical appearance. Although at present the reflectance confocal microscopy cannot replace the current diagnostic standards for tinea incognito, it may be successfully used as in vivo non-invasive screening tool to facilitate the diagnosis and point to the need for further investigation of the patient. © 2012 John Wiley & Sons A/S.

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

PubMed

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.

Digital adaptive optics line-scanning confocal imaging system.

PubMed

Liu, Changgeng; Kim, Myung K

2015-01-01

A digital adaptive optics line-scanning confocal imaging (DAOLCI) system is proposed by applying digital holographic adaptive optics to a digital form of line-scanning confocal imaging system. In DAOLCI, each line scan is recorded by a digital hologram, which allows access to the complex optical field from one slice of the sample through digital holography. This complex optical field contains both the information of one slice of the sample and the optical aberration of the system, thus allowing us to compensate for the effect of the optical aberration, which can be sensed by a complex guide star hologram. After numerical aberration compensation, the corrected optical fields of a sequence of line scans are stitched into the final corrected confocal image. In DAOLCI, a numerical slit is applied to realize the confocality at the sensor end. The width of this slit can be adjusted to control the image contrast and speckle noise for scattering samples. DAOLCI dispenses with the hardware pieces, such as Shack–Hartmann wavefront sensor and deformable mirror, and the closed-loop feedbacks adopted in the conventional adaptive optics confocal imaging system, thus reducing the optomechanical complexity and cost. Numerical simulations and proof-of-principle experiments are presented that demonstrate the feasibility of this idea.

Automated Image Analysis Corrosion Working Group Update: February 1, 2018

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wendelberger, James G.

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).

Small fiber neuropathy in women with fibromyalgia. An in vivo assessment using corneal confocal bio-microscopy.

PubMed

Ramírez, Manuel; Martínez-Martínez, Laura-Aline; Hernández-Quintela, Everardo; Velazco-Casapía, Jorge; Vargas, Angélica; Martínez-Lavín, Manuel

2015-10-01

A consistent line of investigation suggests that fibromyalgia is a neuropathic pain syndrome. This outlook has been recently reinforced by several controlled studies that describe decreased small nerve fiber density in skin biopsies of patients with fibromyalgia. The cornea receives the densest small fiber innervation of the body. Corneal confocal bio-microscopy is a new noninvasive method to evaluate small nerve fiber morphology. Our objective was to assess corneal small nerve fiber morphology in patients with fibromyalgia, and to associate corneal nerve microscopic features with neuropathic pain descriptors and other fibromyalgia symptoms. We studied 17 female patients with fibromyalgia and 17 age-matched healthy control subjects. All the participants completed different questionnaires regarding the symptoms of fibromyalgia, including a neuropathic pain survey. A central corneal thickness scan was obtained with a confocal microscope. Nerve measurements were made by a single ophthalmologist without knowledge of the clinical diagnosis. Stromal nerve thickness was defined as the mean value between the widest and the narrowest portion of each analyzed stromal nerve. Corneal sub-basal plexus nerve density was also assessed. Patients with fibromyalgia had stromal nerve thickness of 5.0 ± 1.0 µm (mean ± standard deviation) significantly different from that of control's values (6.1 ± 1.3) p = 0.01. Patients also had decreased sub-basal plexus nerve density per square millimeter (85 ± 29) vs. 107 ± 26 of controls p = 0.02. When controls and patients were grouped together, there was an association between stromal nerve slenderness and neuropathic pain descriptors (Fisher's exact test p = 0.007). Women suffering from fibromyalgia have thinner corneal stromal nerves and diminished sub-basal plexus nerve density when compared to healthy controls. Nerve scarcity is associated with neuropathic pain descriptors. Small fiber neuropathy may play a role in the pathogenesis of fibromyalgia pain. Corneal confocal microscopy could become a useful test in the study of patients with fibromyalgia. Copyright © 2015. Published by Elsevier Inc.

Optimisation approaches for concurrent transmitted light imaging during confocal microscopy.

PubMed

Collings, David A

2015-01-01

The transmitted light detectors present on most modern confocal microscopes are an under-utilised tool for the live imaging of plant cells. As the light forming the image in this detector is not passed through a pinhole, out-of-focus light is not removed. It is this extended focus that allows the transmitted light image to provide cellular and organismal context for fluorescence optical sections generated confocally. More importantly, the transmitted light detector provides images that have spatial and temporal registration with the fluorescence images, unlike images taken with a separately-mounted camera. Because plants often provide difficulties for taking transmitted light images, with the presence of pigments and air pockets in leaves, this study documents several approaches to improving transmitted light images beginning with ensuring that the light paths through the microscope are correctly aligned (Köhler illumination). Pigmented samples can be imaged in real colour using sequential scanning with red, green and blue lasers. The resulting transmitted light images can be optimised and merged in ImageJ to generate colour images that maintain registration with concurrent fluorescence images. For faster imaging of pigmented samples, transmitted light images can be formed with non-absorbed wavelengths. Transmitted light images of Arabidopsis leaves expressing GFP can be improved by concurrent illumination with green and blue light. If the blue light used for YFP excitation is blocked from the transmitted light detector with a cheap, coloured glass filters, the non-absorbed green light will form an improved transmitted light image. Changes in sample colour can be quantified by transmitted light imaging. This has been documented in red onion epidermal cells where changes in vacuolar pH triggered by the weak base methylamine result in measurable colour changes in the vacuolar anthocyanin. Many plant cells contain visible levels of pigment. The transmitted light detector provides a useful tool for documenting and measuring changes in these pigments while maintaining registration with confocal imaging.

Fluorescence lifetime imaging with near-infrared dyes

NASA Astrophysics Data System (ADS)

Becker, Wolfgang; Shcheslavskiy, Vladislav

2013-02-01

Near-infrared (NIR) dyes are used as fluorescence markers in small-animal imaging and in diffuse optical tomography of the human brain. In these applications it is important to know whether the dyes bind to proteins or other tissue constituents, and whether their fluorescence lifetimes depend on the targets they are bound to. Unfortunately, neither the lasers nor the detectors of commonly used confocal and multiphoton laser scanning microscopes allow for excitation and detection of NIR fluorescence. We therefore upgraded existing confocal TCSPC FLIM systems with NIR lasers and NIR sensitive detectors. In multiphoton systems we used the Ti:Sa laser as a one-photon excitation source in combination with an NIR-sensitive detector in the confocal beam path. We tested a number of NIR dyes in biological tissue. Some of them showed clear lifetime changes depending on the tissue structures they are bound to. We therefore believe that NIR FLIM can deliver supplementary information on the tissue constitution and on local biochemical parameters.

Chromatic confocal microscopy for multi-depth imaging of epithelial tissue

PubMed Central

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

Modulated-alignment dual-axis (MAD) confocal microscopy for deep optical sectioning in tissues

PubMed Central

Leigh, Steven Y.; Chen, Ye; Liu, Jonathan T.C.

2014-01-01

A strategy is presented to enable optical-sectioning microscopy with improved contrast and imaging depth using low-power (0.5 - 1 mW) diode laser illumination. This technology combines the inherent strengths of focal-modulation microscopy and dual-axis confocal (DAC) microscopy for rejecting out-of-focus and multiply scattered background light in tissues. The DAC architecture is unique in that it utilizes an intersecting pair of illumination and collection beams to improve the spatial-filtering and optical-sectioning performance of confocal microscopy while focal modulation selectively ‘labels’ in-focus signals via amplitude modulation. Simulations indicate that modulating the spatial alignment of dual-axis beams at a frequency f generates signals from the focal volume of the microscope that are modulated at 2f with minimal modulation of background signals, thus providing nearly an order-of-magnitude improvement in optical-sectioning contrast compared to DAC microscopy alone. Experiments show that 2f lock-in detection enhances contrast and imaging depth within scattering phantoms and fresh tissues. PMID:24940534

Spatially-controlled illumination with rescan confocal microscopy enhances image quality, resolution and reduces photodamage

NASA Astrophysics Data System (ADS)

Krishnaswami, Venkataraman; De Luca, Giulia M. R.; Breedijk, Ronald M. P.; Van Noorden, Cornelis J. F.; Manders, Erik M. M.; Hoebe, Ron A.

2017-02-01

Fluorescence microscopy is an important tool in biomedical imaging. An inherent trade-off lies between image quality and photodamage. Recently, we have introduced rescan confocal microscopy (RCM) that improves the lateral resolution of a confocal microscope down to 170 nm. Previously, we have demonstrated that with controlled-light exposure microscopy, spatial control of illumination reduces photodamage without compromising image quality. Here, we show that the combination of these two techniques leads to high resolution imaging with reduced photodamage without compromising image quality. Implementation of spatially-controlled illumination was carried out in RCM using a line scanning-based approach. Illumination is spatially-controlled for every line during imaging with the help of a prediction algorithm that estimates the spatial profile of the fluorescent specimen. The estimation is based on the information available from previously acquired line images. As a proof-of-principle, we show images of N1E-115 neuroblastoma cells, obtained by this new setup with reduced illumination dose, improved resolution and without compromising image quality.

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

PubMed

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.

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

NASA Astrophysics Data System (ADS)

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, the results were comparable between the two modalities. Additionally, a method of achieving blood flow maps at different depth using a combination of HiLo and LSI imaging is also discussed. The significance of this combined technique could help categorize blood flow to particular depths; this can help improve outcomes of medical treatments such pulse dye laser and photodynamic therapy treatments.

A versatile optical microscope for time-dependent single-molecule and single-particle spectroscopy

NASA Astrophysics Data System (ADS)

Li, Hao; Yang, Haw

2018-03-01

This work reports the design and implementation of a multi-function optical microscope for time-dependent spectroscopy on single molecules and single nanoparticles. It integrates the now-routine single-object measurements into one standalone platform so that no reconfiguration is needed when switching between different types of sample or spectroscopy modes. The illumination modes include evanescent field through total internal reflection, dark-field illumination, and epi-excitation onto a diffraction-limited spot suitable for confocal detection. The detection modes include spectrally resolved line imaging, wide-field imaging with dual-color capability, and two-color single-element photon-counting detection. The switch between different spectroscopy and data acquisition modes is fully automated and executed through computer programming. The capability of this microscope is demonstrated through selected proof-of-principle experiments.

Microscopic observations of self-healing products in calcareous fly ash mortars.

PubMed

Jóźwiak-Niedźwiedzka, Daria

2015-01-01

The results of microstructural characterization of mortars containing fly ash class C (High Calcium Fly Ash) from combustion of lignite are presented. The evaluation of the microstructure was performed using scanning electron microscope, optical, and confocal microscope. The tested beams were bent till the crack and microcracks opening, which were healed during the different curing time. The results showed that the replacement of cement with fly ash class C influenced the process of crack healing. The addition of HCFA, at both 30% and 60%, speeds up the self-healing process in cracks and particularly in micro-cracks. In the research, the completely filling up of the cracks by new phases has not been observed, only the beginning of such process has been noticed. © 2014 Wiley Periodicals, Inc.

A versatile optical microscope for time-dependent single-molecule and single-particle spectroscopy.

PubMed

Li, Hao; Yang, Haw

2018-03-28

This work reports the design and implementation of a multi-function optical microscope for time-dependent spectroscopy on single molecules and single nanoparticles. It integrates the now-routine single-object measurements into one standalone platform so that no reconfiguration is needed when switching between different types of sample or spectroscopy modes. The illumination modes include evanescent field through total internal reflection, dark-field illumination, and epi-excitation onto a diffraction-limited spot suitable for confocal detection. The detection modes include spectrally resolved line imaging, wide-field imaging with dual-color capability, and two-color single-element photon-counting detection. The switch between different spectroscopy and data acquisition modes is fully automated and executed through computer programming. The capability of this microscope is demonstrated through selected proof-of-principle experiments.

Video-mosaicking of in vivo reflectance confocal microscopy images for noninvasive examination of skin lesion (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kose, Kivanc; Gou, Mengran; Yelamos, Oriol; Cordova, Miguel A.; Rossi, Anthony; Nehal, Kishwer S.; Camps, Octavia I.; Dy, Jennifer G.; Brooks, Dana H.; Rajadhyaksha, Milind

2017-02-01

In this report we describe a computer vision based pipeline to convert in-vivo reflectance confocal microscopy (RCM) videos collected with a handheld system into large field of view (FOV) mosaics. For many applications such as imaging of hard to access lesions, intraoperative assessment of MOHS margins, or delineation of lesion margins beyond clinical borders, raster scan based mosaicing techniques have clinically significant limitations. In such cases, clinicians often capture RCM videos by freely moving a handheld microscope over the area of interest, but the resulting videos lose large-scale spatial relationships. Videomosaicking is a standard computational imaging technique to register, and stitch together consecutive frames of videos into large FOV high resolution mosaics. However, mosaicing RCM videos collected in-vivo has unique challenges: (i) tissue may deform or warp due to physical contact with the microscope objective lens, (ii) discontinuities or "jumps" between consecutive images and motion blur artifacts may occur, due to manual operation of the microscope, and (iii) optical sectioning and resolution may vary between consecutive images due to scattering and aberrations induced by changes in imaging depth and tissue morphology. We addressed these challenges by adapting or developing new algorithmic methods for videomosaicking, specifically by modeling non-rigid deformations, followed by automatically detecting discontinuities (cut locations) and, finally, applying a data-driven image stitching approach that fully preserves resolution and tissue morphologic detail without imposing arbitrary pre-defined boundaries. We will present example mosaics obtained by clinical imaging of both melanoma and non-melanoma skin cancers. The ability to combine freehand mosaicing for handheld microscopes with preserved cellular resolution will have high impact application in diverse clinical settings, including low-resource healthcare systems.

Hyper-dependence of breast cancer cell types on the nuclear transporter Importin β1.

PubMed

Kuusisto, Henna V; Jans, David A

2015-08-01

We previously reported that overexpression of members of the Importin (Imp) superfamily of nuclear transporters results in increased nuclear trafficking through conventional transport pathways in tumour cells. Here we show for the first time that the extent of overexpression of Impβ1 correlates with disease state in the MCF10 human breast tumour progression system. Excitingly, we find that targeting Impβ1 activity through siRNA is >30 times more efficient in decreasing the viability of malignant ductal carcinoma cells compared to isogenic non-transformed counterparts, and is highly potent and tumour selective at subnanomolar concentrations. Tumour cell selectivity of the siRNA effects was unique to Impβ1 and not other Imps, with flow cytometric analysis showing >60% increased cell death compared to controls concomitant with reduced nuclear import efficiency as indicated by confocal microscopic analysis. This hypersensitivity of malignant cell types to Impβ1 knockdown raises the exciting possibility of anti-cancer therapies targeted at Impβ1. Copyright © 2015 Elsevier B.V. All rights reserved.

Resonant Scanning with Large Field of View Reduces Photobleaching and Enhances Fluorescence Yield in STED Microscopy.

PubMed

Wu, Yong; Wu, Xundong; Lu, Rong; Zhang, Jin; Toro, Ligia; Stefani, Enrico

2015-10-01

Photobleaching is a major limitation of superresolution Stimulated Depletion Emission (STED) microscopy. Fast scanning has long been considered an effective means to reduce photobleaching in fluorescence microscopy, but a careful quantitative study of this issue is missing. In this paper, we show that the photobleaching rate in STED microscopy can be slowed down and the fluorescence yield be enhanced by scanning with high speed, enabled by using large field of view in a custom-built resonant-scanning STED microscope. The effect of scanning speed on photobleaching and fluorescence yield is more remarkable at higher levels of depletion laser irradiance, and virtually disappears in conventional confocal microscopy. With ≥6 GW∙cm(-2) depletion irradiance, we were able to extend the fluorophore survival time of Atto 647N and Abberior STAR 635P by ~80% with 8-fold wider field of view. We confirm that STED Photobleaching is primarily caused by the depletion light acting upon the excited fluorophores. Experimental data agree with a theoretical model. Our results encourage further increasing the linear scanning speed for photobleaching reduction in STED microscopy.

Super-Resolution Scanning Laser Microscopy Based on Virtually Structured Detection

PubMed Central

Zhi, Yanan; Wang, Benquan; Yao, Xincheng

2016-01-01

Light microscopy plays a key role in biological studies and medical diagnosis. The spatial resolution of conventional optical microscopes is limited to approximately half the wavelength of the illumination light as a result of the diffraction limit. Several approaches—including confocal microscopy, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy, and structured illumination microscopy—have been established to achieve super-resolution imaging. However, none of these methods is suitable for the super-resolution ophthalmoscopy of retinal structures because of laser safety issues and inevitable eye movements. We recently experimentally validated virtually structured detection (VSD) as an alternative strategy to extend the diffraction limit. Without the complexity of structured illumination, VSD provides an easy, low-cost, and phase artifact–free strategy to achieve super-resolution in scanning laser microscopy. In this article we summarize the basic principles of the VSD method, review our demonstrated single-point and line-scan super-resolution systems, and discuss both technical challenges and the potential of VSD-based instrumentation for super-resolution ophthalmoscopy of the retina. PMID:27480461

Inhomogeneity of Cellulose Microfibril Assembly in Plant Cell Walls Revealed with Sum Frequency Generation Microscopy.

PubMed

Huang, Shixin; Makarem, Mohamadamin; Kiemle, Sarah N; Hamedi, Hossein; Sau, Moujhuri; Cosgrove, Daniel J; Kim, Seong H

2018-05-17

Sum frequency generation (SFG) vibrational spectroscopy can selectively detect and analyze noncentrosymmetric components interspersed in amorphous matrices; this principle has been used for studies of nanoscale structure and mesoscale assembly of cellulose in plant cell walls. However, the spectral information averaged over a large area or volume cannot provide regiospecific or tissue-specific information of different cells in plants. This study demonstrates spatially resolved SFG analysis and imaging by combining a broad-band SFG spectroscopy system with an optical microscope. The system was designed to irradiate both narrow-band 800 nm and broad-band tunable IR beams through a single reflective objective lens, but from opposite sides of the surface normal direction of the sample. The developed technique was used to reveal inhomogeneous distributions of cellulose microfibrils within single cell walls, such as cotton fibers and onion epidermis as well as among different tissues in Arabidopsis inflorescence stems and bamboo culms. SFG microscopy can be used for vibrational spectroscopic imaging of other biological systems in complement to conventional Fourier transform infrared spectroscopy and confocal Raman microscopy.

Laser-Based Surface Modification of Microstructure for Carbon Fiber-Reinforced Plastics

NASA Astrophysics Data System (ADS)

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.

'En face' ex vivo reflectance confocal microscopy to help the surgery of basal cell carcinoma of the eyelid.

PubMed

Espinasse, Marine; Cinotti, Elisa; Grivet, Damien; Labeille, Bruno; Prade, Virginie; Douchet, Catherine; Cambazard, Frédéric; Thuret, Gilles; Gain, Philippe; Perrot, Jean Luc

2017-07-01

Ex vivo confocal microscopy is a recent imaging technique for the perioperative control of skin tumour margins. Up to date, it has been used in the fluorescence mode and with vertical sections of the specimen margins. The aim of this study was to evaluate its use in the reflectance mode and with a horizontal ('en face') scanning of the surgical specimen in a series of basal cell carcinoma of the eyelid. Prospective consecutive cohort study was performed at the University Hospital of Saint-Etienne, France. Forty-one patients with 42 basal cell carcinoma of the eyelid participated in this study. Basal cell carcinomas were excised with a 2-mm-wide clinically safe margin. The surgical specimens were analysed under ex vivo confocal microscopy in the reflectance mode and with an en face scanning in order to control at a microscopic level if the margins were free from tumour invasion. Histopathogical examination was later performed in order to compare the results. Sensitivity and specificity of ex vivo confocal microscopy for the presence of tumour-free margins. Ex vivo confocal microscopy results were consistent with histopathology in all cases (tumour-free margins in 40 out of 42 samples; sensitivity and specificity of 100%). Ex vivo confocal microscopy in the reflectance mode with an 'en face' scanning can control tumour margins of eyelid basal cell carcinomas and optimize their surgical management. This procedure has the advantage on the fluorescent mode of not needing any contrast agent to examine the samples. © 2016 Royal Australian and New Zealand College of Ophthalmologists.

ConfocalVR: Immersive Visualization Applied to Confocal Microscopy.

PubMed

Stefani, Caroline; Lacy-Hulbert, Adam; Skillman, Thomas

2018-06-24

ConfocalVR is a virtual reality (VR) application created to improve the ability of researchers to study the complexity of cell architecture. Confocal microscopes take pictures of fluorescently labeled proteins or molecules at different focal planes to create a stack of 2D images throughout the specimen. Current software applications reconstruct the 3D image and render it as a 2D projection onto a computer screen where users need to rotate the image to expose the full 3D structure. This process is mentally taxing, breaks down if you stop the rotation, and does not take advantage of the eye's full field of view. ConfocalVR exploits consumer-grade virtual reality (VR) systems to fully immerse the user in the 3D cellular image. In this virtual environment the user can: 1) adjust image viewing parameters without leaving the virtual space, 2) reach out and grab the image to quickly rotate and scale the image to focus on key features, and 3) interact with other users in a shared virtual space enabling real-time collaborative exploration and discussion. We found that immersive VR technology allows the user to rapidly understand cellular architecture and protein or molecule distribution. We note that it is impossible to understand the value of immersive visualization without experiencing it first hand, so we encourage readers to get access to a VR system, download this software, and evaluate it for yourself. The ConfocalVR software is available for download at http://www.confocalvr.com, and is free for nonprofits. Copyright © 2018. Published by Elsevier Ltd.

Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains.

PubMed

Müllenbroich, M Caroline; Silvestri, Ludovico; Onofri, Leonardo; Costantini, Irene; Hoff, Marcel Van't; Sacconi, Leonardo; Iannello, Giulio; Pavone, Francesco S

2015-10-01

Comprehensive mapping and quantification of neuronal projections in the central nervous system requires high-throughput imaging of large volumes with microscopic resolution. To this end, we have developed a confocal light-sheet microscope that has been optimized for three-dimensional (3-D) imaging of structurally intact clarified whole-mount mouse brains. We describe the optical and electromechanical arrangement of the microscope and give details on the organization of the microscope management software. The software orchestrates all components of the microscope, coordinates critical timing and synchronization, and has been written in a versatile and modular structure using the LabVIEW language. It can easily be adapted and integrated to other microscope systems and has been made freely available to the light-sheet community. The tremendous amount of data routinely generated by light-sheet microscopy further requires novel strategies for data handling and storage. To complete the full imaging pipeline of our high-throughput microscope, we further elaborate on big data management from streaming of raw images up to stitching of 3-D datasets. The mesoscale neuroanatomy imaged at micron-scale resolution in those datasets allows characterization and quantification of neuronal projections in unsectioned mouse brains.

Three-dimensional molecular mapping of a multiple emulsion by means of CARS microscopy.

PubMed

Meyer, Tobias; Akimov, Denis; Tarcea, Nicolae; Chatzipapadopoulos, Susana; Muschiolik, Gerald; Kobow, Jens; Schmitt, Michael; Popp, Jürgen

2008-02-07

Multiple emulsions consisting of water droplets dispersed in an oil phase containing emulsifier which is emulsified in an outer water phase (W/O/W) are of great interest in pharmacology for developing new drugs, in the nutrition sciences for designing functional food, and in biology as model systems for cell organelles such as liposomes. In the food industry multiple emulsions with high sugar content in the aqueous phase can be used for the production of sweets, because the high sugar content prevents deterioration. However, for these emulsions the refractive indexes of oil and aqueous phase are very similar. This seriously impedes the analysis of these emulsions, e.g., for process monitoring, because microscopic techniques based on transmission or reflection do not provide sufficient contrast. We have characterized the inner dispersed phase of concentrated W/O/W emulsions with the same refractive index of the three phases by micro Raman spectroscopy and investigated the composition and molecular distribution in water-oil-water emulsions by means of three-dimensional laser scanning CARS (coherent anti-Stokes Raman scattering) microscopy. CARS microscopy has been used to study water droplets dispersed in oil droplets at different Raman resonances to visualize different molecular species. Water droplets with a diameter of about 700 nm could clearly be visualized. The advantages of CARS microscopy for studying this particular system are emphasized by comparing this microscopic technique with conventional confocal reflection and transmission microscopies.

Multimodal microscopy and the stepwise multi-photon activation fluorescence of melanin

NASA Astrophysics Data System (ADS)

Lai, Zhenhua

The author's work is divided into three aspects: multimodal microscopy, stepwise multi-photon activation fluorescence (SMPAF) of melanin, and customized-profile lenses (CPL) for on-axis laser scanners, which will be introduced respectively. A multimodal microscope provides the ability to image samples with multiple modalities on the same stage, which incorporates the benefits of all modalities. The multimodal microscopes developed in this dissertation are the Keck 3D fusion multimodal microscope 2.0 (3DFM 2.0), upgraded from the old 3DFM with improved performance and flexibility, and the multimodal microscope for targeting small particles (the "Target" system). The control systems developed for both microscopes are low-cost and easy-to-build, with all components off-the-shelf. The control system have not only significantly decreased the complexity and size of the microscope, but also increased the pixel resolution and flexibility. The SMPAF of melanin, activated by a continuous-wave (CW) mode near-infrared (NIR) laser, has potential applications for a low-cost and reliable method of detecting melanin. The photophysics of melanin SMPAF has been studied by theoretical analysis of the excitation process and investigation of the spectra, activation threshold, and photon number absorption of melanin SMPAF. SMPAF images of melanin in mouse hair and skin, mouse melanoma, and human black and white hairs are compared with images taken by conventional multi-photon fluorescence microscopy (MPFM) and confocal reflectance microscopy (CRM). SMPAF images significantly increase specificity and demonstrate the potential to increase sensitivity for melanin detection compared to MPFM images and CRM images. Employing melanin SMPAF imaging to detect melanin inside human skin in vivo has been demonstrated, which proves the effectiveness of melanin detection using SMPAF for medical purposes. Selective melanin ablation with micrometer resolution has been presented using the Target system. Compared to the traditional selective photothermolysis, this method demonstrates higher precision, higher specificity and deeper penetration. Therefore, the SMPAF guided selective ablation of melanin is a promising tool of removing melanin for both medical and cosmetic purposes. Three CPLs have been designed for low-cost linear-motion scanners, low-cost fast spinning scanners and high-precision fast spinning scanners. Each design has been tailored to the industrial manufacturing ability and market demands.

Pseudomonas aeruginosa Infectious Keratitis in a High Oxygen Transmissible Rigid Contact Lens Rabbit Model

PubMed Central

Wei, Cynthia; Zhu, Meifang; Petroll, W. Matthew; Robertson, Danielle M.

2014-01-01

Purpose. To establish a rabbit model of infectious Pseudomonas aeruginosa keratitis using ultrahigh oxygen transmissible rigid lenses and characterize the frequency and severity of infection when compared to a non–oxygen transmissible lens material. Methods. Rabbits were fit with rigid lenses composed of ultrahigh and non–oxygen transmissible materials. Prior to wear, lenses were inoculated with an invasive corneal isolate of P. aeruginosa stably conjugated to green fluorescent protein (GFP). Corneas were examined before and after lens wear using a modified Heidelberg Rostock Tomograph in vivo confocal microscope. Viable bacteria adherent to unworn and worn lenses were assessed by standard plate counts. The presence of P. aeruginosa-GFP and myeloperoxidase-labeled neutrophils in infected corneal tissue was evaluated using laser scanning confocal microscopy. Results. The frequency and severity of infectious keratitis was significantly greater with inoculated ultrahigh oxygen transmissible lenses. Infection severity was associated with increasing neutrophil infiltration and in severe cases, corneal melting. In vivo confocal microscopic analysis of control corneas following lens wear confirmed that hypoxic lens wear was associated with mechanical surface damage, whereas no ocular surface damage was evident in the high-oxygen lens group. Conclusions. These data indicate that in the absence of adequate tear clearance, the presence of P. aeruginosa trapped under the lens overrides the protective effects of oxygen on surface epithelial cells. These findings also suggest that alternative pathophysiological mechanisms exist whereby changes under the lens in the absence of frank hypoxic damage result in P. aeruginosa infection in the otherwise healthy corneal epithelium. PMID:25125601

Three-dimensional confocal microscopy of the living cornea and ocular lens

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1991-07-01

The three-dimensional reconstruction of the optic zone of the cornea and the ocular crystalline lens has been accomplished using confocal microscopy and volume rendering computer techniques. A laser scanning confocal microscope was used in the reflected light mode to obtain the two-dimensional images from the cornea and the ocular lens of a freshly enucleated rabbit eye. The light source was an argon ion laser with a 488 nm wavelength. The microscope objective was a Leitz X25, NA 0.6 water immersion lens. The 400 micron thick cornea was optically sectioned into 133 three micron sections. The semi-transparent cornea and the in-situ ocular lens was visualized as high resolution, high contrast two-dimensional images. The structures observed in the cornea include: superficial epithelial cells and their nuclei, basal epithelial cells and their 'beaded' cell borders, basal lamina, nerve plexus, nerve fibers, nuclei of stromal keratocytes, and endothelial cells. The structures observed in the in- situ ocular lens include: lens capsule, lens epithelial cells, and individual lens fibers. The three-dimensional data sets of the cornea and the ocular lens were reconstructed in the computer using volume rendering techniques. Stereo pairs were also created of the two- dimensional ocular images for visualization. The stack of two-dimensional images was reconstructed into a three-dimensional object using volume rendering techniques. This demonstration of the three-dimensional visualization of the intact, enucleated eye provides an important step toward quantitative three-dimensional morphometry of the eye. The important aspects of three-dimensional reconstruction are discussed.

Detection of endolithic spatial distribution in marble stone.

PubMed

Casanova Municchia, A; Percario, Z; Caneva, G

2014-10-01

The penetration of endolithic microorganisms, which develop to depths of several millimetres or even centimetres into the stone, and the diffusion of their extracellular substances speeds up the stone deterioration process. The aim of this study was to investigate, using a confocal laser scanning microscopy with a double-staining, a marble rock sample by observing the endolithic spatial distribution and quantifying the volume they occupied within the stone, in order to understand the real impact of these microorganisms on the conservation of stone monuments. Often the only factors taken into account by biodeterioration studies regarding endolithic microorganisms, are spread and depth of penetration. Despite the knowledge of three-dimensional spatial distribution and quantification of volume, it is indispensable to understand the real damage caused by endolithic microorganisms to stone monuments. In this work, we analyze a marble rock sample using a confocal laser scanning microscopy stained with propidium iodide and Concavalin-A conjugate with the fluorophore Alexa Fluor 488, comparing these results with other techniques (SEM microscope, microphotographs of polished cross-sections and thin-section, PAS staining methods), An image analysis approach has also been applied. The use of confocal laser scanning microscopy with double staining shows clear evidence of the presence of endolithic microorganisms (cyanobacteria and fungi) as well as the extracellular polymeric substance matrix in a three-dimensional architecture as part of the rock sample, this technique, therefore, seems very useful when applied to restoration interventions on stone monuments when endolithic growth is suspected. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

Improving Axial Resolution in Confocal Microscopy with New High Refractive Index Mounting Media

PubMed Central

Fouquet, Coralie; Gilles, Jean-François; Heck, Nicolas; Dos Santos, Marc; Schwartzmann, Richard; Cannaya, Vidjeacoumary; Morel, Marie-Pierre; Davidson, Robert Stephen; Trembleau, Alain; Bolte, Susanne

2015-01-01

Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required. PMID:25822785

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

PubMed

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 < 0.05) when compared to the control group. Various qualitative changes were noted, such as cell degeneration, micronuclei, binucleation, intracytoplasmic 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.

A comparison of caveolae and caveolin-1 to folate receptor α in retina and retinal pigment epithelium

PubMed Central

Bridges, Christy C.; El-Sherbeny, Amira; Roon, Penny; Ola, M. Shamsul; Kekuda, Ramesh; Ganapathy, Vadivel; Cameron, Richard S.; Cameron, Patricia L.

2015-01-01

Summary Caveolae are flask-shaped membrane invaginations present in most mammalian cells. They are distinguished by the presence of a striated coat composed of the protein, caveolin. Caveolae have been implicated in numerous cellular processes, including potocytosis in which caveolae are hypothesized to co-localize with folate receptor α and participate in folate uptake. Our laboratory has recently localized folate receptor α to the basolateral surface of the retinal pigment epithelium (RPE). It is present also in many other cells of the retina. In the present study, we asked whether caveolae were present in the RPE, and if so, whether their pattern of distribution was similar to folate receptor α. We also examined the distribution pattern of caveolin-1, which can be a marker of caveolae. Extensive electron microscopical analysis revealed caveolae associated with endothelial cells. However, none were detected in intact or cultured RPE. Laser scanning confocal microscopical analysis of intact RPE localized caveolin-1 to the apical and basal surfaces, a distribution unlike folate receptor α. Western analysis confirmed the presence of caveolin-1 in cultured RPE cells and laser scanning confocal microscopy localized the protein to the basal plasma membrane of the RPE, a distribution like that of folate receptor α. This distribution was confirmed by electron microscopic immunolocalization. The lack of caveolae in the RPE suggests that these structures may not be essential for folate internalization in the RPE. PMID:11508338

Application of reflectance confocal microscopy to evaluate skin damage after irradiation with an yttrium-scandium-gallium-garnet (YSGG) laser.

PubMed

Yue, Xueping; Wang, Hongwei; Li, Qing; Li, Linfeng

2017-02-01

The objective of this study was to observe the characteristics of the skin after irradiation with a 2790-nm yttrium-scandium-gallium-garnet (YSGG) laser using reflectance confocal microscopy (RCM). A 2790-nm YSGG laser was used to irradiate fresh foreskin (four doses, at spot density 3) in vitro. The characteristics of microscopic ablative columns (MAC), thermal coagulation zone (TCZ), and microscopic treatment zones (MTZ) were observed immediately after irradiation using digital microscope and RCM. The characteristics of MAC, TCZ, and MTZ with variations in pulse energy were comparatively analyzed. After irradiation, MAC, TCZ, and MTZ characteristics and undamaged skin between MTZs can be observed by RCM. The depth and width of MTZ obviously increased with the increase in pulse energy. At 80, 120, and 160 mJ/microbeam (MB), the MTZ actual area and proportion were about two times that of the theoretical value and three times at 200 mJ/MB. With increases in depth, the single MAC gradually decreased in a fingertip-shaped model, with TCZ slowly increasing, and MTZ slightly decreasing in a columnar shape. RCM was able to determine the characteristics of thermal injury on the skin after the 2790-nm YSGG laser irradiation with different pulse energies. Pulse energy higher than 200 mJ/MB may have much larger thermal injury and side effect. RCM could be used in the clinic in future.

A comparison of two micro-beam X-ray emission techniques for actinide elemental distribution in microscopic particles originating from the hydrogen bombs involved in the Palomares (Spain) and Thule (Greenland) accidents

NASA Astrophysics Data System (ADS)

Jimenez-Ramos, M. C.; Eriksson, M.; García-López, J.; Ranebo, Y.; García-Tenorio, R.; Betti, M.; Holm, E.

2010-09-01

In order to validate and to gain confidence in two micro-beam techniques: particle induced X-ray emission with nuclear microprobe technique (μ-PIXE) and synchrotron radiation induced X-ray fluorescence in a confocal alignment (confocal SR μ-XRF) for characterization of microscopic particles containing actinide elements (mixed plutonium and uranium) a comparative study has been performed. Inter-comparison of the two techniques is essential as the X-ray production cross-sections for U and Pu are different for protons and photons and not well defined in the open literature, especially for Pu. The particles studied consisted of nuclear weapons material, and originate either in the so called Palomares accident in Spain, 1966 or in the Thule accident in Greenland, 1968. In the determination of the average Pu/U mass ratios (not corrected by self-absorption) in the analysed microscopic particles the results from both techniques show a very good agreement. In addition, the suitability of both techniques for the analysis with good resolution (down to a few μm) of the Pu/U distribution within the particles has been proved. The set of results obtained through both techniques has allowed gaining important information concerning the characterization of the remaining fissile material in the areas affected by the aircraft accidents. This type of information is essential for long-term impact assessments of contaminated sites.

Revealing organization of cellulose in wood cell walls by Raman imaging

Treesearch

Umesh P. Agarwal; Sally A. Ralph

2007-01-01

Anisotropy of cellulose organization in mature black spruce wood cell wall was investigated by Raman imaging using a 1 [mu]m lateral-resolution capable confocal Raman microscope. In these studies, wood cross sections (CS) and radial longitudinal sections (LS) that were partially delignified by acid chlorite treatment were used. In the case of CS where latewood cells...

Axial tomography in 3D live cell microscopy

NASA Astrophysics Data System (ADS)

Richter, Verena; Bruns, Sarah; Bruns, Thomas; Piper, Mathis; Weber, Petra; Wagner, Michael; Cremer, Christoph; Schneckenburger, Herbert

2017-07-01

A miniaturized setup for sample rotation on a microscope stage has been developed, combined with light sheet, confocal or structured illumination microscopy and applied to living cells as well as to small organisms. This setup permits axial tomography with improved visualization of single cells or small cell clusters as well as an enhanced effective 3D resolution upon sample rotation.

In Situ Observations of Agglomeration of Non-metallic Inclusions at Steel/Ar and Steel/Slag Interfaces by High-Temperature Confocal Laser Scanning Microscope: A Review

NASA Astrophysics Data System (ADS)

Mu, Wangzhong; Dogan, Neslihan; Coley, Kenneth S.

2018-05-01

The agglomeration behavior of non-metallic inclusions in the steelmaking process is important for controlling the cleanliness of the steel. In this work, the observation of agglomeration behaviors of inclusions at steel/Ar and steel/slag interfaces using a high-temperature confocal laser scanning microscope (HT-CLSM) is summarized. This HT-CLSM technique has been applied to observe phase transformation during solidification and heat treatment and the engulfment and pushing behavior of inclusions in front of the solidified interface. In the current work, the inclusion agglomeration behavior at steel/Ar and steel/slag interfaces is summarized and discussed. Subsequently, the development of the theoretical work investigating inclusion agglomeration at steel/Ar and steel/slag interfaces including the initial capillary force model and Kralchevsky-Paunov model is described. Finally, the Kralchevsky-Paunov model is applied to investigating nitride inclusion agglomeration at high-manganese steel/Ar interfaces. This work aims to give a critical review of the application of HT-CLSM in secondary refining as well as a better control of inclusion elimination for clean steel production.

Confocal laser scanning microscopy in study of bone calcification

NASA Astrophysics Data System (ADS)

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.

Crystallization Behavior of the CaO-Al2O3-MgO System Studied with a Confocal Laser Scanning Microscope

NASA Astrophysics Data System (ADS)

Jung, Sung Suk; Sohn, Il

2012-12-01

The crystallization behavior of a calcium-aluminate system with various MgO content from 2.5 to 7.5 wt pct and CaO/Al2O3 ratios between 0.8 and 1.2 has been examined using a confocal laser scanning microscope (CLSM). CCT (continuous cooling transformation) and time temperature transformation (TTT) diagrams were constructed to identify the primary crystal phase of slag at different compositions and at cooling rates between 25 and 800 K/minutes. In the slag at a CaO/Al2O3 ratio of 1.0, crystallization temperature increased during isothermal and continuous cooling with higher MgO content, and the shortest incubation time was observed at 5 wt pct MgO. When MgO content was fixed to be 5 wt pct, crystallization temperature increased with lower CaO/Al2O3 ratio. According to the slag composition, cooling rates and temperature, the primary phase could be CA, or C5A3, or C3A, or C3MA2, or MgO, and the crystal morphology changes from dendrites to faceted crystals to columnar crystals in this composition range.

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

NASA Astrophysics Data System (ADS)

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.

Evanescent-Wave Filtering in Images Using Remote Terahertz Structured Illumination

NASA Astrophysics Data System (ADS)

Flammini, M.; Pontecorvo, E.; Giliberti, V.; Rizza, C.; Ciattoni, A.; Ortolani, M.; DelRe, E.

2017-11-01

Imaging with structured illumination allows for the retrieval of subwavelength features of an object by conversion of evanescent waves into propagating waves. In conditions in which the object plane and the structured-illumination plane do not coincide, this conversion process is subject to progressive filtering of the components with high spatial frequency when the distance between the two planes increases, until the diffraction-limited lateral resolution is restored when the distance exceeds the extension of evanescent waves. We study the progressive filtering of evanescent waves by developing a remote super-resolution terahertz imaging system operating at a wavelength λ =1.00 mm , based on a freestanding knife edge and a reflective confocal terahertz microscope. In the images recorded with increasing knife-edge-to-object-plane distance, we observe the transition from a super-resolution of λ /17 ≃60 μ m to the diffraction-limited lateral resolution of Δ x ≃λ expected for our confocal microscope. The extreme nonparaxial conditions are analyzed in detail, exploiting the fact that, in the terahertz frequency range, the knife edge can be positioned at a variable subwavelength distance from the object plane. Electromagnetic simulations of radiation scattering by the knife edge reproduce the experimental super-resolution achieved.

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

NASA Astrophysics Data System (ADS)

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.

Isolation and Structural Studies of Mitochondria from Pea Roots.

PubMed

Vishwakarma, Abhaypratap; Gupta, Kapuganti Jagadis

2017-01-01

For structural and respiratory studies, isolation of intact and active mitochondria is essential. Here, we describe an isolation method which gave good yield and intact mitochondria from 2-week-old pea (Pisum sativum) roots grown hydroponically under standard growth conditions. We used Percoll gradient centrifugation for this isolation procedure. The yield of purified mitochondria was 50 μg/g FW. Isolated mitochondria maintained their structure which was observed by using MitoTracker green in confocal microscope and scanning electron microscopy (SEM). Intact mitochondria are clearly visible in SCM images. Taken together this isolation method can be used for physiological and microscopic studies on mitochondria.

Microscopic Approach to the Nonlinear Elasticity of Compressed Emulsions

NASA Astrophysics Data System (ADS)

Jorjadze, Ivane; Pontani, Lea-Laetitia; Brujic, Jasna

2013-01-01

Using confocal microscopy, we measure the packing geometry and interdroplet forces as a function of the osmotic pressure in a 3D emulsion system. We assume a harmonic interaction potential over a wide range of volume fractions and attribute the observed nonlinear elastic response of the pressure with density to the first corrections to the scaling laws of the microstructure away from the critical point. The bulk modulus depends on the excess contacts created under compression, which leads to the correction exponent α=1.5. Microscopically, the nonlinearities manifest themselves as a narrowing of the distribution of the pressure per particle as a function of the global pressure.

SERS substrate based on silver nanoparticles and graphene: Dependence on the layer number of graphene

NASA Astrophysics Data System (ADS)

Garg, Preeti; Soni, R. K.; Raman, R.

2018-05-01

In this report, we describe a low-cost fabrication process for highly sensitive SERS substrate by using thermal evaporation technique. The SERS substrate structure consists of silver nanoparticles deposited on monolayer, bilayer and few layer graphene. The fabricated SERS substrates are investigated by field emission scanning electron microscope (FE-SEM), atomic force microscope (AFM), and confocal Raman spectroscope. From the surface morphology we have verified that the fabricated SERS substrate consist of high-density of silver nanoparticles with their size distribution varies from 10 to 150 nm. The surface-enhanced Raman scattering activities of these nanostructures is highest for monolayer graphene.

Evaluation of a completely robotized neurosurgical operating microscope.

PubMed

Kantelhardt, Sven R; Finke, Markus; Schweikard, Achim; Giese, Alf

2013-01-01

Operating microscopes are essential for most neurosurgical procedures. Modern robot-assisted controls offer new possibilities, combining the advantages of conventional and automated systems. We evaluated the prototype of a completely robotized operating microscope with an integrated optical coherence tomography module. A standard operating microscope was fitted with motors and control instruments, with the manual control mode and balance preserved. In the robot mode, the microscope was steered by a remote control that could be fixed to a surgical instrument. External encoders and accelerometers tracked microscope movements. The microscope was additionally fitted with an optical coherence tomography-scanning module. The robotized microscope was tested on model systems. It could be freely positioned, without forcing the surgeon to take the hands from the instruments or avert the eyes from the oculars. Positioning error was about 1 mm, and vibration faded in 1 second. Tracking of microscope movements, combined with an autofocus function, allowed determination of the focus position within the 3-dimensional space. This constituted a second loop of navigation independent from conventional infrared reflector-based techniques. In the robot mode, automated optical coherence tomography scanning of large surface areas was feasible. The prototype of a robotized optical coherence tomography-integrated operating microscope combines the advantages of a conventional manually controlled operating microscope with a remote-controlled positioning aid and a self-navigating microscope system that performs automated positioning tasks such as surface scans. This demonstrates that, in the future, operating microscopes may be used to acquire intraoperative spatial data, volume changes, and structural data of brain or brain tumor tissue.

Reflectance confocal microscopy and features of melanocytic lesions: an internet-based study of the reproducibility of terminology.

PubMed

Pellacani, Giovanni; Vinceti, Marco; Bassoli, Sara; Braun, Ralph; Gonzalez, Salvador; Guitera, Pascale; Longo, Caterina; Marghoob, Ashfaq A; Menzies, Scott W; Puig, Susana; Scope, Alon; Seidenari, Stefania; Malvehy, Josep

2009-10-01

To test the interobserver and intraobserver reproducibility of the standard terminology for description and diagnosis of melanocytic lesions in in vivo confocal microscopy. A dedicated Web platform was developed to train the participants and to allow independent distant evaluations of confocal images via the Internet. Department of Dermatology, University of Modena and Reggio Emilia, Modena, Italy. The study population was composed of 15 melanomas, 30 nevi, and 5 Spitz/Reed nevi. Six expert centers were invited to participate at the study. Intervention Evaluation of 36 features in 345 confocal microscopic images from melanocytic lesions. Interobserved and intraobserved agreement, by calculating the Cohen kappa statistics measure for each descriptor. High overall levels of reproducibility were shown for most of the evaluated features. In both the training and test sets there was a parallel trend of decreasing kappa values as deeper anatomic skin levels were evaluated. All of the features, except 1, used for melanoma diagnosis, including roundish pagetoid cells, nonedged papillae, atypical cells in basal layer, cerebriform clusters, and nucleated cells infiltrating dermal papillae, showed high overall levels of reproducibility. However, less-than-ideal reproducibility was obtained for some descriptors, such as grainy appearance of the epidermis, junctional thickening, mild atypia in basal layer, plump bright cells, small bright cells, and reticulated fibers in the dermis. Conclusion The standard consensus confocal terminology useful for the evaluation of melanocytic lesions was reproducibly recognized by independent observers.

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

PubMed

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.

In vivo laser confocal microscopy findings in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy

PubMed Central

Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2012-01-01

Background: The purpose of this study was to investigate pathological changes of the corneal cell layer in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy by in vivo laser corneal confocal microscopy. Methods: Two patients were evaluated using a cornea-specific in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph 2 Rostock Cornea Module, HRT 2-RCM). The affected corneal areas of both patients were examined. Image analysis was performed to identify corneal epithelial and stromal deposits correlated with this dystrophy. Results: Variously shaped (linear, multilaminar, curvilinear, ring-shape, geographic) highly reflective materials were observed in the “map” area, mainly in the basal epithelial cell layer. In “fingerprint” lesions, multiple linear and curvilinear hyporeflective lines were observed. Additionally, in the affected corneas, infiltration of possible Langerhans cells and other inflammatory cells was observed as highly reflective Langerhans cell-like or dot images. Finally, needle-shaped materials were observed in one patient. Conclusion: HRT 2-RCM laser confocal microscopy is capable of identifying corneal microstructural changes related to map-dot-fingerprint corneal dystrophy in vivo. The technique may be useful in elucidating the pathogenesis and natural course of map-dot-fingerprint corneal dystrophy and other similar basement membrane abnormalities. PMID:22888214

The application of dermal papillary rings in dermatology by in vivo confocal laser scanning microscopy

NASA Astrophysics Data System (ADS)

Xiang, W. Z.; Xu, A. E.; Xu, J.; Bi, Z. G.; Shang, Y. B.; Ren, Q. S.

2010-08-01

Confocal laser scanning microscopy (CLSM) allows noninvasive visualization of human skin in vivo, without needing to fix or section the tissue. Melanocytes and pigmented keratinocytes at the level of the basal layer form bright dermal papillary rings which are readily amenable to identify in confocal images. Our purpose was to explore the role of dermal papillary rings in assessment of lesion location, the diagnosis, differential diagnosis of lesions and assessment of therapeutic efficacy by in vivo CLSM. Seventy-one patients were imaged with the VivaScope 1500 reflectance confocal microscope provided by Lucid, Inc. The results indicate that dermal papillary rings can assess the location of lesion; the application of dermal papillary rings can provide diagnostic support and differential diagnosis for vitiligo, nevus depigmentosus, tinea versicolor, halo nevus, common nevi, and assess the therapeutic efficacy of NBUVB phototherapy plus topical 0.1 percent tacrolimus ointment for vitiligo. In conclusion, our findings indicate that the dermal papillary rings play an important role in the assessment the location of lesion, diagnosis, differential diagnosis of lesions and assessment of therapeutic efficacy by in vivo CLSM. CLSM may be a promising tool for noninvasive examination in dermatology. However, larger studies are needed to expand the application of dermal papillary rings in dermatology.

Granulocytes of the red claw crayfish Cherax quadricarinatus can endocytose beads, E. coli and WSSV, but in different ways.

PubMed

Duan, Hu; Jin, Songjun; Zhang, Yan; Li, Fuhua; Xiang, Jianhai

2014-10-01

The hemocytes of the red claw crayfish Cherax quadricarinatus are classified by morphologic observation into the following types: hyalinocytes (H), semi-granulocytes (SG) and granulocytes (G). Density gradient centrifugation with Percoll was developed to separate these three subpopulations of hemocytes. Beads, Escherichia coli, and FITC labeling WSSV were used to investigate the characteristics of granulocytes by using scanning electron microscope, transmission electron microscope, and laser scan confocal microscope. Results showed that granulocytes could phagocytose beads and E. coli by endocytic pathways. WSSV could rely on caveolae-mediated endocytosis to mainly enter into granulocytes. These results could elucidate the mechanism of the innate immunity function of granulocytes, and it also showed the mechanism by which WSSV invaded granulocytes in the red claw crayfish. Copyright © 2014 Elsevier Ltd. All rights reserved.

Sample holder for axial rotation of specimens in 3D microscopy.

PubMed

Bruns, T; Schickinger, S; Schneckenburger, H

2015-10-01

In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three-dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three-dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three-dimensional specimens is essential. Fluorescence z-projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Intraoperative confocal microscopy in the visualization of 5-aminolevulinic acid fluorescence in low-grade gliomas.

PubMed

Sanai, Nader; Snyder, Laura A; Honea, Norissa J; Coons, Stephen W; Eschbacher, Jennifer M; Smith, Kris A; Spetzler, Robert F

2011-10-01

Greater extent of resection (EOR) for patients with low-grade glioma (LGG) corresponds with improved clinical outcome, yet remains a central challenge to the neurosurgical oncologist. Although 5-aminolevulinic acid (5-ALA)-induced tumor fluorescence is a strategy that can improve EOR in gliomas, only glioblastomas routinely fluoresce following 5-ALA administration. Intraoperative confocal microscopy adapts conventional confocal technology to a handheld probe that provides real-time fluorescent imaging at up to 1000× magnification. The authors report a combined approach in which intraoperative confocal microscopy is used to visualize 5-ALA tumor fluorescence in LGGs during the course of microsurgical resection. Following 5-ALA administration, patients with newly diagnosed LGG underwent microsurgical resection. Intraoperative confocal microscopy was conducted at the following points: 1) initial encounter with the tumor; 2) the midpoint of tumor resection; and 3) the presumed brain-tumor interface. Histopathological analysis of these sites correlated tumor infiltration with intraoperative cellular tumor fluorescence. Ten consecutive patients with WHO Grades I and II gliomas underwent microsurgical resection with 5-ALA and intraoperative confocal microscopy. Macroscopic tumor fluorescence was not evident in any patient. However, in each case, intraoperative confocal microscopy identified tumor fluorescence at a cellular level, a finding that corresponded to tumor infiltration on matched histological analyses. Intraoperative confocal microscopy can visualize cellular 5-ALA-induced tumor fluorescence within LGGs and at the brain-tumor interface. To assess the clinical value of 5-ALA for high-grade gliomas in conjunction with neuronavigation, and for LGGs in combination with intraoperative confocal microscopy and neuronavigation, a Phase IIIa randomized placebo-controlled trial (BALANCE) is underway at the authors' institution.

Surface imaging microscope

NASA Astrophysics Data System (ADS)

Rogala, Eric W.; Bankman, Isaac N.

2008-04-01

The three-dimensional shapes of microscopic objects are becoming increasingly important for battlespace CBRNE sensing. Potential applications of microscopic 3D shape observations include characterization of biological weapon particles and manufacturing of micromechanical components. Aerosol signatures of stand-off lidar systems, using elastic backscatter or polarization, are dictated by the aerosol particle shapes and sizes that must be well characterized in the lab. A low-cost, fast instrument for 3D surface shape microscopy will be a valuable point sensor for biological particle sensing applications. Both the cost and imaging durations of traditional techniques such as confocal microscopes, atomic force microscopes, and electron scanning microscopes are too high. We investigated the feasibility of a low-cost, fast interferometric technique for imaging the 3D surface shape of microscopic objects at frame rates limited only by the camera in the system. The system operates at two laser wavelengths producing two fringe images collected simultaneously by a digital camera, and a specialized algorithm we developed reconstructs the surface map of the microscopic object. The current implementation assembled to test the concept and develop the new 3D reconstruction algorithm has 0.25 micron resolution in the x and y directions, and about 0.1 micron accuracy in the z direction, as tested on a microscopic glass test object manufactured with etching techniques. We describe the interferometric instrument, present the reconstruction algorithm, and discuss further development.

Registration procedure for spatial correlation of physical energy deposition of particle irradiation and cellular response utilizing cell-fluorescent ion track hybrid detectors

NASA Astrophysics Data System (ADS)

Niklas, M.; Zimmermann, F.; Schlegel, J.; Schwager, C.; Debus, J.; Jäkel, O.; Abdollahi, A.; Greilich, S.

2016-09-01

The hybrid technology cell-fluorescent ion track hybrid detector (Cell-Fit-HD) enables the investigation of radiation-related cellular events along single ion tracks on the subcellular scale in clinical ion beams. The Cell-Fit-HD comprises a fluorescent nuclear track detector (FNTD, the physical compartment), a device for individual particle detection and a substrate for viable cell-coating, i.e. the biological compartment. To date both compartments have been imaged sequentially in situ by confocal laser scanning microscopy (CLSM). This is yet in conflict with a functional read-out of the Cell-Fit-HD utilizing a fast live-cell imaging of the biological compartment with low phototoxicity on greater time scales. The read-out of the biological from the physical compartment was uncoupled. A read-out procedure was developed to image the cell layer by conventional widefield microscopy whereas the FNTD was imaged by CLSM. Point mapping registration of the confocal and widefield imaging data was performed. Non-fluorescent crystal defects (spinels) visible in both read-outs were used as control point pairs. The accuracy achieved was on the sub-µm scale. The read-out procedure by widefield microscopy does not impair the unique ability of spatial correlation by the Cell-Fit-HD. The uncoupling will enlarge the application potential of the hybrid technology significantly. The registration allows for an ultimate correlation of microscopic physical beam parameters and cell kinetics on greater time scales. The method reported herein will be instrumental for the introduction of a novel generation of compact detectors facilitating biodosimetric research towards high-throughput analysis.

Optimization of confocal laser induced fluorescence for long focal length applications

NASA Astrophysics Data System (ADS)

Jemiolo, Andrew J.; Henriquez, Miguel F.; Thompson, Derek S.; Scime, Earl E.

2017-10-01

Laser induced fluorescence (LIF) is a non-perturbative diagnostic for measuring ion and neutral particle velocities and temperatures in a plasma. The conventional method for single-photon LIF requires intersecting optical paths for light injection and collection. The multiple vacuum windows needed for such measurements are unavailable in many plasma experiments. Confocal LIF eliminates the need for perpendicular intersecting optical paths by using concentric injection and collection paths through a single window. One of the main challenges with using confocal LIF is achieving high resolution measurements at the longer focal lengths needed for many plasma experiments. We present confocal LIF measurements in HELIX, a helicon plasma experiment at West Virginia University, demonstrating spatial resolution dependence on focal length and spatial filtering. By combining aberration mitigating optics with spatial filtering, our results show high resolution measurements at focal lengths of 0.5 m, long enough to access the interiors of many laboratory plasma experiments. This work was supported by U.S. National Science Foundation Grant No. PHY-1360278.

Raman imaging of lignin and cellulose distribution in black spruce wood (Picea mariana) cell walls

Treesearch

Umesh P. Agarwal

2005-01-01

A detailed understanding of wood cell wall structure and organization is important from both fundamental and practical point of views. A state-of- the-art 633-nm laser based confocal Raman microscope was used in situ to investigate the cell wall organization of black spruce wood. Chemical information on lignin and cellulose from morphologically distinct cell wall...

An Unconventional Approach to Reducing Retinal Degeneration After Traumatic Ocular Injury

DTIC Science & Technology

2017-09-01

AWARD NUMBER: W81XWH-15-1-0138 TITLE: An Unconventional Approach to Reducing Retinal Degeneration After Traumatic Ocular Injury PRINCIPAL...2015 - 30 Jun 2017 4. TITLE AND SUBTITLE An Unconventional Approach to Reducing Retinal Degeneration After Traumatic Ocular Injury 5 a . CONTRACT...optic confocal microscope system , test it, and establish protocols for the first successful in vivo retinal microvessel and pericyte advanced

Neutron-induced defects in optical fibers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rizzolo, S., E-mail: serena.rizzolo@univ-st-etienne.fr; Dipartimento di Fisica e Chimica, Università di Palermo, Palermo; and Areva Centre Technique, Le Creusot

2014-10-21

We present a study on 0.8 MeV neutron-induced defects up to fluences of 10{sup 17} n/cm{sup 2} in fluorine doped optical fibers by using electron paramagnetic resonance, optical absorption and confocal micro-luminescence techniques. Our results allow to address the microscopic mechanisms leading to the generation of Silica-related point-defects such as E', H(I), POR and NBOH Centers.

Confocal microscopy of fluid inclusions reveals fluid-pressure histories of sediments and an unexpected origin of gas condensate

NASA Astrophysics Data System (ADS)

Aplin, Andrew C.; Larter, Steve R.; Bigge, M. Ashley; MacLeod, Gordon; Swarbrick, Richard E.; Grunberger, Daniel

2000-11-01

We present two examples of how fluid inclusion data can be used to determine geologic pressure histories and to quantify the compositional evolution of petroleum in oil reservoirs. Volumetric liquid: vapor ratios generated with a confocal laser scanning microscope are used along with pressure-vapor-temperature (P-V-T) modeling software to estimate the composition, P-T phase envelope, and isochore of single petroleum inclusions in the North Sea's Judy and Alwyn fields. In both cases, the gas condensates currently in the reservoirs formed by the emplacement of gas into preexisting oil accumulations. Pressure histories of individual units in each field are also revealed, providing the kind of data needed to determine the permeability and fluid flow histories of sedimentary basins.

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

PubMed

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.

Reflectance confocal microscopy of red blood cells: simulation and experiment (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zeidan, Adel; Yeheskely-Hayon, Daniella; Minai, Limor; Yelin, Dvir

2016-03-01

The properties of red blood cells are a remarkable indicator of the body's physiological condition; their density could indicate anemia or polycythemia, their absorption spectrum correlates with blood oxygenation, and their morphology is highly sensitive to various pathologic states including iron deficiency, ovalocytosis, and sickle cell disease. Therefore, measuring the morphology of red blood cells is important for clinical diagnosis, providing valuable indications on a patient's health. In this work, we simulated the appearance of normal red blood cells under a reflectance confocal microscope and discovered unique relations between the cells' morphological parameters and the resulting characteristic interference patterns. The simulation results showed good agreement with in vitro reflectance confocal images of red blood cells, acquired using spectrally encoded flow cytometry (SEFC) that imaged the cells during linear flow and without artificial staining. By matching the simulated patterns to the SEFC images of the cells, the cells' three-dimensional shapes were evaluated and their volumes were calculated. Potential applications include measurement of the mean corpuscular volume, cell morphological abnormalities, cell stiffness under mechanical stimuli, and the detection of various hematological diseases.

Design and demonstration of multimodal optical scanning microscopy for confocal and two-photon imaging

NASA Astrophysics Data System (ADS)

Chun, Wanhee; Do, Dukho; Gweon, Dae-Gab

2013-01-01

We developed a multimodal microscopy based on an optical scanning system in order to obtain diverse optical information of the same area of a sample. Multimodal imaging researches have mostly depended on a commercial microscope platform, easy to use but restrictive to extend imaging modalities. In this work, the beam scanning optics, especially including a relay lens, was customized to transfer broadband (400-1000 nm) lights to a sample without any optical error or loss. The customized scanning optics guarantees the best performances of imaging techniques utilizing the lights within the design wavelength. Confocal reflection, confocal fluorescence, and two-photon excitation fluorescence images were obtained, through respective implemented imaging channels, to demonstrate imaging feasibility for near-UV, visible, near-IR continuous light, and pulsed light in the scanning optics. The imaging performances for spatial resolution and image contrast were verified experimentally; the results were satisfactory in comparison with theoretical results. The advantages of customization, containing low cost, outstanding combining ability and diverse applications, will contribute to vitalize multimodal imaging researches.

Sealing ability of three root-end filling materials prepared using an erbium: Yttrium aluminium garnet laser and endosonic tip evaluated by confocal laser scanning microscopy

PubMed Central

Nanjappa, A Salin; Ponnappa, KC; Nanjamma, KK; Ponappa, MC; Girish, Sabari; Nitin, Anita

2015-01-01

Aims: (1) To compare the sealing ability of mineral trioxide aggregate (MTA), Biodentine, and Chitra-calcium phosphate cement (CPC) when used as root-end filling, evaluated under confocal laser scanning microscope using Rhodamine B dye. (2) To evaluate effect of ultrasonic retroprep tip and an erbium:yttrium aluminium garnet (Er:YAG) laser on the integrity of three different root-end filling materials. Materials and Methods: The root canals of 80 extracted teeth were instrumented and obturated with gutta-percha. The apical 3 mm of each tooth was resected and 3 mm root-end preparation was made using ultrasonic tip (n = 30) and Er:YAG laser (n = 30). MTA, Biodentine, and Chitra-CPC were used to restore 10 teeth each. The samples were coated with varnish and after drying, they were immersed in Rhodamine B dye for 24 h. The teeth were then rinsed, sectioned longitudinally, and observed under confocal laser scanning microscope. Statistical Analysis Used: Data were analyzed using one-way analysis of variance (ANOVA) and a post-hoc Tukey's test at P < 0.05 (R software version 3.1.0). Results: Comparison of microleakage showed maximum peak value of 0.45 mm for Biodentine, 0.85 mm for MTA, and 1.05 mm for Chitra-CPC. The amount of dye penetration was found to be lesser in root ends prepared using Er:YAG laser when compared with ultrasonics, the difference was found to be statistically significant (P < 0.05). Conclusions: Root-end cavities prepared with Er:YAG laser and restored with Biodentine showed superior sealing ability compared to those prepared with ultrasonics. PMID:26180420

An in vitro Comparative Evaluation of Three Remineralizing Agents using Confocal Microscopy

PubMed Central

Chokshi, Achala; Konde, Sapna; Shetty, Sunil Raj; Chandra, Kumar Narayan; Jana, Sinjana; Mhambrey, Sanjana; Thakur, Sneha

2016-01-01

Introduction The caries process has been thought to be irreversible, resulting in the permanent loss of tooth substance and eventually the development of a cavity. Recent approaches focused on application of remineralizing agents to incipient carious lesions, aim at controlling demineralization and promoting remineralization. Remineralizing agents create a supersaturated environment around the lesion; thus, preventing mineral loss and forces calcium and phosphate ions in the vacant areas. Aim To compare and evaluate the remineralization potential of Fluoride Varnish, CPP-ACP Paste (Casein Phosphopeptide-Amorphous Calcium Phosphate) and fTCP Paste (functionalized Tricalcium Phosphate) using confocal microscope. Materials and Methods Two windows of 3X3mm were created on the labial cervical and incisal thirds in 60 permanent maxillary central incisors. The teeth were demineralized to create artificial caries and divided into three groups of 20 each. Group I specimens were coated with Fluoride Varnish once whereas those in CPP-ACP paste group and fTCP group were brushed for 2 minutes, twice daily for 20 and 40 days. The specimens were stored in artificial saliva during the study period and were later sectioned and observed under confocal microscope. Data obtained was statistically analyzed using Fischer’s exact test, ANOVA and post-hoc Bonferroni’s test. Results Fluoride Varnish, CPP-ACP Paste and fTCP Paste showed remineralization of artificial carious lesions at both the time intervals. Fluoride varnish showed the highest remineralization followed by CPP-ACP Paste and fTCP Paste. A statistically significant increase in remineralization potential of CPP-ACP Paste and fTCP Paste was observed at the end of 40 days as compared to 20 days. Conclusion Fluoride varnish showed the greatest remineralization potential of artificial carious lesions followed by CPP-ACP Paste and fTCP Paste respectively. PMID:27504408

Color-coded Imaging Enables Fluorescence-guided Surgery to Resect the Tumor Along with the Tumor Microenvironment in a Syngeneic Mouse Model of EL-4 Lymphoma.

PubMed

Hasegawa, Kosuke; Suetsugu, Atsushi; Nakamura, Miki; Matsumoto, Takuro; Kunisada, Takahiro; Shimizu, Masahito; Saji, Shigetoyo; Moriwaki, Hisataka; Bouvet, Michael; Hoffman, Robert M

2016-09-01

Fluorescence-guided surgery (FGS) of cancer is an emerging technology. We have previously shown the importance of resecting both the tumor and the tumor microenvironment (TME) for curative FGS. We also previously developed a syngeneic model using the mouse lymphoma cell line EL-4, expressing red fluorescent protein (EL-4-RFP), growing in green fluorescent protein (GFP) transgenic mice, which we have used in the present report to develop FGS of the tumor microenvironment. EL-4-RFP lymphoma cells were injected subcutaneously in C57/BL6 GFP transgenic mice. EL-4-RFP cells subsequently formed tumors by 35 days after cell transplantation. Using the portable hand-held Dino-Lite digital imaging system, subcutaneous tumors were resected by FGS. Resected tumor tissues were visualized with the Olympus FV1000 confocal microscope. Using the Dino-Lite, subcutaneous tumors and the tumor microenvironment were clearly visualized and resected. In the resected tumor, host stromal cells, including adipocyte-like cells and blood vessels with lymphocytes, were observed by confocal microscopy in addition to cancer cells by color-coded confocal imaging. The cancer cells and stromal cells in the TME were deeply intermingled in a highly-complex pattern. Color-coded FGS is an effective method to completely resect cancer cells along with the stromal cells in the TME which interact in a highly-complex pattern. Microscopically, cancer cells invade the TME and vice versa. To prevent tumor recurrence, it is necessary to resect the TME along with the tumor. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Light and Life in Baltimore—and Beyond

PubMed Central

Edidin, Michael

2015-01-01

Baltimore has been the home of numerous biophysical studies using light to probe cells. One such study, quantitative measurement of lateral diffusion of rhodopsin, set the standard for experiments in which recovery after photobleaching is used to measure lateral diffusion. Development of this method from specialized microscopes to commercial scanning confocal microscopes has led to widespread use of the technique to measure lateral diffusion of membrane proteins and lipids, and as well diffusion and binding interactions in cell organelles and cytoplasm. Perturbation of equilibrium distributions by photobleaching has also been developed into a robust method to image molecular proximity in terms of fluorescence resonance energy transfer between donor and acceptor fluorophores. PMID:25650914

Feasibility of confocal endomicroscopy in the diagnosis of pediatric gastrointestinal disorders

PubMed Central

Venkatesh, Krishnappa; Cohen, Marta; Evans, Clair; Delaney, Peter; Thomas, Steven; Taylor, Christopher; Abou-Taleb, Ashraf; Kiesslich, Ralf; Thomson, Mike

2009-01-01

AIM: To evaluate the feasibility and utility of confocal laser endomicroscopy (CLE) in the description of normal gastrointestinal (GI) mucosa and in the diagnosis of GI disorders in children, in comparison to histology. METHODS: Forty-four patients (19 female) median age 10.9 years (range 0.7-16.6 years) with suspected or known GI pathology underwent esophago-gastro-duodenoscopy (OGD) (n = 36) and/or ileocolonoscopy (IC) (n = 31) with CLE using sodium fluorescein and acriflavine as contrast agents. Histological sections were compared with same site confocal images by two experienced pediatric and GI histopathologists and endoscopists, respectively. RESULTS: Duodenum and ileum were intubated in all but one patient undergoing OGD and IC. The median procedure time was 16.4 min (range 7-25 min) for OGD and 27.9 min (range 15-45 min) for IC. A total of 4798 confocal images were compared with 153 biopsies from the upper GI tract from 36 procedures, and 4661 confocal images were compared with 188 biopsies from the ileocolon from 31 procedures. Confocal images were comparable to conventional histology both in normal and in pathological conditions such as esophagitis, Helicobacter pylori gastritis, celiac disease, inflammatory bowel disease, colonic heterotopia, and graft versus host disease. CONCLUSION: CLE offers the prospect of targeting biopsies to abnormal mucosa, thereby increasing diagnostic yield, reducing the number of biopsies, decreasing the burden on the histopathological services, and reducing costs. PMID:19437560

Confocal multispot microscope for fast and deep imaging in semicleared tissues

NASA Astrophysics Data System (ADS)

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.

A stereo-compound hybrid microscope for combined intracellular and optical recording of invertebrate neural network activity.

PubMed

Frost, William N; Wang, Jean; Brandon, Christopher J

2007-05-15

Optical recording studies of invertebrate neural networks with voltage-sensitive dyes seldom employ conventional intracellular electrodes. This may in part be due to the traditional reliance on compound microscopes for such work. While such microscopes have high light-gathering power, they do not provide depth of field, making working with sharp electrodes difficult. Here we describe a hybrid microscope design, with switchable compound and stereo objectives, that eases the use of conventional intracellular electrodes in optical recording experiments. We use it, in combination with a voltage-sensitive dye and photodiode array, to identify neurons participating in the swim motor program of the marine mollusk Tritonia. This microscope design should be applicable to optical recording studies in many preparations.

Biological applications of confocal fluorescence polarization microscopy

NASA Astrophysics Data System (ADS)

Bigelow, Chad E.

Fluorescence polarization microscopy is a powerful modality capable of sensing changes in the physical properties and local environment of fluorophores. In this thesis we present new applications for the technique in cancer diagnosis and treatment and explore the limits of the modality in scattering media. We describe modifications to our custom-built confocal fluorescence microscope that enable dual-color imaging, optical fiber-based confocal spectroscopy and fluorescence polarization imaging. Experiments are presented that indicate the performance of the instrument for all three modalities. The limits of confocal fluorescence polarization imaging in scattering media are explored and the microscope parameters necessary for accurate polarization images in this regime are determined. A Monte Carlo routine is developed to model the effect of scattering on images. Included in it are routines to track the polarization state of light using the Mueller-Stokes formalism and a model for fluorescence generation that includes sampling the excitation light polarization ellipse, Brownian motion of excited-state fluorophores in solution, and dipole fluorophore emission. Results from this model are compared to experiments performed on a fluorophore-embedded polymer rod in a turbid medium consisting of polystyrene microspheres in aqueous suspension. We demonstrate the utility of the fluorescence polarization imaging technique for removal of contaminating autofluorescence and for imaging photodynamic therapy drugs in cell monolayers. Images of cells expressing green fluorescent protein are extracted from contaminating fluorescein emission. The distribution of meta-tetrahydroxypheny1chlorin in an EMT6 cell monolayer is also presented. A new technique for imaging enzyme activity is presented that is based on observing changes in the anisotropy of fluorescently-labeled substrates. Proof-of-principle studies are performed in a model system consisting of fluorescently labeled bovine serum albumin attached to sepharose beads. The action of trypsin and proteinase K on the albumin is monitored to demonstrate validity of the technique. Images of the processing of the albumin in J774 murine macrophages are also presented indicating large intercellular differences in enzyme activity. Future directions for the technique are also presented, including the design of enzyme probes specific for prostate specific antigen based on fluorescently-labeled dendrimers. A technique for enzyme imaging based on extracellular autofluorescence is also proposed.

Multi-spectral confocal microendoscope for in-vivo imaging

NASA Astrophysics Data System (ADS)

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.

The development of confocal arthroscopy as optical histology for rotator cuff tendinopathy.

PubMed

Wu, J-P; Walton, M; Wang, A; Anderson, P; Wang, T; Kirk, T B; Zheng, M H

2015-09-01

MRI, ultrasound and video arthroscopy are traditional imaging technologies for noninvasive or minimal invasive assessment of the rotator cuff tendon pathology. However, these imaging modalities do not have sufficient resolution to demonstrate the pathology of rotator cuff tendons at a microstructural level. Therefore, they are insensitive to low-level tendon diseases. Although traditional histology can be used to analyze the physiology of rotator cuff tendons, it requires biopsy that traumatizes the rotator cuff, thus, potentially comprising the mechanical properties of tendons. Besides, it cannot offer real-time histological information. Confocal endoscopy offers a way to assess the microstructural disorder in tissues without biopsy. However, the application of this useful technique for detecting low-level tendon diseases has been restricted by using clinical grade fluorescent contrast agent to acquire high-resolution microstructural images of tendons. In this study, using a clinical grade sodium fluorescein contrast agent, we have reported the development of confocal arthroscopy for optical histological assessment without biopsy. The confocal arthroscopic technique was able to demonstrate rotator cuff tendinopathy in human cadavers, which appeared macroscopically normal under video arthroscopic examinations. The tendinopathy status of the rotator cuff tendons was confirmed by corresponding traditional histology. The development of confocal arthroscopy may provide a minimally invasive imaging technique for real-time histology of rotator cuff without the need for tissue biopsy. This technique has the potential for surgeons to gain in real time the histological information of rotator cuff tendons, which may assist planning repair strategies and potentially improve intervention outcomes. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum).

PubMed

Gao, Wei; Nan, Tiegui; Tan, Guiyu; Zhao, Hongwei; Tan, Weiming; Meng, Fanyun; Li, Zhaohu; Li, Qing X; Wang, Baomin

2015-01-01

The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC) imaging method for cadmium ions (Cd2+) was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl)-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA) was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions.

Comparisons between conventional optical imaging and parametric indirect microscopic imaging on human skin detection

NASA Astrophysics Data System (ADS)

Liu, Guoyan; Gao, Kun; Liu, Xuefeng; Ni, Guoqiang

2016-10-01

We report a new method, polarization parameters indirect microscopic imaging with a high transmission infrared light source, to detect the morphology and component of human skin. A conventional reflection microscopic system is used as the basic optical system, into which a polarization-modulation mechanics is inserted and a high transmission infrared light source is utilized. The near-field structural characteristics of human skin can be delivered by infrared waves and material coupling. According to coupling and conduction physics, changes of the optical wave parameters can be calculated and curves of the intensity of the image can be obtained. By analyzing the near-field polarization parameters in nanoscale, we can finally get the inversion images of human skin. Compared with the conventional direct optical microscope, this method can break diffraction limit and achieve a super resolution of sub-100nm. Besides, the method is more sensitive to the edges, wrinkles, boundaries and impurity particles.

Imaging Single Cells in the Living Retina

PubMed Central

Williams, David R.

2011-01-01

A quarter century ago, we were limited to a macroscopic view of the retina inside the living eye. Since then, new imaging technologies, including confocal scanning laser ophthalmoscopy, optical coherence tomography, and adaptive optics fundus imaging, transformed the eye into a microscope in which individual cells can now be resolved noninvasively. These technologies have enabled a wide range of studies of the retina that were previously impossible. PMID:21596053

Quantum Computing in Diamond

DTIC Science & Technology

2007-05-28

104 N2 103 N2 (a) (b) (c) Fig. 1: Confocal microscope images of NV centers created in bulk diamond through ion implantation of (a) gallium ions...nitrogen defects in diamond by chemical vapour deposition, J. R. Rabeau, S. Prawer, Y.L. Chin, F. Jelezko, T. Gaebel, and J. Wrachtrup, Applied...Physics Letters, 86, 31926, (2005) 2. Diamond Chemical Vapour Deposition on Opitcal Fibres for Fluorescence Waveguiding, J.R. Rabeau, S.T

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

PubMed Central

Wolenski, Joseph S.; Julich, Doerthe

2014-01-01

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy. PMID:24600334

Multicolor Three-Dimensional Tracking for Single-Molecule Fluorescence Resonance Energy Transfer Measurements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keller, Aaron M.; DeVore, Matthew S.; Stich, Dominik G.

Single-molecule fluorescence resonance energy transfer (smFRET) remains a widely utilized and powerful tool for quantifying heterogeneous interactions and conformational dynamics of biomolecules. However, traditional smFRET experiments either are limited to short observation times (typically less than 1 ms) in the case of “burst” confocal measurements or require surface immobilization which usually has a temporal resolution limited by the camera framing rate. We developed a smFRET 3D tracking microscope that is capable of observing single particles for extended periods of time with high temporal resolution. The confocal tracking microscope utilizes closed-loop feedback to follow the particle in solution by recentering itmore » within two overlapping tetrahedral detection elements, corresponding to donor and acceptor channels. We demonstrated the microscope’s multicolor tracking capability via random walk simulations and experimental tracking of 200 nm fluorescent beads in water with a range of apparent smFRET efficiency values, 0.45-0.69. We also demonstrated the microscope’s capability to track and quantify double-stranded DNA undergoing intramolecular smFRET in a viscous glycerol solution. In future experiments, the smFRET 3D tracking system will be used to study protein conformational dynamics while diffusing in solution and native biological environments with high temporal resolution.« less

Multicolor Three-Dimensional Tracking for Single-Molecule Fluorescence Resonance Energy Transfer Measurements

DOE PAGES

Keller, Aaron M.; DeVore, Matthew S.; Stich, Dominik G.; ...

2018-04-19

Single-molecule fluorescence resonance energy transfer (smFRET) remains a widely utilized and powerful tool for quantifying heterogeneous interactions and conformational dynamics of biomolecules. However, traditional smFRET experiments either are limited to short observation times (typically less than 1 ms) in the case of “burst” confocal measurements or require surface immobilization which usually has a temporal resolution limited by the camera framing rate. We developed a smFRET 3D tracking microscope that is capable of observing single particles for extended periods of time with high temporal resolution. The confocal tracking microscope utilizes closed-loop feedback to follow the particle in solution by recentering itmore » within two overlapping tetrahedral detection elements, corresponding to donor and acceptor channels. We demonstrated the microscope’s multicolor tracking capability via random walk simulations and experimental tracking of 200 nm fluorescent beads in water with a range of apparent smFRET efficiency values, 0.45-0.69. We also demonstrated the microscope’s capability to track and quantify double-stranded DNA undergoing intramolecular smFRET in a viscous glycerol solution. In future experiments, the smFRET 3D tracking system will be used to study protein conformational dynamics while diffusing in solution and native biological environments with high temporal resolution.« less

Red blood cell transport mechanisms in polyester thread-based blood typing devices.

PubMed

Nilghaz, Azadeh; Ballerini, David R; Guan, Liyun; Li, Lizi; Shen, Wei

2016-02-01

A recently developed blood typing diagnostic based on a polyester thread substrate has shown great promise for use in medical emergencies and in impoverished regions. The device is easy to use and transport, while also being inexpensive, accurate, and rapid. This study used a fluorescent confocal microscope to delve deeper into how red blood cells were behaving within the polyester thread-based diagnostic at the cellular level, and how plasma separation could be made to visibly occur on the thread, making it possible to identify blood type in a single step. Red blood cells were stained and the plasma phase dyed with fluorescent compounds to enable them to be visualised under the confocal microscope at high magnification. The mechanisms uncovered were in surprising contrast with those found for a similar, paper-based method. Red blood cell aggregates did not flow over each other within the thread substrate as expected, but suffered from a restriction to their flow which resulted in the chromatographic separation of the RBCs from the liquid phase of the blood. It is hoped that these results will lead to the optimisation of the method to enable more accurate and sensitive detection, increasing the range of blood systems that can be detected.

Video-rate in vivo fluorescence imaging with a line-scanned dual-axis confocal microscope.

PubMed

Chen, Ye; Wang, Danni; Khan, Altaz; Wang, Yu; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T C

2015-10-01

Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.

Video-rate in vivo fluorescence imaging with a line-scanned dual-axis confocal microscope

NASA Astrophysics Data System (ADS)

Chen, Ye; Wang, Danni; Khan, Altaz; Wang, Yu; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T. C.

2015-10-01

Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.

Real-Time Analysis of Magnetic Hyperthermia Experiments on Living Cells under a Confocal Microscope.

PubMed

Connord, Vincent; Clerc, Pascal; Hallali, Nicolas; El Hajj Diab, Darine; Fourmy, Daniel; Gigoux, Véronique; Carrey, Julian

2015-05-01

Combining high-frequency alternating magnetic fields (AMF) and magnetic nanoparticles (MNPs) is an efficient way to induce biological responses through several approaches: magnetic hyperthermia, drug release, controls of gene expression and neurons, or activation of chemical reactions. So far, these experiments cannot be analyzed in real-time during the AMF application. A miniaturized electromagnet fitting under a confocal microscope is built, which produces an AMF of frequency and amplitude similar to the ones used in magnetic hyperthermia. AMF application induces massive damages to tumoral cells having incorporated nanoparticles into their lysosomes without affecting the others. Using this setup, real-time analyses of molecular events occurring during AMF application are performed. Lysosome membrane permeabilization and reactive oxygen species production are detected after only 30 min of AMF application, demonstrating they occur at an early stage in the cascade of events leading eventually to cell death. Additionally, lysosomes self-assembling into needle-shaped organization under the influence of AMF is observed in real-time. This experimental approach will permit to get a deeper insight into the physical, molecular, and biological process occurring in several innovative techniques used in nanomedecine based on the combined use of MNPs and high-frequency magnetic fields. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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.

Microscopic and spectroscopic evaluation of novel PLGA-chitosan Nanoplexes as an ocular delivery system.

PubMed

Jain, Gaurav K; Pathan, Shadab A; Akhter, Sohail; Jayabalan, Nirmal; Talegaonkar, Sushma; Khar, Roop K; Ahmad, Farhan J

2011-02-01

The interaction of PLGA-chitosan Nanoplexes with ocular mucosa was investigated ex vivo and in vivo to assess their potential as ocular delivery system. Fluorescent Rhodamine Nanoplexes (Rd-Nanoplexes) were prepared by ionotropic gelation method. The size and morphology of Nanoplexes was investigated by TEM, SEM and PCS. The corneal retention, uptake and penetration of Nanoplexes were analyzed by spectrofluorimetry and confocal microscopy. Corneas from Rd-Nanoplexes-treated rabbits were evaluated for the in vivo uptake and ocular tolerance. The Nanoplexes prepared were round with a mean diameter of 115.6±17nm and the encapsulation efficiency of Rd was 59.4±2.5%. Data from ex vivo and in vivo studies showed that the amounts of Rd in the cornea were significantly higher for Nanoplexes than for a control Rd solution, these amounts being fairly constant for up to 24h. Confocal microscopy of the corneas revealed paracellular and transcellular uptake of the Nanoplexes. The uptake mechanism postulated was adsorptive-mediated endocytosis and opening of the tight junctions between epithelial cells. No alteration was microscopically observed after ocular surface exposure to Nanoplexes. Taken together, these data demonstrate that Nanoplexes are potentially useful as ocular drug carriers. Copyright © 2010 Elsevier B.V. All rights reserved.

Immunodetection and intracellular localization of caldesmon-like proteins in Amoeba proteus.

PubMed

Gagola, M; Kłopocka, W; Greebecki, A; Makuch, R

2003-09-01

Caldesmon immunoanalogues were detected in Amoeba proteus cell homogenates by the Western blot technique. Three immunoreactive bands were recognized by polyclonal antibodies against the whole molecule of chicken gizzard caldesmon as well as by a monoclonal antibody against its C-terminal domain: one major and two minor bands corresponding to proteins with apparent molecular masses of 150, 69, and 60 kDa. The presence of caldesmon-like protein(s) in amoebae was revealed as well in single cells after their fixation, staining with the same antibodies, and recording their total fluorescence in a confocal laser scanning microscope. Proteins recognized by the antibodies bind to filamentous actin. This was established by a cosedimentation assay in cell homogenates and by colocalization of the caldesmon-related immunofluorescence with the fluorescence of filamentous actin stained with rhodamine-labelled phalloidin, demonstrated in optical sections of single cells in a confocal microscope. Caldesmon is colocalized with filamentous actin in the withdrawn cell regions where the cortical actomyosin network contracts and actin is depolymerized, in the frontal zone where actin is polymerized again and the cortical cytoskeleton is reconstructed, inside the nucleus and in the perinuclear cytoskeleton, and probably at the cell-to-substratum adhesion sites. The regulatory role of caldesmon in these functionally different regions of locomoting amoebae is discussed.

Spectrally resolved chromatic confocal interferometry for one-shot nano-scale surface profilometry with several tens of micrometric depth range

NASA Astrophysics Data System (ADS)

Chen, Liang-Chia; Chen, Yi-Shiuan; Chang, Yi-Wei; Lin, Shyh-Tsong; Yeh, Sheng Lih

2013-01-01

In this research, new nano-scale measurement methodology based on spectrally-resolved chromatic confocal interferometry (SRCCI) was successfully developed by employing integration of chromatic confocal sectioning and spectrally-resolve white light interferometry (SRWLI) for microscopic three dimensional surface profilometry. The proposed chromatic confocal method (CCM) using a broad band while light in combination with a specially designed chromatic dispersion objective is capable of simultaneously acquiring multiple images at a large range of object depths to perform surface 3-D reconstruction by single image shot without vertical scanning and correspondingly achieving a high measurement depth range up to hundreds of micrometers. A Linnik-type interferometric configuration based on spectrally resolved white light interferometry is developed and integrated with the CCM to simultaneously achieve nanoscale axis resolution for the detection point. The white-light interferograms acquired at the exit plane of the spectrometer possess a continuous variation of wavelength along the chromaticity axis, in which the light intensity reaches to its peak when the optical path difference equals to zero between two optical arms. To examine the measurement accuracy of the developed system, a pre-calibrated accurate step height target with a total step height of 10.10 μm was measured. The experimental result shows that the maximum measurement error was verified to be less than 0.3% of the overall measuring height.

Confocal fluorescence microscopy to evaluate changes in adipocytes in the tumor microenvironment associated with invasive ductal carcinoma and ductal carcinoma in situ.

PubMed

Dobbs, Jessica L; Shin, Dongsuk; Krishnamurthy, Savitri; Kuerer, Henry; Yang, Wei; Richards-Kortum, Rebecca

2016-09-01

Adipose tissue is a dynamic organ that provides endocrine, inflammatory and angiogenic factors, which can assist breast carcinoma cells with invasion and metastasis. Previous studies have shown that adipocytes adjacent to carcinoma, known as cancer-associated adipocytes, undergo extensive changes that correspond to an "activated phenotype," such as reduced size relative to adipocytes in non-neoplastic breast tissue. Optical imaging provides a tool that can be used to characterize adipocyte morphology and other features of the tumor microenvironment. In this study, we used confocal fluorescence microscopy to acquire images of freshly excised breast tissue stained topically with proflavine. We developed a computerized algorithm to identify and quantitatively measure phenotypic properties of adipocytes located adjacent to and far from normal collagen, ductal carcinoma in situ and invasive ductal carcinoma. Adipocytes were measured in confocal fluorescence images of fresh breast tissue collected from 22 patients. Results show that adipocytes adjacent to neoplastic tissue margins have significantly smaller area compared to adipocytes far from the margins of neoplastic lesions and compared to adipocytes adjacent to non-neoplastic collagenous stroma. These findings suggest that confocal microscopic images can be utilized to evaluate phenotypic properties of adipocytes in breast stroma which may be useful in defining alterations in microenvironment that may aid in the development and progression of neoplastic lesions. © 2016 UICC.

Confocal absorption spectral imaging of MoS2: optical transitions depending on the atomic thickness of intrinsic and chemically doped MoS2.

PubMed

Dhakal, Krishna P; Duong, Dinh Loc; Lee, Jubok; Nam, Honggi; Kim, Minsu; Kan, Min; Lee, Young Hee; Kim, Jeongyong

2014-11-07

We performed a nanoscale confocal absorption spectral imaging to obtain the full absorption spectra (over the range 1.5-3.2 eV) within regions having different numbers of layers and studied the variation of optical transition depending on the atomic thickness of the MoS2 film. Three distinct absorption bands corresponding to A and B excitons and a high-energy background (BG) peak at 2.84 eV displayed a gradual redshift as the MoS2 film thickness increased from the monolayer, to the bilayer, to the bulk MoS2 and this shift was attributed to the reduction of the gap energy in the Brillouin zone at the K-point as the atomic thickness increased. We also performed n-type chemical doping of MoS2 films using reduced benzyl viologen (BV) and the confocal absorption spectra modified by the doping showed a strong dependence on the atomic thickness: A and B exciton peaks were greatly quenched in the monolayer MoS2 while much less effect was shown in larger thickness and the BG peak either showed very small quenching for 1 L MoS2 or remained constant for larger thicknesses. Our results indicate that confocal absorption spectral imaging can provide comprehensive information on optical transitions of microscopic size intrinsic and doped two-dimensional layered materials.

The Scanning Optical Microscope.

ERIC Educational Resources Information Center

Sheppard, C. J. R.

1978-01-01

Describes the principle of the scanning optical microscope and explains its advantages over the conventional microscope in the improvement of resolution and contrast, as well as the possibility of producing a picture from optical harmonies generated within the specimen.

Quantum dots conjugated zinc oxide nanosheets: Impeder of microbial growth and biofilm

NASA Astrophysics Data System (ADS)

Patil, Rajendra; Gholap, Haribhau; Warule, Sambhaji; Banpurkar, Arun; Kulkarni, Gauri; Gade, Wasudeo

2015-01-01

The grieving problem of the 21st century has been the antimicrobial resistance in pathogenic microorganisms to conventional antibiotics. Therefore, developments of novel antibacterial materials which effectively inhibit or kill such resistant microorganisms have become the need of the hour. In the present study, we communicate the synthesis of quantum dots conjugated zinc oxide nanostructures (ZnO/CdTe) as an impeder of microbial growth and biofilm. The as-synthesized nanostructures were characterized by X-ray diffraction, ultraviolet-visible spectroscopy, photoluminescence spectroscopy, field emission scanning electron microscopy and high resolution transmission electron microscopy. The growth impedance property of ZnO and ZnO/CdTe on Gram positive organism, Bacillus subtilis NCIM 2063 and Gram negative, Escherichia coli NCIM 2931 and biofilm impedance activity in Pseudomonas aeruginosa O1 was found to occur due to photocatalytical action on the cell biofilm surfaces. The impedance in microbial growth and biofilm formation was further supported by ruptured appearances of cells and dettrered biofilm under field emission scanning electron and confocal laser scanning microscope. The ZnO/CdTe nanostructures array synthesized by hydrothermal method has an advantage of low growth temperature, and opportunity to fabricate inexpensive material for nano-biotechnological applications.

A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici☆

PubMed Central

Kilaru, S.; Schuster, M.; Studholme, D.; Soanes, D.; Lin, C.; Talbot, N.J.; Steinberg, G.

2015-01-01

Fluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein localization. Cytoplasmic expression of FPs in fungal pathogens allows greater insight into invasion strategies and the host-pathogen interaction. Detection of their fluorescent signal depends on the right combination of microscopic setup and signal brightness. Slow rates of photo-bleaching are pivotal for in vivo observation of FPs over longer periods of time. Here, we test green-fluorescent proteins, including Aequorea coerulescens GFP (AcGFP), enhanced GFP (eGFP) from Aequorea victoria and a novel Zymoseptoria tritici codon-optimized eGFP (ZtGFP), for their usage in conventional and laser-enhanced epi-fluorescence, and confocal laser-scanning microscopy. We show that eGFP, expressed cytoplasmically in Z. tritici, is significantly brighter and more photo-stable than AcGFP. The codon-optimized ZtGFP performed even better than eGFP, showing significantly slower bleaching and a 20–30% further increase in signal intensity. Heterologous expression of all GFP variants did not affect pathogenicity of Z. tritici. Our data establish ZtGFP as the GFP of choice to investigate intracellular protein dynamics in Z. tritici, but also infection stages of this wheat pathogen inside host tissue. PMID:26092799

Wide-Field Imaging Using Nitrogen Vacancies

NASA Technical Reports Server (NTRS)

Englund, Dirk Robert (Inventor); Trusheim, Matthew Edwin (Inventor)

2017-01-01

Nitrogen vacancies in bulk diamonds and nanodiamonds can be used to sense temperature, pressure, electromagnetic fields, and pH. Unfortunately, conventional sensing techniques use gated detection and confocal imaging, limiting the measurement sensitivity and precluding wide-field imaging. Conversely, the present sensing techniques do not require gated detection or confocal imaging and can therefore be used to image temperature, pressure, electromagnetic fields, and pH over wide fields of view. In some cases, wide-field imaging supports spatial localization of the NVs to precisions at or below the diffraction limit. Moreover, the measurement range can extend over extremely wide dynamic range at very high sensitivity.

Ex vivo measurement of postmortem tissue changes in the crystalline lens by Brillouin spectroscopy and confocal reflectance microscopy.

PubMed

Reiss, Stephan; Sperlich, K; Hovakimyan, M; Martius, P; Guthoff, R F; Stolz, H; Stachs, O

2012-08-01

Use of Brillouin spectroscopy in ophthalmology enables noninvasive, spatially resolved determination of the rheological properties of crystalline lens tissue. Furthermore, the Brillouin shift correlates with the protein concentration inside the lens. In vitro measurements on extracted porcine lenses demonstrate that results obtained with Brillouin spectroscopy depend strongly on time after death. The intensity of the Brillouin signal decreases significantly as early as 5 h postmortem. Moreover, the fluctuation of the Brillouin frequency shift inside the lens increases with postmortem time. Images of lens tissue taken with a confocal reflectance microscope between measurements reveal a degenerative aging process. These tissue changes correlate with our results from Brillouin spectroscopy. It is concluded that only in vivo measurements appropriately reflect the rheological properties of the eye lens and its protein concentration.

Improving high resolution retinal image quality using speckle illumination HiLo imaging

PubMed Central

Zhou, Xiaolin; Bedggood, Phillip; Metha, Andrew

2014-01-01

Retinal image quality from flood illumination adaptive optics (AO) ophthalmoscopes is adversely affected by out-of-focus light scatter due to the lack of confocality. This effect is more pronounced in small eyes, such as that of rodents, because the requisite high optical power confers a large dioptric thickness to the retina. A recently-developed structured illumination microscopy (SIM) technique called HiLo imaging has been shown to reduce the effect of out-of-focus light scatter in flood illumination microscopes and produce pseudo-confocal images with significantly improved image quality. In this work, we adopted the HiLo technique to a flood AO ophthalmoscope and performed AO imaging in both (physical) model and live rat eyes. The improvement in image quality from HiLo imaging is shown both qualitatively and quantitatively by using spatial spectral analysis. PMID:25136486

Improving high resolution retinal image quality using speckle illumination HiLo imaging.

PubMed

Zhou, Xiaolin; Bedggood, Phillip; Metha, Andrew

2014-08-01

Retinal image quality from flood illumination adaptive optics (AO) ophthalmoscopes is adversely affected by out-of-focus light scatter due to the lack of confocality. This effect is more pronounced in small eyes, such as that of rodents, because the requisite high optical power confers a large dioptric thickness to the retina. A recently-developed structured illumination microscopy (SIM) technique called HiLo imaging has been shown to reduce the effect of out-of-focus light scatter in flood illumination microscopes and produce pseudo-confocal images with significantly improved image quality. In this work, we adopted the HiLo technique to a flood AO ophthalmoscope and performed AO imaging in both (physical) model and live rat eyes. The improvement in image quality from HiLo imaging is shown both qualitatively and quantitatively by using spatial spectral analysis.

Live cell refractometry using Hilbert phase microscopy and confocal reflectance microscopy.

PubMed

Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Yaqoob, Zahid; Badizadegan, Kamran; Dasari, Ramachandra R; Feld, Michael S

2009-11-26

Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ.

Live Cell Refractometry Using Hilbert Phase Microscopy and Confocal Reflectance Microscopy†

PubMed Central

Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Yaqoob, Zahid; Badizadegan, Kamran; Dasari, Ramachandra R.; Feld, Michael S.

2010-01-01

Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ. PMID:19803506

Reflectance confocal microscopy of optical phantoms

PubMed Central

Jacques, Steven L.; Wang, Bo; Samatham, Ravikant

2012-01-01

A reflectance confocal scanning laser microscope (rCSLM) operating at 488-nm wavelength imaged three types of optical phantoms: (1) 100-nm-dia. polystyrene microspheres in gel at 2% volume fraction, (2) solid polyurethane phantoms (INO BiomimicTM), and (3) common reflectance standards (SpectralonTM). The noninvasive method measured the exponential decay of reflected signal as the focus (zf) moved deeper into the material. The two experimental values, the attenuation coefficient μ and the pre-exponential factor ρ, were mapped into the material optical scattering properties, the scattering coefficient μs and the anisotropy of scattering g. Results show that μs varies as 58, 8–24, and 130–200 cm-1 for phantom types (1), (2) and (3), respectively. The g varies as 0.112, 0.53–0.67, and 0.003–0.26, respectively. PMID:22741065

Rotary-scanning optical resolution photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Qi, Weizhi; Xi, Lei

2016-10-01

Optical resolution photoacoustic microscopy (ORPAM) is currently one of the fastest evolving photoacoustic imaging modalities. It has a comparable spatial resolution to pure optical microscopic techniques such as epifluorescence microscopy, confocal microscopy, and two-photon microscopy, but also owns a deeper penetration depth. In this paper, we report a rotary-scanning (RS)-ORPAM that utilizes a galvanometer scanner integrated with objective to achieve rotary laser scanning. A 15 MHz cylindrically focused ultrasonic transducer is mounted onto a motorized rotation stage to follow optical scanning traces synchronously. To minimize the loss of signal to noise ratio, the acoustic focus is precisely adjusted to reach confocal with optical focus. Black tapes and carbon fibers are firstly imaged to evaluate the performance of the system, and then in vivo imaging of vasculature networks inside the ears and brains of mice is demonstrated using this system.

A stereo-compound hybrid microscope for combined intracellular and optical recording of invertebrate neural network activity

PubMed Central

Frost, William N.; Wang, Jean; Brandon, Christopher J.

2007-01-01

Optical recording studies of invertebrate neural networks with voltage-sensitive dyes seldom employ conventional intracellular electrodes. This may in part be due to the traditional reliance on compound microscopes for such work. While such microscopes have high light-gathering power, they do not provide depth of field, making working with sharp electrodes difficult. Here we describe a hybrid microscope design, with switchable compound and stereo objectives, that eases the use of conventional intracellular electrodes in optical recording experiments. We use it, in combination with a voltage-sensitive dye and photodiode array, to identify neurons participating in the swim motor program of the marine mollusk Tritonia. This microscope design should be applicable to optical recording studies in many preparations. PMID:17306887

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue.

PubMed

Yoshitake, Tadayuki; Giacomelli, Michael G; Cahill, Lucas C; Schmolze, Daniel B; Vardeh, Hilde; Faulkner-Jones, Beverly E; Connolly, James L; Fujimoto, James G

2016-12-01

Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue.

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue

PubMed Central

Yoshitake, Tadayuki; Giacomelli, Michael G.; Cahill, Lucas C.; Schmolze, Daniel B.; Vardeh, Hilde; Faulkner-Jones, Beverly E.; Connolly, James L.; Fujimoto, James G.

2016-01-01

Abstract. Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue. PMID:28032121

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue

NASA Astrophysics Data System (ADS)

Yoshitake, Tadayuki; Giacomelli, Michael G.; Cahill, Lucas C.; Schmolze, Daniel B.; Vardeh, Hilde; Faulkner-Jones, Beverly E.; Connolly, James L.; Fujimoto, James G.

2016-12-01

Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue.

[Cytocompatibility of nanophase hydroxyapatite ceramics].

PubMed

Wen, Bo; Chen, Zhi-qing; Jiang, Yin-shan; Yang, Zheng-wen; Xu, Yong-zhong

2004-12-01

To evaluate the cytocompatibility of nanophase hydroxyapatite ceramics in vitro. Hydroxyapatite (HA) was prepared via wet method. The grain size of the hydroxyapatite in the study was determined by scanning electron microscope and atomic force microscope with image analysis software. Primary osteoblast culture was established from rat calvaria. Cell adherence and proliferation on nanophase hydroxyapatite ceramics and conventional hydroxyapatite ceramics were examined at 1, 3, 5, 7 days. Morphology of the cells was observed by microscope. The average grain size of the nanophase and conventional HA was 55 nm and 780 nm, respectively. Throughout 7 days period, osteoblast proliferation on the HA was similar to that on tissue culture borosilicate glass controls, osteoblasts could attach, spread and proliferate on HA. However, compared to conventional ceramics, osteoblast proliferation on nanophase HA was significantly better after 1, 3, 5 and 7 days. Cytocompatibility of nanophase HA was significantly better than conventional ceramics.

Elemental and topographical imaging of microscopic variations in deposition on NSTX-U and DIII-D samples

NASA Astrophysics Data System (ADS)

Skinner, C. H.; Kaita, R.; Koel, B. E.; Chrobak, C. P.; Wampler, W. R.

2017-10-01

Tokamak plasma facing components (PFCs) have surface roughness that can cause microscopic spatial variations in erosion and deposition and hence influence material migration. Previous RBS measurements showed indirect evidence for this but the spatial (0.5mm) resolution was insufficient for direct imaging. We will present elemental images at sub-micron resolution of deposition on NSTX-U and DiMES samples that show strong microscopic variations and correlate this with 3D topographical maps of surface irregularities. The elemental imaging is performed with a Scanning Auger Microprobe (SAM) that measures element-specific Auger electrons excited by an SEM electron beam. 3D topographical maps of the samples are performed with a Leica DCM 3D confocal light microscope and compared to the elemental deposition pattern. The initial results appear consistent with erosion at the downstream edges of the surface pores exposed to the incident ion flux, whereas the deeper regions are shadowed and serve as deposition traps. Support was provided through DOE Contract Numbers DE-AC02-09CH11466, DE-FC02-04ER54698 and DE-NA0003525.

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

PubMed Central

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.

Raman imaging to investigate ultrastructure and composition of plant cell walls : distribution of lignin and cellulose in black spruce wood (Picea mariana)

Treesearch

Umesh P. Agarwal

2006-01-01

A detailed understanding of the structural organization of the cell wall of vascular plants is important from both the perspectives of plant biology and chemistry and of commercial utilization. A state-of-the-art 633-nm laser-based confocal Raman microscope was used to determine the distribution of cell wall components in the cross section of black spruce wood in situ...

Three-dimensional behavior of ice crystals and biological cells during freezing of cell suspensions.

PubMed

Ishiguro, H; Koike, K

1998-09-11

Behavior of ice crystals and human red blood cells during extracellular-freezing was investigated in three-dimensions using a confocal laser scanning microscope(CLSM), which noninvasively produces tomograms of biological materials. Physiological saline and physiological saline with 2.4 M glycerol were used for suspension. Various cooling rates for directional solidification were used for distinctive morphology of the ice crystals. Addition of acridine orange as a fluorescent dye into the cell suspension enabled ice crystal, cells and unfrozen solution to be distinguished by different colors. The results indicate that the microscopic structure is three-dimensional for flat, cellular, and dendritic solid-liquid interfaces and that a CLSM is very effective in studying three-dimensional structure during the freezing of cell suspensions.

Light and life in Baltimore--and beyond.

PubMed

Edidin, Michael

2015-02-03

Baltimore has been the home of numerous biophysical studies using light to probe cells. One such study, quantitative measurement of lateral diffusion of rhodopsin, set the standard for experiments in which recovery after photobleaching is used to measure lateral diffusion. Development of this method from specialized microscopes to commercial scanning confocal microscopes has led to widespread use of the technique to measure lateral diffusion of membrane proteins and lipids, and as well diffusion and binding interactions in cell organelles and cytoplasm. Perturbation of equilibrium distributions by photobleaching has also been developed into a robust method to image molecular proximity in terms of fluorescence resonance energy transfer between donor and acceptor fluorophores. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A cell-free assay to determine the stoichiometry of plasma membrane proteins.

PubMed

Trigo, Cesar; Vivar, Juan P; Gonzalez, Carlos B; Brauchi, Sebastian

2013-04-01

Plasma membrane receptors, transporters, and ion channel molecules are often found as oligomeric structures that participate in signaling cascades essential for cell survival. Different states of protein oligomerization may play a role in functional control and allosteric regulation. Stochastic GFP-photobleaching (SGP) has emerged as an affordable and simple method to determine the stoichiometry of proteins at the plasma membrane. This non-invasive optical approach can be useful for total internal reflection of fluorescence microscopy (TIRFM), where signal-to-noise ratio is very high at the plasma membrane. Here, we report an alternative methodology implemented on a standard laser scanning confocal microscope (LSCM). The simplicity of our method will allow for its implementation in any epifluorescence microscope of choice.

Chronic Chagas disease: PCR-xenodiagnosis without previous microscopic observation is a useful tool to detect viable Trypanosoma cruzi.

PubMed

Saavedra, Miguel; Zulantay, Inés; Apt, Werner; Martínez, Gabriela; Rojas, Antonio; Rodríguez, Jorge

2013-01-01

We evaluate the elimination of the microscopic stage of conventional xenodiagnosis (XD) to optimize the parasitological diagnosis of Trypanosoma cruzi in chronic Chagas disease. To this purpose we applied under informed consent two XD cages to 150 Chilean chronic chagasic patients. The fecal samples (FS) of the triatomines at 30, 60 and 90 days post feeding were divided into two parts: in one a microscopic search for mobile trypomastigote and/or epimastigote forms was performed. In the other part, DNA extraction-purification for PCR directed to the conserved region of kDNA minicircles of trypanosomes (PCR-XD), without previous microscopic observation was done. An XD was considered positive when at least one mobile T. cruzi parasite in any one of three periods of incubation was observed, whereas PCR-XD was considered positive when the 330 bp band specific for T. cruzi was detected. 25 of 26 cases with positive conventional XD were PCR-XD positive (concordance 96.2%), whereas 85 of 124 cases with negative conventional XD were positive by PCR-XD (68.5%). Human chromosome 12 detected by Real-time PCR used as exogenous internal control of PCR-XD reaction allowed to discounting of PCR inhibition and false negative in 40 cases with negative PCR-XD. PCR-XD performed without previous microscopic observation is a useful tool for detection of viable parasites with higher efficiency then conventional XD.

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

NASA Astrophysics Data System (ADS)

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.