MAMMALIAN APOPTOSIS IN WHOLE NEONATAL OVARIES, EMBRYOS AND FETAL LIMBS USING CONFOCAL MICROSCOPY

EPA Science Inventory

The emergence of confocal laser scanning microscopy (CLSM) as a technique capable of optically generating serial sections of whole-mount tissue and then reassembling the computer-stored images as a virtual 3-dimensional structure offers a viable alternative to traditional section...

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

EPA Science Inventory

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

Confocal microscopy of thick tissue sections: 3D visualizaiton of rat kidney glomeruli

EPA Science Inventory

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

Confocal Microscopy of thick tissue sections: 3D Visualization of rat kidney glomeruli

EPA Science Inventory

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

Comparative measurement of collagen bundle orientation by Fourier analysis and semiquantitative evaluation: reliability and agreement in Masson's trichrome, Picrosirius red and confocal microscopy techniques.

PubMed

Marcos-Garcés, V; Harvat, M; Molina Aguilar, P; Ferrández Izquierdo, A; Ruiz-Saurí, A

2017-08-01

Measurement of collagen bundle orientation in histopathological samples is a widely used and useful technique in many research and clinical scenarios. Fourier analysis is the preferred method for performing this measurement, but the most appropriate staining and microscopy technique remains unclear. Some authors advocate the use of Haematoxylin-Eosin (H&E) and confocal microscopy, but there are no studies comparing this technique with other classical collagen stainings. In our study, 46 human skin samples were collected, processed for histological analysis and stained with Masson's trichrome, Picrosirius red and H&E. Five microphotographs of the reticular dermis were taken with a 200× magnification with light microscopy, polarized microscopy and confocal microscopy, respectively. Two independent observers measured collagen bundle orientation with semiautomated Fourier analysis with the Image-Pro Plus 7.0 software and three independent observers performed a semiquantitative evaluation of the same parameter. The average orientation for each case was calculated with the values of the five pictures. We analyzed the interrater reliability, the consistency between Fourier analysis and average semiquantitative evaluation and the consistency between measurements in Masson's trichrome, Picrosirius red and H&E-confocal. Statistical analysis for reliability and agreement was performed with the SPSS 22.0 software and consisted of intraclass correlation coefficient (ICC), Bland-Altman plots and limits of agreement and coefficient of variation. Interrater reliability was almost perfect (ICC > 0.8) with all three histological and microscopy techniques and always superior in Fourier analysis than in average semiquantitative evaluation. Measurements were consistent between Fourier analysis by one observer and average semiquantitative evaluation by three observers, with an almost perfect agreement with Masson's trichrome and Picrosirius red techniques (ICC > 0.8) and a strong agreement with H&E-confocal (0.7 < ICC < 0.8). Comparison of measurements between the three techniques for the same observer showed an almost perfect agreement (ICC > 0.8), better with Fourier analysis than with semiquantitative evaluation (single and average). These results in nonpathological skin samples were also confirmed in a preliminary analysis in eight scleroderma skin samples. Our results show that Masson's trichrome and Picrosirius red are consistent with H&E-confocal for measuring collagen bundle orientation in histological samples and could thus be used indistinctly for this purpose. Fourier analysis is superior to average semiquantitative evaluation and should keep being used as the preferred method. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

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.

Imaging of endodontic biofilms by combined microscopy (FISH/cLSM - SEM).

PubMed

Schaudinn, C; Carr, G; Gorur, A; Jaramillo, D; Costerton, J W; Webster, P

2009-08-01

Scanning electron microscopy is a useful imaging approach for the visualization of bacterial biofilms in their natural environments including their medical and dental habitats, because it allows for the exploration of large surfaces with excellent resolution of topographic features. Most biofilms in nature, however, are embedded in a thick layer of extracellular matrix that prevents a clear identification of individual bacteria by scanning electron microscopy. The use of confocal laser scanning microscopy on the other hand in combination with fluorescence in situ hybridization enables the visualization of matrix embedded bacteria in multi-layered biofilms. In our study, fluorescence in situ hybridization/confocal laser scanning microscopy and scanning electron microscopy were applied to visualize bacterial biofilm in endodontic root canals. The resulting fluorescence in situ hybridization /confocal laser scanning microscopy and scanning electron microscopy and pictures were subsequently combined into one single image to provide high-resolution information on the location of hidden bacteria. The combined use of scanning electron microscopy and fluorescence in situ hybridization / confocal laser scanning microscopy has the potential to overcome the limits of each single technique.

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

Simple fiber-optic confocal microscopy with nanoscale depth resolution beyond the diffraction barrier.

PubMed

Ilev, Ilko; Waynant, Ronald; Gannot, Israel; Gandjbakhche, Amir

2007-09-01

A novel fiber-optic confocal approach for ultrahigh depth-resolution (

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.

Multiple excitation nano-spot generation and confocal detection for far-field microscopy.

PubMed

Mondal, Partha Pratim

2010-03-01

An imaging technique is developed for the controlled generation of multiple excitation nano-spots for far-field microscopy. The system point spread function (PSF) is obtained by interfering two counter-propagating extended depth-of-focus PSF (DoF-PSF), resulting in highly localized multiple excitation spots along the optical axis. The technique permits (1) simultaneous excitation of multiple planes in the specimen; (2) control of the number of spots by confocal detection; and (3) overcoming the point-by-point based excitation. Fluorescence detection from the excitation spots can be efficiently achieved by Z-scanning the detector/pinhole assembly. The technique complements most of the bioimaging techniques and may find potential application in high resolution fluorescence microscopy and nanoscale imaging.

Multiple excitation nano-spot generation and confocal detection for far-field microscopy

NASA Astrophysics Data System (ADS)

Mondal, Partha Pratim

2010-03-01

An imaging technique is developed for the controlled generation of multiple excitation nano-spots for far-field microscopy. The system point spread function (PSF) is obtained by interfering two counter-propagating extended depth-of-focus PSF (DoF-PSF), resulting in highly localized multiple excitation spots along the optical axis. The technique permits (1) simultaneous excitation of multiple planes in the specimen; (2) control of the number of spots by confocal detection; and (3) overcoming the point-by-point based excitation. Fluorescence detection from the excitation spots can be efficiently achieved by Z-scanning the detector/pinhole assembly. The technique complements most of the bioimaging techniques and may find potential application in high resolution fluorescence microscopy and nanoscale imaging.

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

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.

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.

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.

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.

Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells.

PubMed

Zhang, Yuejin; Wei, Fuxiang; Poh, Yeh-Chuin; Jia, Qiong; Chen, Junjian; Chen, Junwei; Luo, Junyu; Yao, Wenting; Zhou, Wenwen; Huang, Wei; Yang, Fang; Zhang, Yao; Wang, Ning

2017-07-01

Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only a few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. 3D-magnetic twisting cytometry (3D-MTC) is a technique for applying local mechanical stresses to living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors, followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic-field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super-resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real-time acquisition of a living cell's mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC-microscopy platform takes ∼20 d to construct, and the experimental procedures require ∼4 d when carried out by a life sciences graduate student.

Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells

PubMed Central

Zhang, Yuejin; Wei, Fuxiang; Poh, Yeh-Chuin; Jia, Qiong; Chen, Junjian; Chen, Junwei; Luo, Junyu; Yao, Wenting; Zhou, Wenwen; Huang, Wei; Yang, Fang; Zhang, Yao; Wang, Ning

2017-01-01

Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. Three dimensional-Magnetic Twisting Cytometry (3D-MTC) is a technique for applying local mechanical stresses on living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real time acquisition of a living cell’s mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC – microscopy platform takes around 20 days to construct and the experimental procedures require ~4 days when carried out by a life sciences graduate student. PMID:28686583

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.

Advanced chemical imaging and comparison of human and porcine hair follicles for drug delivery by confocal Raman microscopy.

PubMed

Franzen, Lutz; Mathes, Christiane; Hansen, Steffi; Windbergs, Maike

2013-06-01

Hair follicles have recently gained a lot of interest for dermal drug delivery. They provide facilitated penetration into the skin and a high potential to serve as a drug depot. In this area of research, excised pig ear is a widely accepted in vitro model to evaluate penetration of drug delivery into hair follicles. However, a comparison of human and porcine follicles in terms of chemical composition has not been performed so far. In this study, we applied confocal Raman microscopy as a chemically selective imaging technique to compare human and porcine follicle composition and to visualize component distribution within follicle cross-sections. Based on the evaluation of human and porcine Raman spectra optical similarity for both species was successfully confirmed. Furthermore, cyanoacrylate skin surface biopsies, which are generally used to determine the extent of follicular penetration, were imaged by a novel complementary analytical approach combining confocal Raman microscopy and optical profilometry. This all-encompassing analysis allows investigation of intactness and component distribution of the excised hair bulb in three dimensions. Confocal Raman microscopy shows a high potential as a noninvasive and chemically selective technique for the analysis of trans-follicular drug delivery.

Advanced chemical imaging and comparison of human and porcine hair follicles for drug delivery by confocal Raman microscopy

NASA Astrophysics Data System (ADS)

Franzen, Lutz; Mathes, Christiane; Hansen, Steffi; Windbergs, Maike

2013-06-01

Hair follicles have recently gained a lot of interest for dermal drug delivery. They provide facilitated penetration into the skin and a high potential to serve as a drug depot. In this area of research, excised pig ear is a widely accepted in vitro model to evaluate penetration of drug delivery into hair follicles. However, a comparison of human and porcine follicles in terms of chemical composition has not been performed so far. In this study, we applied confocal Raman microscopy as a chemically selective imaging technique to compare human and porcine follicle composition and to visualize component distribution within follicle cross-sections. Based on the evaluation of human and porcine Raman spectra optical similarity for both species was successfully confirmed. Furthermore, cyanoacrylate skin surface biopsies, which are generally used to determine the extent of follicular penetration, were imaged by a novel complementary analytical approach combining confocal Raman microscopy and optical profilometry. This all-encompassing analysis allows investigation of intactness and component distribution of the excised hair bulb in three dimensions. Confocal Raman microscopy shows a high potential as a noninvasive and chemically selective technique for the analysis of trans-follicular drug delivery.

High speed wavefront sensorless aberration correction in digital micromirror based confocal microscopy.

PubMed

Pozzi, P; Wilding, D; Soloviev, O; Verstraete, H; Bliek, L; Vdovin, G; Verhaegen, M

2017-01-23

The quality of fluorescence microscopy images is often impaired by the presence of sample induced optical aberrations. Adaptive optical elements such as deformable mirrors or spatial light modulators can be used to correct aberrations. However, previously reported techniques either require special sample preparation, or time consuming optimization procedures for the correction of static aberrations. This paper reports a technique for optical sectioning fluorescence microscopy capable of correcting dynamic aberrations in any fluorescent sample during the acquisition. This is achieved by implementing adaptive optics in a non conventional confocal microscopy setup, with multiple programmable confocal apertures, in which out of focus light can be separately detected, and used to optimize the correction performance with a sampling frequency an order of magnitude faster than the imaging rate of the system. The paper reports results comparing the correction performances to traditional image optimization algorithms, and demonstrates how the system can compensate for dynamic changes in the aberrations, such as those introduced during a focal stack acquisition though a thick sample.

3D Image Analysis of Geomaterials using Confocal Microscopy

NASA Astrophysics Data System (ADS)

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

2009-05-01

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

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.

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.

Phase sensitive optical coherence microscopy for photothermal imaging of gold nanorods

NASA Astrophysics Data System (ADS)

Hu, Yong; Podoleanu, Adrian G.; Dobre, George

2018-03-01

We describe a swept source based phase sensitive optical coherence microscopy (OCM) system for photothermal imaging of gold nanorods (GNR). The phase sensitive OCM system employed in the study has a displacement sensitivity of 0.17 nm to vibrations at single frequencies below 250 Hz. We demonstrate the generation of phase maps and confocal phase images. By displaying the difference between successive confocal phase images, we perform the confocal photothermal imaging of accumulated GNRs behind a glass coverslip and behind the scattering media separately. Compared with two-photon luminescence (TPL) detection techniques reported in literature, the technique in this study has the advantage of a simplified experimental setup and provides a more efficient method for imaging the aggregation of GNR. However, the repeatability performance of this technique suffers due to jitter noise from the swept laser source.

Evaluation of Characterization Techniques for Carbon-Carbon Composites

DTIC Science & Technology

1992-05-01

Enhancement of Resin (50X) 51 28 Confocal Image of Reticulated , Vitreous Carbon Foam 53 29 Schemmtic Principle of Backscattered Electron Microscopy for...future. 7.2 Confocal Microscopy Both carbon - carbon composites and reticulated vitreous carbon foams were submitted to Sarastro, Inc. to evaluate...indicate 1-micron resolutions are possible; however, the depth penetration is limited even further at these parameters. Six reticulated vitreous carbon

Fibered Confocal Fluorescence Microscopy for the Noninvasive Imaging of Langerhans Cells in Macaques.

PubMed

Todorova, Biliana; Salabert, Nina; Tricot, Sabine; Boisgard, Raphaël; Rathaux, Mélanie; Le Grand, Roger; Chapon, Catherine

2017-01-01

We developed a new approach to visualize skin Langerhans cells by in vivo fluorescence imaging in nonhuman primates. Macaques were intradermally injected with a monoclonal, fluorescently labeled antibody against HLA-DR molecule and were imaged for up to 5 days by fibered confocal microscopy (FCFM). The network of skin Langerhans cells was visualized by in vivo fibered confocal fluorescence microscopy. Quantification of Langerhans cells revealed no changes to cell density with time. Ex vivo experiments confirmed that injected fluorescent HLA-DR antibody specifically targeted Langerhans cells in the epidermis. This study demonstrates the feasibility of single-cell, in vivo imaging as a noninvasive technique to track Langerhans cells in nontransgenic animals.

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.

Confocal filtering in cathodoluminescence microscopy of nanostructures

NASA Astrophysics Data System (ADS)

Narváez, Angela C.; Weppelman, I. Gerward C.; Moerland, Robert J.; Hoogenboom, Jacob P.; Kruit, Pieter

2014-06-01

Cathodoluminescence (CL) microscopy allows optical characterization of nanostructures at high spatial resolution. At the nanoscale, a main challenge of the technique is related to the background CL generated within the sample substrate. Here, we implement confocal detection of the CL signal to minimize the background contribution to the measurement. Nano-phosphors were used as point sources to evaluate the filtering capabilities of our confocal CL system, obtaining an axial intensity profile with 2.7 μm full width at half maximum for the central peak, in good correspondence with theoretical expectations. Considering the electron interaction volume, we found that the confocal filter becomes effective for electron energies above 20 keV, when using a 25 μm pinhole (0.86 Airy units). To illustrate our approach, we present confocal CL imaging of gold nanowires and triangular shaped plates deposited on an indium-tin oxide covered glass substrate, comparing the images with those obtained in standard unfiltered CL detection. The results show that confocal CL microscopy is a valuable tool for the investigation of nanostructures on highly cathodoluminescent substrates, widely used in biological and optical applications.

Capturing the Surface Texture and Shape of Pollen: A Comparison of Microscopy Techniques

PubMed Central

Sivaguru, Mayandi; Mander, Luke; Fried, Glenn; Punyasena, Surangi W.

2012-01-01

Research on the comparative morphology of pollen grains depends crucially on the application of appropriate microscopy techniques. Information on the performance of microscopy techniques can be used to inform that choice. We compared the ability of several microscopy techniques to provide information on the shape and surface texture of three pollen types with differing morphologies. These techniques are: widefield, apotome, confocal and two-photon microscopy (reflected light techniques), and brightfield and differential interference contrast microscopy (DIC) (transmitted light techniques). We also provide a first view of pollen using super-resolution microscopy. The three pollen types used to contrast the performance of each technique are: Croton hirtus (Euphorbiaceae), Mabea occidentalis (Euphorbiaceae) and Agropyron repens (Poaceae). No single microscopy technique provided an adequate picture of both the shape and surface texture of any of the three pollen types investigated here. The wavelength of incident light, photon-collection ability of the optical technique, signal-to-noise ratio, and the thickness and light absorption characteristics of the exine profoundly affect the recovery of morphological information by a given optical microscopy technique. Reflected light techniques, particularly confocal and two-photon microscopy, best capture pollen shape but provide limited information on very fine surface texture. In contrast, transmitted light techniques, particularly differential interference contrast microscopy, can resolve very fine surface texture but provide limited information on shape. Texture comprising sculptural elements that are spaced near the diffraction limit of light (∼250 nm; NDL) presents an acute challenge to optical microscopy. Super-resolution structured illumination microscopy provides data on the NDL texture of A. repens that is more comparable to textural data from scanning electron microscopy than any other optical microscopy technique investigated here. Maximizing the recovery of morphological information from pollen grains should lead to more robust classifications, and an increase in the taxonomic precision with which ancient vegetation can be reconstructed. PMID:22720050

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

PubMed

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

2015-05-01

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

Imaging intracellular protein dynamics by spinning disk confocal microscopy

PubMed Central

Stehbens, Samantha; Pemble, Hayley; Murrow, Lindsay; Wittmann, Torsten

2012-01-01

The palette of fluorescent proteins has grown exponentially over the last decade, and as a result live imaging of cells expressing fluorescently tagged proteins is becoming more and more main stream. Spinning disk confocal microscopy (SDC) is a high speed optical sectioning technique, and a method of choice to observe and analyze intracellular fluorescent protein dynamics at high spatial and temporal resolution. In an SDC system, a rapidly rotating pinhole disk generates thousands of points of light that scan the specimen simultaneously, which allows direct capture of the confocal image with low noise scientific grade cooled charged-coupled device (CCD) cameras, and can achieve frame rates of up 1000 frames per second. In this chapter we describe important components of a state-of-the-art spinning disk system optimized for live cell microscopy, and provide a rationale for specific design choices. We also give guidelines how other imaging techniques such as total internal reflection (TIRF) microscopy or spatially controlled photoactivation can be coupled with SDC imaging, and provide a short protocol on how to generate cell lines stably expressing fluorescently tagged proteins by lentivirus-mediated transduction. PMID:22264541

Longitudinal in vivo two-photon fluorescence imaging

PubMed Central

Crowe, Sarah E.; Ellis-Davies, Graham C.R.

2014-01-01

Fluorescence microscopy is an essential technique for the basic sciences, especially biomedical research. Since the invention of laser scanning confocal microscopy in 1980s, that enabled imaging both fixed and living biological tissue with three-dimensional precision, high-resolution fluorescence imaging has revolutionized biological research. Confocal microscopy, by its very nature, has one fundamental limitation. Due to the confocal pinhole, deep tissue fluorescence imaging is not practical. In contrast (no pun intended), two-photon fluorescence microscopy allows, in principle, the collection of all emitted photons from fluorophores in the imaged voxel, dramatically extending our ability to see deep into living tissue. Since the development of transgenic mice with genetically encoded fluorescent protein in neocortical cells in 2000, two-photon imaging has enabled the dynamics of individual synapses to be followed for up to two years. Since the initial landmark contributions to this field in 2002, the technique has been used to understand how neuronal structure are changed by experience, learning and memory and various diseases. Here we provide a basic summary of the crucial elements that are required for such studies, and discuss many applications of longitudinal two-photon fluorescence microscopy that have appeared since 2002. PMID:24214350

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

In vivo confocal microscopy in dermatology: from research to clinical application

NASA Astrophysics Data System (ADS)

Ulrich, Martina; Lange-Asschenfeldt, Susanne

2013-06-01

Confocal laser scanning microscopy (CLSM) represents an emerging technique for the noninvasive histomorphological analysis of skin in vivo and has shown its applicability for dermatological research as well as its value as an adjunct tool in the clinical management of skin cancer patients. Herein, we aim to give an overview on the current clinical indications for CLSM in dermatology and also highlight the diverse applications of CLSM in dermatological research.

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

CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography).

PubMed

Siegel, Nisan; Storrie, Brian; Bruce, Marc; Brooker, Gary

2015-02-07

FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called "CINCH". An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution.

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.

Cornea and anterior eye assessment with slit lamp biomicroscopy, specular microscopy, confocal microscopy, and ultrasound biomicroscopy

PubMed Central

Martin, Raul

2018-01-01

Current corneal assessment technologies make the process of corneal evaluation extremely fast and simple, and several devices and technologies show signs that help in identification of different diseases thereby, helping in diagnosis, management, and follow-up of patients. The purpose of this review is to present and update readers on the evaluation of cornea and ocular surface. This first part reviews a description of slit lamp biomicroscopy (SLB), endothelial specular microscopy, confocal microscopy, and ultrasound biomicroscopy examination techniques and the second part describes the corneal topography and tomography, providing up-to-date information on the clinical recommendations of these techniques in eye care practice. Although the SLB is a traditional technique, it is of paramount importance in clinical diagnosis and compulsory when an eye test is conducted in primary or specialist eye care practice. Different techniques allow the early diagnosis of many diseases, especially when clinical signs have not yet become apparent and visible with SLB. These techniques also allow for patient follow-up in several clinical conditions or diseases, facilitating clinical decisions and improving knowledge regarding the corneal anatomy. PMID:29380757

Realistic representation of Bacillus subtilis biofilms architecture using combined microscopy (CLSM, ESEM and FESEM).

PubMed

Bridier, A; Meylheuc, T; Briandet, R

2013-05-01

In this contribution, we used a set of microscopic techniques including confocal laser scanning microscopy (CLSM), environmental scanning electron microscopy (ESEM) and field emission scanning electron microscopy (FESEM) to analyze the three-dimensional spatial arrangement of cells and their surrounding matrix in Bacillus subtilis biofilm. The combination of the different techniques enabled a deeper and realistic deciphering of biofilm architecture by providing the opportunity to overcome the limits of each single technique. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

Applications of microscopy to genetic therapy of cystic fibrosis and other human diseases.

PubMed

Moninger, Thomas O; Nessler, Randy A; Moore, Kenneth C

2006-01-01

Gene therapy has become an extremely important and active field of biomedical research. Microscopy is an integral component of this effort. This chapter presents an overview of imaging techniques used in our facility in support of cystic fibrosis gene therapy research. Instrumentation used in these studies includes light and confocal microscopy, transmission electron microscopy, and scanning electron microscopy. Techniques outlined include negative staining, cryo-electron microscopy, three-dimentional reconstruction, enzyme cytochemistry, immunocytochemistry, and fluorescence imaging.

Correlation mapping microscopy

NASA Astrophysics Data System (ADS)

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

2015-03-01

Changes in the microcirculation are associated with conditions such as Raynauds disease. Current modalities used to assess the microcirculation such as nailfold capillaroscopy are limited due to their depth ambiguity. A correlation mapping technique was recently developed to extend the capabilities of Optical Coherence Tomography to generate depth resolved images of the microcirculation. Here we present the extension of this technique to microscopy modalities, including confocal microscopy. It is shown 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.

Confocal microscopy as a useful approach to describe gill rakers of Asian species of carp and native filter-feeding fishes of the upper Mississippi River system

USGS Publications Warehouse

Liza R. Walleser,; D.R. Howard,; Sandheinrich, Mark B.; Gaikowski, Mark P.; Amberg, Jon J.

2014-01-01

To better understand potential diet overlap among exotic Asian species of carp and native species of filter-feeding fishes of the upper Mississippi River system, microscopy was used to document morphological differences in the gill rakers. Analysing samples first with light microscopy and subsequently with confocal microscopy, the three-dimensional structure of gill rakers in Hypophthalmichthys molitrix,Hypophthalmichthys nobilis and Dorosoma cepedianum was more thoroughly described and illustrated than previous work with traditional microscopy techniques. The three-dimensional structure of gill rakers in Ictiobus cyprinellus was described and illustrated for the first time.

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.

Confocal laser scanning microscopy of porcine skin: implications for human wound healing studies

PubMed Central

VARDAXIS, N. J.; BRANS, T. A.; BOON, M. E.; KREIS, R. W.; MARRES, L. M.

1997-01-01

The structure of porcine skin as examined by light microscopy is reviewed and its similarities to and differences from human skin are highlighted. Special imaging techniques and staining procedures are described and their use in gathering morphological information in porcine skin is discussed. Confocal laser scanning microscopy (CLSM) was employed to examine the structure of porcine skin and the findings are presented as an adjunct to the information already available in the literature. It is concluded that CLSM provides valuable additional morphological information to material examined by conventional microscopy and is useful for wound healing studies in the porcine model. PMID:9183682

En-face Flying Spot OCT/Ophthalmoscope

NASA Astrophysics Data System (ADS)

Rosen, Richard B.; Garcia, Patricia; Podoleanu, Adrian Gh.; Cucu, Radu; Dobre, George; Trifanov, Irina; van Velthoven, Mirjam E. J.; de Smet, Marc D.; Rogers, John A.; Hathaway, Mark; Pedro, Justin; Weitz, Rishard

This is a review of a technique for high-resolution imaging of the eye that allows multiple sample sectioning perspectives with different axial resolutions. The technique involves the flying spot approach employed in confocal scanning laser ophthalmoscopy which is extended to OCT imaging via time domain en face fast lateral scanning. The ability of imaging with multiple axial resolutions stimulated the development of the dual en face OCT-confocal imaging technology. Dual imaging also allows various other imaging combinations, such as OCT with confocal microscopy for imaging the eye anterior segment and OCT with fluorescence angiography imaging.

'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.

High-resolution measurements of the multilayer ultra-structure of articular cartilage and their translational potential

PubMed Central

2014-01-01

Current musculoskeletal imaging techniques usually target the macro-morphology of articular cartilage or use histological analysis. These techniques are able to reveal advanced osteoarthritic changes in articular cartilage but fail to give detailed information to distinguish early osteoarthritis from healthy cartilage, and this necessitates high-resolution imaging techniques measuring cells and the extracellular matrix within the multilayer structure of articular cartilage. This review provides a comprehensive exploration of the cellular components and extracellular matrix of articular cartilage as well as high-resolution imaging techniques, including magnetic resonance image, electron microscopy, confocal laser scanning microscopy, second harmonic generation microscopy, and laser scanning confocal arthroscopy, in the measurement of multilayer ultra-structures of articular cartilage. This review also provides an overview for micro-structural analysis of the main components of normal or osteoarthritic cartilage and discusses the potential and challenges associated with developing non-invasive high-resolution imaging techniques for both research and clinical diagnosis of early to late osteoarthritis. PMID:24946278

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.

Confocal filtering in cathodoluminescence microscopy of nanostructures

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

Narváez, Angela C., E-mail: a.c.narvaez@tudelft.nl, E-mail: j.p.hoogenboom@tudelft.nl; Weppelman, I. Gerward C.; Moerland, Robert J.

2014-06-23

Cathodoluminescence (CL) microscopy allows optical characterization of nanostructures at high spatial resolution. At the nanoscale, a main challenge of the technique is related to the background CL generated within the sample substrate. Here, we implement confocal detection of the CL signal to minimize the background contribution to the measurement. Nano-phosphors were used as point sources to evaluate the filtering capabilities of our confocal CL system, obtaining an axial intensity profile with 2.7 μm full width at half maximum for the central peak, in good correspondence with theoretical expectations. Considering the electron interaction volume, we found that the confocal filter becomes effectivemore » for electron energies above 20 keV, when using a 25 μm pinhole (0.86 Airy units). To illustrate our approach, we present confocal CL imaging of gold nanowires and triangular shaped plates deposited on an indium-tin oxide covered glass substrate, comparing the images with those obtained in standard unfiltered CL detection. The results show that confocal CL microscopy is a valuable tool for the investigation of nanostructures on highly cathodoluminescent substrates, widely used in biological and optical applications.« less

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.

Imaging of Scleral Collagen Deformation Using Combined Confocal Raman Microspectroscopy and Polarized Light Microscopy Techniques.

PubMed

Chakraborty, Nilay; Wang, Mian; Solocinski, Jason; Kim, Wonsuk; Argento, Alan

2016-01-01

This work presents an optospectroscopic characterization technique for soft tissue microstructure using site-matched confocal Raman microspectroscopy and polarized light microscopy. Using the technique, the microstructure of soft tissue samples is directly observed by polarized light microscopy during loading while spatially correlated spectroscopic information is extracted from the same plane, verifying the orientation and arrangement of the collagen fibers. Results show the response and orientation of the collagen fiber arrangement in its native state as well as during tensile and compressive loadings in a porcine sclera model. An example is also given showing how the data can be used with a finite element program to estimate the strain in individual collagen fibers. The measurements demonstrate features that indicate microstructural reorganization and damage of the sclera's collagen fiber arrangement under loading. The site-matched confocal Raman microspectroscopic characterization of the tissue provides a qualitative measure to relate the change in fibrillar arrangement with possible chemical damage to the collagen microstructure. Tests and analyses presented here can potentially be used to determine the stress-strain behavior, and fiber reorganization of the collagen microstructure in soft tissue during viscoelastic response.

CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography)

PubMed Central

Siegel, Nisan; Storrie, Brian; Bruce, Marc

2016-01-01

FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called “CINCH”. An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution. PMID:26839443

Spinning Disk Confocal Imaging of Neutrophil Migration in Zebrafish

PubMed Central

Lam, Pui-ying; Fischer, Robert S; Shin, William D.; Waterman, Clare M; Huttenlocher, Anna

2014-01-01

Live-cell imaging techniques have been substantially improved due to advances in confocal microscopy instrumentation coupled with ultrasensitive detectors. The spinning disk confocal system is capable of generating images of fluorescent live samples with broad dynamic range and high temporal and spatial resolution. The ability to acquire fluorescent images of living cells in vivo on a millisecond timescale allows the dissection of biological processes that have not previously been visualized in a physiologically relevant context. In vivo imaging of rapidly moving cells such as neutrophils can be technically challenging. In this chapter, we describe the practical aspects of imaging neutrophils in zebrafish embryos using spinning disk confocal microscopy. Similar setups can also be applied to image other motile cell types and signaling processes in translucent animals or tissues. PMID:24504955

Learning reflectance confocal microscopy of melanocytic skin lesions through histopathologic transversal sections.

PubMed

Braga, Juliana Casagrande Tavoloni; Macedo, Mariana Petaccia; Pinto, Clovis; Duprat, João; Begnami, Maria Dirlei; Pellacani, Giovanni; Rezze, Gisele Gargantini

2013-01-01

Histopathologic interpretation of dermoscopic and reflectance confocal microscopy (RCM) features of cutaneous melanoma was timidly carried out using perpendicular histologic sections, which does not mimic the same plane of the image achieved at both techniques (horizontal plane). The aim of this study was to describe the transverse histologic sections research technique and correlate main dermoscopic features characteristic of cutaneous melanoma (atypical network, irregular globules and pseudopods) with RCM and histopathology in perpendicular and transverse sections in order to offer a more precise interpretation of in vivo detectable features. Four melanomas and 2 nevi with different dermoscopic clues have been studied. Lesion areas that showed characteristic dermoscopic features were imaged by dermoscopy and confocal microscopy and directly correlated with histopathology in perpendicular and transverse sections. We presented the possibility to perform transverse sections as a new approach to understand RCM features. Atypical network showed different aspects in the 2 melanomas: in one case it was characterized by pleomorphic malignant melanocytes with tendency to form aggregates, whereas in the other elongated dendritic cells crowded around dermal papillae, some of them forming bridges that resembled the mitochondrial aspect at confocal and histopathology transversal sections. Pigment globules in melanomas and nevi differed for the presence of large atypical cells in the former, and pseudopods showed up as elongated nests protruded toward the periphery of the lesion. Transverse histologic research sections have a consistent dermoscopic and confocal correlate, and it may represent an help in confocal feature interpretation and an advance in improving melanoma diagnosis and knowledge of the biology of melanocytic lesions.

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.

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.

Lipids in cheese

USDA-ARS?s Scientific Manuscript database

Lipids are present in cheese at levels above 20 percent and are analyzed by several techniques. Scanning electron microscopy and confocal laser scanning microscopy are used to examine the microstructure, gas chromatography is employed to look at fatty acid composition, and differential scanning cal...

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.

In vivo confocal microscopy, an inner vision of the cornea - a major review.

PubMed

Guthoff, Rudolf F; Zhivov, Andrey; Stachs, Oliver

2009-01-01

The demands of modern ophthalmology have evolved from descriptive findings from the slit lamp to in vivo assessment of cellular level changes. Nowadays, the latter can be provided by in vivo confocal microscopy. This article gives an overview of confocal principles using tandem scanning, scanning slit and laser scanning techniques used in ophthalmology. The main part of the paper describes the clinical applications emphasizing the anatomy of the normal and pathological cornea, and illustrates side-effects of topical medication, contact lens wear, cross-linking and refractive surgery. Finally, a summary about experimental applications, including animal studies, surface characterization and volume rendering as well as future developments, is given.

In vivo confocal microscopy of the cornea: New developments in image acquisition, reconstruction and analysis using the HRT-Rostock Corneal Module

PubMed Central

Petroll, W. Matthew; Robertson, Danielle M.

2015-01-01

The optical sectioning ability of confocal microscopy allows high magnification images to be obtained from different depths within a thick tissue specimen, and is thus ideally suited to the study of intact tissue in living subjects. In vivo confocal microscopy has been used in a variety of corneal research and clinical applications since its development over 25 years ago. In this article we review the latest developments in quantitative corneal imaging with the Heidelberg Retinal Tomograph with Rostock Corneal Module (HRT-RCM). We provide an overview of the unique strengths and weaknesses of the HRT-RCM. We discuss techniques for performing 3-D imaging with the HRT-RCM, including hardware and software modifications that allow full thickness confocal microscopy through focusing (CMTF) of the cornea, which can provide quantitative measurements of corneal sublayer thicknesses, stromal cell and extracellular matrix backscatter, and depth dependent changes in corneal keratocyte density. We also review current approaches for quantitative imaging of the subbasal nerve plexus, which require a combination of advanced image acquisition and analysis procedures, including wide field mapping and 3-D reconstruction of nerve structures. The development of new hardware, software, and acquisition techniques continues to expand the number of applications of the HRT-RCM for quantitative in vivo corneal imaging at the cellular level. Knowledge of these rapidly evolving strategies should benefit corneal clinicians and basic scientists alike. PMID:25998608

Inserting ex vivo fluorescence confocal microscopy perioperatively in Mohs micrographic surgery expedites bedside assessment of excision margins in recurrent basal cell carcinoma.

PubMed

Longo, Caterina; Ragazzi, Moira; Castagnetti, Fabio; Gardini, Stefano; Palmieri, Tamara; Lallas, Aimilios; Moscarella, Elvira; Piana, Simonetta; Pellacani, Giovanni; Zalaudek, Iris; Argenziano, Giuseppe

2013-01-01

Mohs micrographic surgery can be employed in recurrent basal cell carcinoma, although it is a time-consuming technique. Recently, ex vivo fluorescence confocal microscopy (FCM) has been employed to obtain a fast assessment of tumor margins at the bedside. In our case we successfully employed ex vivo FCM to assess the tumor margins and we treated the persistent tumor with intensity-modulated radiation therapy. Our case demonstrates that a multidisciplinary approach is very efficient in managing complex and recurrent tumors and highlights the benefits of FCM as a new technique that can be used in the surgical theater to speed up the entire procedure.

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

Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.

PubMed

Jemielita, Matthew; Taormina, Michael J; Delaurier, April; Kimmel, Charles B; Parthasarathy, Raghuveer

2013-12-01

The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

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.

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

NASA Astrophysics Data System (ADS)

Han, Wenfeng; Xiao, Zexin; Wang, Xiaofen

2009-11-01

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

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.

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.

Confocal Raman microscopy to monitor extracellular matrix during dental pulp stem cells differentiation

NASA Astrophysics Data System (ADS)

Salehi, Hamideh; Collart-Dutilleul, Pierre-Yves; Gergely, Csilla; Cuisinier, Frédéric J. G.

2015-07-01

Regenerative medicine brings promising applications for mesenchymal stem cells, such as dental pulp stem cells (DPSCs). Confocal Raman microscopy, a noninvasive technique, is used to study osteogenic differentiation of DPSCs. Integrated Raman intensities in the 2800 to 3000 cm-1 region (C-H stretching) and the 960 cm-1 peak (ν1 PO43-) were collected (to image cells and phosphate, respectively), and the ratio of two peaks 1660 over 1690 cm-1 (amide I bands) to measure the collagen cross-linking has been calculated. Raman spectra of DPSCs after 21 days differentiation reveal several phosphate peaks: ν1 (first stretching mode) at 960 cm-1, ν2 at 430 cm-1, and ν4 at 585 cm-1 and collagen cross-linking can also be calculated. Confocal Raman microscopy enables monitoring osteogenic differentiation in vitro and can be a credible tool for clinical stem cell based research.

Reflectance Confocal Microscopy in Lentigo Maligna.

PubMed

Gamo, R; Pampín, A; Floristán, U

2016-12-01

Lentigo maligna is the most common type of facial melanoma. Diagnosis is complicated, however, as it shares clinical and dermoscopic characteristics with other cutaneous lesions of the face. Reflectance confocal microscopy is an imaging technique that permits the visualization of characteristic features of lentigo maligna. These include a disrupted honeycomb pattern and pagetoid cells with a tendency to show folliculotropism. These cells typically have a dendritic morphology, although they may also appear as round cells measuring over 20μm with atypical nuclei. Poorly defined dermal papillae and atypical cells may be seen at the dermal-epidermal junction and can form bridges resembling mitochondrial structures. Other characteristic findings include junctional swelling with atypical cells located around the follicles, resembling caput medusae. Reflectance confocal microscopy is a very useful tool for diagnosing lentigo maligna. Copyright © 2016 AEDV. Publicado por Elsevier España, S.L.U. All rights reserved.

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.

Determining the size of nanoparticles in the example of magnetic iron oxide core-shell systems

NASA Astrophysics Data System (ADS)

Jarzębski, Maciej; Kościński, Mikołaj; Białopiotrowicz, Tomasz

2017-08-01

The size of nanoparticles is one of the most important factors for their possible applications. Various techniques for the nanoparticle size characterization are available. In this paper selected techniques will be considered base on the prepared core-shell magnetite nanoparticles. Magnetite is one of the most investigated and developed magnetic material. It shows interesting magnetic properties which can be used for biomedical applications, such as drug delivery, hypothermia and also as a contrast agent. To reduce the toxic effects of Fe3O4, magnetic core was covered by dextran and gelatin. Moreover, the shell was doped by fluorescent dye for confocal microscopy investigation. The main investigation focused on the methods for particles size determination of modified magnetite nanoparticles prepared with different techniques. The size distribution were obtained by nanoparticle tracking analysis, dynamic light scattering and transmission electron microscopy. Furthermore, fluorescent correlation spectroscopy (FCS) and confocal microscopy were used to compare the results for particle size determination of core-shell systems.

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.

Quantification of confocal fluorescence microscopy for the detection of cervical intraepithelial neoplasia.

PubMed

Sheikhzadeh, Fahime; Ward, Rabab K; Carraro, Anita; Chen, Zhao Yang; van Niekerk, Dirk; Miller, Dianne; Ehlen, Tom; MacAulay, Calum E; Follen, Michele; Lane, Pierre M; Guillaud, Martial

2015-10-24

Cervical cancer remains a major health problem, especially in developing countries. Colposcopic examination is used to detect high-grade lesions in patients with a history of abnormal pap smears. New technologies are needed to improve the sensitivity and specificity of this technique. We propose to test the potential of fluorescence confocal microscopy to identify high-grade lesions. We examined the quantification of ex vivo confocal fluorescence microscopy to differentiate among normal cervical tissue, low-grade Cervical Intraepithelial Neoplasia (CIN), and high-grade CIN. We sought to (1) quantify nuclear morphology and tissue architecture features by analyzing images of cervical biopsies; and (2) determine the accuracy of high-grade CIN detection via confocal microscopy relative to the accuracy of detection by colposcopic impression. Forty-six biopsies obtained from colposcopically normal and abnormal cervical sites were evaluated. Confocal images were acquired at different depths from the epithelial surface and histological images were analyzed using in-house software. The features calculated from the confocal images compared well with those features obtained from the histological images and histopathological reviews of the specimens (obtained by a gynecologic pathologist). The correlations between two of these features (the nuclear-cytoplasmic ratio and the average of three nearest Delaunay-neighbors distance) and the grade of dysplasia were higher than that of colposcopic impression. The sensitivity of detecting high-grade dysplasia by analysing images collected at the surface of the epithelium, and at 15 and 30 μm below the epithelial surface were respectively 100, 100, and 92 %. Quantitative analysis of confocal fluorescence images showed its capacity for discriminating high-grade CIN lesions vs. low-grade CIN lesions and normal tissues, at different depth of imaging. This approach could be used to help clinicians identify high-grade CIN in clinical settings.

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.

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.

Review of advanced imaging techniques

PubMed Central

Chen, Yu; Liang, Chia-Pin; Liu, Yang; Fischer, Andrew H.; Parwani, Anil V.; Pantanowitz, Liron

2012-01-01

Pathology informatics encompasses digital imaging and related applications. Several specialized microscopy techniques have emerged which permit the acquisition of digital images (“optical biopsies”) at high resolution. Coupled with fiber-optic and micro-optic components, some of these imaging techniques (e.g., optical coherence tomography) are now integrated with a wide range of imaging devices such as endoscopes, laparoscopes, catheters, and needles that enable imaging inside the body. These advanced imaging modalities have exciting diagnostic potential and introduce new opportunities in pathology. Therefore, it is important that pathology informaticists understand these advanced imaging techniques and the impact they have on pathology. This paper reviews several recently developed microscopic techniques, including diffraction-limited methods (e.g., confocal microscopy, 2-photon microscopy, 4Pi microscopy, and spatially modulated illumination microscopy) and subdiffraction techniques (e.g., photoactivated localization microscopy, stochastic optical reconstruction microscopy, and stimulated emission depletion microscopy). This article serves as a primer for pathology informaticists, highlighting the fundamentals and applications of advanced optical imaging techniques. PMID:22754737

Corneal Confocal Microscopy Detects Corneal Nerve Damage in Patients Admitted With Acute Ischemic Stroke.

PubMed

Khan, Adnan; Akhtar, Naveed; Kamran, Saadat; Ponirakis, Georgios; Petropoulos, Ioannis N; Tunio, Nahel A; Dargham, Soha R; Imam, Yahia; Sartaj, Faheem; Parray, Aijaz; Bourke, Paula; Khan, Rabia; Santos, Mark; Joseph, Sujatha; Shuaib, Ashfaq; Malik, Rayaz A

2017-11-01

Corneal confocal microscopy can identify corneal nerve damage in patients with peripheral and central neurodegeneration. However, the use of corneal confocal microscopy in patients presenting with acute ischemic stroke is unknown. One hundred thirty patients (57 without diabetes mellitus [normal glucose tolerance], 32 with impaired glucose tolerance, and 41 with type 2 diabetes mellitus) admitted with acute ischemic stroke, and 28 age-matched healthy control participants underwent corneal confocal microscopy to quantify corneal nerve fiber density, corneal nerve branch density, and corneal nerve fiber length. There was a significant reduction in corneal nerve fiber density, corneal nerve branch density, and corneal nerve fiber length in stroke patients with normal glucose tolerance ( P <0.001, P <0.001, P <0.001), impaired glucose tolerance ( P =0.004, P <0.001, P =0.002), and type 2 diabetes mellitus ( P <0.001, P <0.001, P <0.001) compared with controls. HbA1c and triglycerides correlated with corneal nerve fiber density ( r =-0.187, P =0.03; r =-0.229 P =0.01), corneal nerve fiber length ( r =-0.228, P =0.009; r =-0.285; P =0.001), and corneal nerve branch density ( r =-0.187, P =0.033; r =-0.229, P =0.01). Multiple linear regression showed no independent associations between corneal nerve fiber density, corneal nerve branch density, and corneal nerve fiber length and relevant risk factors for stroke. Corneal confocal microscopy is a rapid noninvasive ophthalmic imaging technique that identifies corneal nerve fiber loss in patients with acute ischemic stroke. © 2017 American Heart Association, Inc.

DURIP: Super-Resolution Module for Confocal Microscopy of Reconfigurable Matter

DTIC Science & Technology

2014-09-28

Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 superresolution microscopy, colloidal particles, self-assembly REPORT...previously have been resolved by optical microscopy. Results of Super Resolution Technique Evaluation Commercially available superresolution imaging...Weaknesses of the method are that is fundamentally a measurement that can only be deployed for fixed samples. Because superresolution is obtained by

Subsurface microscopy of interconnect layers of an integrated circuit.

PubMed

Köklü, F Hakan; Unlü, M Selim

2010-01-15

We apply the NA-increasing lens technique to confocal and wide-field backside microscopy of integrated circuits. We demonstrate 325 nm (lambda(0)/4) lateral spatial resolution while imaging metal structures located inside the interconnect layer of an integrated circuit. Vectorial field calculations are presented justifying our findings.

Exploiting chromatic aberration to spectrally encode depth in reflectance confocal microscopy

NASA Astrophysics Data System (ADS)

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

2011-06-01

We present chromatic confocal microscopy as a technique to axially scan the sample by spectrally encoding depth information to avoid mechanical scanning of the lens or sample. We have achieved an 800 μm focal shift over a range of 680-1080 nm using a hyperchromat lens as the imaging lens. A more complex system that incorporates a water immersion objective to improve axial resolution was built and tested. We determined that increasing objective magnification decreases chromatic shift while improving axial resolution. Furthermore, collimating after the hyperchromat at longer wavelengths yields an increase in focal shift.

In vivo confocal microscopy of human cornea covered with human amniotic membrane.

PubMed

Mimura, Tatsuya; Yamagami, Satoru; Usui, Tomohiko; Honda, Norihiko; Araki, Fumiyuki; Amano, Shiro

2008-01-01

Amniotic membrane transplantation has been widely performed to reconstruct the surface of the eye and treat chemical burns or epithelial defects. However, we have difficulty observing the cornea through the opaque transplanted amniotic membrane by slit-lamp biomicroscopy. We investigated the use of confocal microscopy for observation of human corneas covered with amniotic membrane. Human amniotic membrane was placed onto the normal corneas of five volunteers aged 22-24 years. Then, all layers of the covered corneas were observed by in vivo confocal microscopy. Confocal microscopy displayed the epithelium, basement membrane, and stroma of the amniotic membrane. It also displayed the corneal epithelium. Furthermore, corneal stromal keratocytes and the corneal endothelium were clearly observed through the amniotic membrane by confocal microscopy. We demonstrated that in vivo confocal microscopy enabled us to observe all layers of corneas covered with amniotic membrane in normal human eyes. Our findings suggest that confocal microscopy may have advantages for clinical examination of the ocular surface, including all layers of the cornea.

Progress in the Correlative Atomic Force Microscopy and Optical Microscopy

PubMed Central

Zhou, Lulu; Cai, Mingjun; Tong, Ti; Wang, Hongda

2017-01-01

Atomic force microscopy (AFM) has evolved from the originally morphological imaging technique to a powerful and multifunctional technique for manipulating and detecting the interactions between molecules at nanometer resolution. However, AFM cannot provide the precise information of synchronized molecular groups and has many shortcomings in the aspects of determining the mechanism of the interactions and the elaborate structure due to the limitations of the technology, itself, such as non-specificity and low imaging speed. To overcome the technical limitations, it is necessary to combine AFM with other complementary techniques, such as fluorescence microscopy. The combination of several complementary techniques in one instrument has increasingly become a vital approach to investigate the details of the interactions among molecules and molecular dynamics. In this review, we reported the principles of AFM and optical microscopy, such as confocal microscopy and single-molecule localization microscopy, and focused on the development and use of correlative AFM and optical microscopy. PMID:28441775

Volumetry of human taste buds using laser scanning microscopy.

PubMed

Just, T; Srur, E; Stachs, O; Pau, H W

2009-10-01

In vivo laser scanning confocal microscopy is a relatively new, non-invasive method for assessment of oral cavity epithelia. The penetration depth of approximately 200-400 microm allows visualisation of fungiform papillae and their taste buds. This paper describes the technique of in vivo volumetry of human taste buds. Confocal laser scanning microscopy used a diode laser at 670 nm for illumination. Digital laser scanning confocal microscopy equipment consisted of the Heidelberg Retina Tomograph HRTII and the Rostock Cornea Module. Volume scans of fungiform papillae were used for three-dimensional reconstruction of the taste bud. This technique supplied information on taste bud structure and enabled measurement and calculation of taste bud volume. Volumetric data from a 23-year-old man over a nine-day period showed only a small deviation in values. After three to four weeks, phenomenological changes in taste bud structures were found (i.e. a significant increase in volume, followed by disappearance of the taste bud and appearance of a new taste bud). The data obtained indicate the potential application of this non-invasive imaging modality: to evaluate variation of taste bud volume in human fungiform papillae with ageing; to study the effects of chorda tympani nerve transection on taste bud volume; and to demonstrate recovery of taste buds in patients with a severed chorda tympani nerve who show recovery of gustatory sensibility after surgery.

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

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.

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.

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

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.

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.

Corneal confocal microscopy detects small fiber neuropathy in CMT1A patients

PubMed Central

Tavakoli, Mitra; Marshall, Andy; Banka, Siddharth; Petropoulos, Ioannis N; Fadavi, Hassan; Kingston, Helen; Malik, Rayaz A

2012-01-01

Although unmyelinated nerve fibers are affected in CMT1A, they have not been studied in detail due to the invasive nature of the techniques needed to study them. We established alterations in C-fiber bundles of the cornea in patients with CMT1A using non-invasive corneal confocal microscopy (CCM). Twelve patients with CMT1A and twelve healthy control subjects underwent assessment of neuropathic symptoms and deficits, electrophysiology, quantitative sensory testing, corneal sensitivity and corneal confocal microscopy. Corneal sensitivity, corneal nerve fiber density, corneal nerve branch density, corneal nerve fiber length and corneal nerve fiber tortuosity were significantly reduced in CMT1A patients compared to controls. There was a significant correlation between corneal sensation and CCM parameters with the severity of painful neuropathic symptoms, cold and warm thresholds and median nerve CMAP amplitude. CCM demonstrates significant damage to C-fiber bundles, which relates to some measures of neuropathy in CMT1A patients. PMID:22996176

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.

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.

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.

In Vivo and Ex Vivo Confocal Microscopy for Dermatologic and Mohs Surgeons.

PubMed

Longo, Caterina; Ragazzi, Moira; Rajadhyaksha, Milind; Nehal, Kishwer; Bennassar, Antoni; Pellacani, Giovanni; Malvehy Guilera, Josep

2016-10-01

Confocal microscopy is a modern imaging device that has been extensively applied in skin oncology. More specifically, for tumor margin assessment, it has been used in two modalities: reflectance mode (in vivo on skin patient) and fluorescence mode (on freshly excised specimen). Although in vivo reflectance confocal microscopy is an add-on tool for lentigo maligna mapping, fluorescence confocal microscopy is far superior for basal cell carcinoma and squamous cell carcinoma margin assessment in the Mohs setting. This article provides a comprehensive overview of the use of confocal microscopy for skin cancer margin evaluation. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Spontaneous confocal Raman microscopy--a tool to study the uptake of nanoparticles and carbon nanotubes into cells

NASA Astrophysics Data System (ADS)

Romero, Gabriela; Rojas, Elena; Estrela-Lopis, Irina; Donath, Edwin; Moya, Sergio Enrique

2011-06-01

Confocal Raman microscopy as a label-free technique was applied to study the uptake and internalization of poly(lactide- co-glycolide) (PLGA) nanoparticles (NPs) and carbon nanotubes (CNTs) into hepatocarcinoma human HepG2 cells. Spontaneous confocal Raman spectra was recorded from the cells exposed to oxidized CNTs and to PLGA NPs. The Raman spectra showed bands arising from the cellular environment: lipids, proteins, nucleic acids, as well as bands characteristic for either PLGA NPs or CNTs. The simultaneous generation of Raman bands from the cell and nanomaterials from the same spot proves internalization, and also indicates the cellular region, where the nanomaterial is located. For PLGA NPs, it was found that they preferentially co-localized with lipid bodies, while the oxidized CNTs are located in the cytoplasm.

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

PubMed

Guo, H X; Heinämäki, J; Yliruusi, J

1999-09-20

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

Recent progress in tissue optical clearing for spectroscopic application

NASA Astrophysics Data System (ADS)

Sdobnov, A. Yu.; Darvin, M. E.; Genina, E. A.; Bashkatov, A. N.; Lademann, J.; Tuchin, V. V.

2018-05-01

This paper aims to review recent progress in optical clearing of the skin and over naturally turbid biological tissues and blood using this technique in vivo and in vitro with multiphoton microscopy, confocal Raman microscopy, confocal microscopy, NIR spectroscopy, optical coherence tomography, and laser speckle contrast imaging. Basic principles of the technique, its safety, advantages and limitations are discussed. The application of optical clearing agent on a tissue allows for controlling the optical properties of tissue. Optical clearing-induced reduction of tissue scattering significantly facilitates the observation of deep-located tissue regions, at the same time improving the resolution and image contrast for a variety of optical imaging methods suitable for clinical applications, such as diagnostics and laser treatment of skin diseases, mucosal tumor imaging, laser disruption of pathological abnormalities, etc. Structural images of different skin layers obtained ex vivo for porcine ear skin samples at application of Omnipaque™ and glycerol solutions during 60 min. Red color corresponds to TPEAF signal channel. Green color corresponds to SHG signal channel.

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

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.

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

High-speed bioimaging with frequency-division-multiplexed fluorescence confocal microscopy

NASA Astrophysics Data System (ADS)

Mikami, Hideharu; Harmon, Jeffrey; Ozeki, Yasuyuki; Goda, Keisuke

2017-04-01

We present methods of fluorescence confocal microscopy that enable unprecedentedly high frame rate of > 10,000 fps. The methods are based on a frequency-division multiplexing technique, which was originally developed in the field of communication engineering. Specifically, we achieved a broad bandwidth ( 400 MHz) of detection signals using a dual- AOD method and overcame limitations in frame rate, due to a scanning device, by using a multi-line focusing method, resulting in a significant increase in frame rate. The methods have potential biomedical applications such as observation of sub-millisecond dynamics in biological tissues, in-vivo three-dimensional imaging, and fluorescence imaging flow cytometry.

Characterization of Polymer Blends: Optical Microscopy (*Polarized, Interference and Phase Contrast Microscopy*) and Confocal Microscopy

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

Ramanathan, Nathan Muruganathan; Darling, Seth B.

2015-01-01

Chapter 15 surveys the characterization of macro, micro and meso morphologies of polymer blends by optical microscopy. Confocal Microscopy offers the ability to view the three dimensional morphology of polymer blends, popular in characterization of biological systems. Confocal microscopy uses point illumination and a spatial pinhole to eliminate out-of focus light in samples that are thicker than the focal plane.

Morphological and ultrastructural characterization of ionoregulatory cells in the teleost Oreochromis niloticus following salinity challenge combining complementary confocal scanning laser microscopy and transmission electron microscopy using a novel prefixation immunogold labeling technique.

PubMed

Fridman, Sophie; Rana, Krishen J; Bron, James E

2013-10-01

Aspects of ionoregulatory or mitochondria-rich cell (MRC) differentiation and adaptation in Nile tilapia yolk-sac larvae following transfer from freshwater to elevated salinities, that is, 12.5 and 20 ppt are described. Investigations using immunohistochemistry on whole-mount Nile tilapia larvae using anti- Na⁺/K⁺-ATPase as a primary antibody and Fluoronanogold™ (Nanoprobes) as a secondary immunoprobe allowed fluorescent labeling with the high resolution of confocal scanning laser microscopy combined with the detection of immunolabeled target molecules at an ultrastructural level using transmission electron microscopy (TEM). It reports, for the first time, various developmental stages of MRCs within the epithelial layer of the tail of yolk-sac larvae, corresponding to immature, developing, and mature MRCs, identifiable by their own characteristic ultrastructure and form. Following transfer to hyperosmotic salinities the density of immunogold particles and well as the intricacy of the tubular system appeared to increase. In addition, complementary confocal scanning laser microscopy allowed identification of immunopositive ramifying extensions that appeared to emanate from the basolateral portion of the cell that appeared to be correlated with the localization of subsurface tubular areas displaying immunogold labeled Na⁺/K⁺-ATPase. This integrated approach describes a reliable and repeatable prefixation immunogold labeling technique allowing precise visualization of NaK within target cells combined with a 3D imaging that offers valuable insights into MRC dynamics at an ultrastructural level. Copyright © 2013 Wiley Periodicals, Inc.

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.

A Simple Model for Nonlinear Confocal Ultrasonic Beams

NASA Astrophysics Data System (ADS)

Zhang, Dong; Zhou, Lin; Si, Li-Sheng; Gong, Xiu-Fen

2007-01-01

A confocally and coaxially arranged pair of focused transmitter and receiver represents one of the best geometries for medical ultrasonic imaging and non-invasive detection. We develop a simple theoretical model for describing the nonlinear propagation of a confocal ultrasonic beam in biological tissues. On the basis of the parabolic approximation and quasi-linear approximation, the nonlinear Khokhlov-Zabolotskaya-Kuznetsov (KZK) equation is solved by using the angular spectrum approach. Gaussian superposition technique is applied to simplify the solution, and an analytical solution for the second harmonics in the confocal ultrasonic beam is presented. Measurements are performed to examine the validity of the theoretical model. This model provides a preliminary model for acoustic nonlinear microscopy.

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.

Ex vivo fluorescence confocal microscopy for fast evaluation of tumour margins during Mohs surgery.

PubMed

Bennàssar, A; Vilata, A; Puig, S; Malvehy, J

2014-02-01

Ex vivo fluorescence confocal microscopy (FCM) enables real-time imaging of skin morphology directly in freshly excised tissue. FCM displays wide field-of-view mosaics with cellular resolution, thus enabling a rapid bedside pathology. An application of interest is rapid detection of residual basal cell carcinoma (BCC) in skin excisions during Mohs surgery. We sought to evaluate the sensitivity and specificity of ex vivo imaging with FCM for the detection of residual BCC in Mohs tissue excisions, and to calculate the time invested up to the diagnosis for both FCM and frozen sections. Eighty consecutive BCCs were prospectively collected and the margins scanned with ex vivo FCM, including excisions with and without residual BCC of all major subtypes. Each mosaic was divided into two or four, resulting in 480 submosaics for study. Every confocal submosaic was assessed for the presence or absence of BCC and compared with standard frozen sections as the gold standard. Furthermore, the time spent for each technique was calculated and compared. The overall sensitivity and specificity of detecting residual BCC were 88% and 99%, respectively. Moreover, the new technique reduced by almost two-thirds the time invested when compared with the processing of a frozen section (P < 0·001). The results demonstrate the feasibility of confocal mosaicing microscopy in fresh tissue for rapid surgical pathology, potentially to expedite and guide Mohs surgery with high accuracy. This observation is an important step towards the goal of using real-time surgical pathology for skin tumours. © 2013 British Association of Dermatologists.

EVALUATION OF CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: PRETTY PICTURES OR CONFOCAL QA

EPA Science Inventory

Evaluation of confocal microscopy system performance: Pretty pictures or confocal QA?

Robert M. Zucker

Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, N...

Evaluation of conjunctival inflammatory status by confocal scanning laser microscopy and conjunctival brush cytology in patients with atopic keratoconjunctivitis (AKC)

PubMed Central

Wakamatsu, Tais Hitomi; Okada, Naoko; Kojima, Takashi; Matsumoto, Yukihiro; Ibrahim, Osama M.A.; Adan, Enrique Sato; Fukagawa, Kazumi; Katakami, Chikako; Tsubota, Kazuo; Shimazaki, Jun; Fujishima, Hiroshi

2009-01-01

Purpose To elucidate the status of the conjunctival inflammation in atopic keratoconjunctivitis (AKC) using laser scanning confocal microscopy and compare the relevant findings with conjunctival brush cytology in a prospective controlled study. Methods Twenty eyes from 20 AKC patients as well as 16 eyes from 16 age and sex matched normal subjects were studied. The subjects underwent tear film break-up time (BUT), fluorescein and Rose Bengal staining of the ocular surface, conjunctival confocal microscopy, Schirmer test, and brush cytology. Brush cytology specimens and in vivo confocal microscopy scans underwent evaluation for inflammatory cell densities. Results Brush cytology specimens and in vivo confocal microscopy scans from AKC patients revealed significantly higher numbers of inflammatory cells (p<0.05). Conjunctival inflammatory cell density showed a negative correlation with tear stability and a positive correlation with vital staining scores and conjunctival injection grades. The extent of conjunctival inflammation assessed by in vivo confocal microscopy showed a strong positive linear correlation with the inflammation status evaluated by brush cytology. The corneal inflammatory cell density assessed by in vivo confocal microscopy showed a significant negative correlation with tear stability and a positive linear correlation with corneal fluorescein staining. Conclusions Confocal scanning laser microscopy is an efficient, noninvasive, and a promising tool for the quantitative assessment of conjunctival inflammation, a parameter of this new technology which correlated well with subjective and objective ocular surface clinical findings. PMID:19693288

Advances in combined endoscopic fluorescence confocal microscopy and optical coherence tomography

NASA Astrophysics Data System (ADS)

Risi, Matthew D.

Confocal microendoscopy provides real-time high resolution cellular level images via a minimally invasive procedure. Results from an ongoing clinical study to detect ovarian cancer with a novel confocal fluorescent microendoscope are presented. As an imaging modality, confocal fluorescence microendoscopy typically requires exogenous fluorophores, has a relatively limited penetration depth (100 μm), and often employs specialized aperture configurations to achieve real-time imaging in vivo. Two primary research directions designed to overcome these limitations and improve diagnostic capability are presented. Ideal confocal imaging performance is obtained with a scanning point illumination and confocal aperture, but this approach is often unsuitable for real-time, in vivo biomedical imaging. By scanning a slit aperture in one direction, image acquisition speeds are greatly increased, but at the cost of a reduction in image quality. The design, implementation, and experimental verification of a custom multi-point-scanning modification to a slit-scanning multi-spectral confocal microendoscope is presented. This new design improves the axial resolution while maintaining real-time imaging rates. In addition, the multi-point aperture geometry greatly reduces the effects of tissue scatter on imaging performance. Optical coherence tomography (OCT) has seen wide acceptance and FDA approval as a technique for ophthalmic retinal imaging, and has been adapted for endoscopic use. As a minimally invasive imaging technique, it provides morphological characteristics of tissues at a cellular level without requiring the use of exogenous fluorophores. OCT is capable of imaging deeper into biological tissue (˜1-2 mm) than confocal fluorescence microscopy. A theoretical analysis of the use of a fiber-bundle in spectral-domain OCT systems is presented. The fiber-bundle enables a flexible endoscopic design and provides fast, parallelized acquisition of the optical coherence tomography data. However, the multi-mode characteristic of the fibers in the fiber-bundle affects the depth sensitivity of the imaging system. A description of light interference in a multi-mode fiber is presented along with numerical simulations and experimental studies to illustrate the theoretical analysis.

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

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.

Monosynaptic convergence of chorda tympani and glossopharyngeal afferents onto ascending relay neurons in the nucleus of the solitary tract: A high-resolution confocal and correlative electron microscopy approach

PubMed Central

Corson, James A.; Erisir, Alev

2014-01-01

While physiological studies suggested convergence of chorda tympani and glossopharyngeal afferent axons onto single neurons of the rostral nucleus of the solitary tract (rNTS), anatomical evidence has been elusive. The current study uses high-magnification confocal microscopy to identify putative synaptic contacts from afferent fibers of the two nerves onto individual projection neurons. Imaged tissue is re-visualized with electron microscopy, confirming that overlapping fluorescent signals in confocal z-stacks accurately identify appositions between labeled terminal and dendrite pairs. Monte Carlo modeling reveals that the probability of overlapping fluorophores is stochastically unrelated to the density of afferent label suggesting that convergent innervation in the rNTS is selective rather than opportunistic. Putative synaptic contacts from each nerve are often compartmentalized onto dendrite segments of convergently innervated neurons. These results have important implications for orosensory processing in the rNTS, and the techniques presented here have applications in investigations of neural microcircuitry with an emphasis on innervation patterning. PMID:23640852

Biomimetic Coating on Porous Alumina for Tissue Engineering: Characterisation by Cell Culture and Confocal Microscopy

PubMed Central

Kolos, Elizabeth; Ruys, Andrew J

2015-01-01

In this study porous alumina samples were prepared and then coated using the biomimetic coating technique using a five times Simulated Body Fluid (5.0SBF) as the growth solution. A coating was achieved after pre-treatment with concentrated acid. From elemental analysis, the coating contained calcium and phosphorous, but also sodium and chlorine. Halite was identified by XRD, a sodium chloride phase. Sintering was done to remove the halite phase. Once halite was burnt off, the calcium phosphate crystals were not covered with halite and, therefore, the apatite phases can be clearly observed. Cell culturing showed sufficient cell attachment to the less porous alumina, Sample B, that has more calcium phosphate growth, while the porous alumina, Sample A, with minimal calcium phosphate growth attained very little cell attachment. This is likely due to the contribution that calcium phosphate plays in the attachment of bone-like cells to a bioinert ceramic such as alumina. These results were repeated on both SEM and confocal microscopy analysis. Confocal microscopy was a novel characterisation approach which gave useful information and was a visual aid.

Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy.

PubMed

Siegel, Nisan; Brooker, Gary

2014-09-22

FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called "CINCH".

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

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.

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.

Correlating confocal microscopy and atomic force indentation reveals metastatic cancer cells stiffen during invasion into collagen I matrices

NASA Astrophysics Data System (ADS)

Staunton, Jack R.; Doss, Bryant L.; Lindsay, Stuart; Ros, Robert

2016-01-01

Mechanical interactions between cells and their microenvironment dictate cell phenotype and behavior, calling for cell mechanics measurements in three-dimensional (3D) extracellular matrices (ECM). Here we describe a novel technique for quantitative mechanical characterization of soft, heterogeneous samples in 3D. The technique is based on the integration of atomic force microscopy (AFM) based deep indentation, confocal fluorescence microscopy, finite element (FE) simulations and analytical modeling. With this method, the force response of a cell embedded in 3D ECM can be decoupled from that of its surroundings, enabling quantitative determination of the elastic properties of both the cell and the matrix. We applied the technique to the quantification of the elastic properties of metastatic breast adenocarcinoma cells invading into collagen hydrogels. We found that actively invading and fully embedded cells are significantly stiffer than cells remaining on top of the collagen, a clear example of phenotypical change in response to the 3D environment. Treatment with Rho-associated protein kinase (ROCK) inhibitor significantly reduces this stiffening, indicating that actomyosin contractility plays a major role in the initial steps of metastatic invasion.

Insights into esophagus tissue architecture using two-photon confocal microscopy

NASA Astrophysics Data System (ADS)

Liu, Nenrong; Wang, Yue; Feng, Shangyuan; Chen, Rong

2013-08-01

In this paper, microstructures of human esophageal mucosa were evaluated using the two-photon laser scanning confocal microscopy (TPLSCM), based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). The distribution of epithelial cells, muscle fibers of muscularis mucosae has been distinctly obtained. Furthermore, esophageal submucosa characteristics with cancer cells invading into were detected. The variation of collagen, elastin and cancer cells is very relevant to the pathology in esophagus, especially early esophageal cancer. Our experimental results indicate that the MPM technique has the much more advantages for label-free imaging, and has the potential application in vivo in the clinical diagnosis and monitoring of early esophageal cancer.

Results of a phase I-II study on intraductal confocal microscopy (IDCM) in patients with common bile duct (CBD) stenosis.

PubMed

Giovannini, M; Bories, E; Monges, G; Pesenti, C; Caillol, F; Delpero, J R

2011-07-01

Confocal endomicroscopy is an emergent technique and allows real optical biopsies in the gastrointestinal (GI) tract. The aim of this study was to evaluate a new intraductal confocal miniprobe in patients with a normal common bile duct (CBD) or with a suspicion of a malignant stenosis (cholangiocarcinoma). Thirty-seven patients (23 males) underwent endoscopic retrograde cholangiopancreatography (ERCP) for bile duct stone removal (7 cases) or bile duct stenosis (30 cases). Intraductal confocal microscopy (IDCM) was performed during the ERCP using a probe-based confocal laser endomicroscopy (pCLE) technique. IDCM was done with the CholangioFlex probe with Cellvizio (Mauna Kea Technologies, Paris, France). The depth of penetration of theCholangioFlex probe was 40-70 μm and magnification was 400×. Images were reviewed by an experienced pathologist in GI disease and compared to ERCP findings, CBD biopsies performed during ERCP or EUS, and in 15 patients to the resected specimen (Wipple resection). No complications related to the CholangioFlex insertion occurred after the ERCP. Good images were obtained in 33 patients. Final histology diagnosis was a normal CBD in 7 cases, 23 malignant stenoses (4 ampullary carcinomas, 13 cholangiocarcinomas, and 6 pancreatic cancer), and 7 inflammatory stenoses (4 chronic pancreatitis, 1 stenosis of hepaticojejunal anastomosis, 1 postcholecystectomy CBD stenosis, and 1 primary sclerosing cholangitis). IDCM of a normal CBD showed a thin black band (<20 μm), normal vessels (thin and regular), and no visible glands. IDCM of malignant strictures revealed irregular vessels with lack of contrast in the CBD wall, large black band (>20 μm), and an aggregate of irregular black cells (black clumps). These aspects were seen in all malignant stenoses and none were seen in benign or normal CBD. The presence of irregular vessels, large black bands, and black clumps seen with confocal laser microscopy enabled prediction of neoplasia with an accuracy rate of 86%, sensitivity of 83%, and specificity of 75%. The respective numbers for standard histopathology were 53, 65, and 53%. This phase I-II study on IDCM showed that IDCM is feasible. This new technique will open a new door for optical biopsy of the CBD.

Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy.

PubMed

Kress, Alla; Wang, Xiao; Ranchon, Hubert; Savatier, Julien; Rigneault, Hervé; Ferrand, Patrick; Brasselet, Sophie

2013-07-02

Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Detecting cells in time varying intensity images in confocal microscopy for gene expression studies in living cells

NASA Astrophysics Data System (ADS)

Mitra, Debasis; Boutchko, Rostyslav; Ray, Judhajeet; Nilsen-Hamilton, Marit

2015-03-01

In this work we present a time-lapsed confocal microscopy image analysis technique for an automated gene expression study of multiple single living cells. Fluorescence Resonance Energy Transfer (FRET) is a technology by which molecule-to-molecule interactions are visualized. We analyzed a dynamic series of ~102 images obtained using confocal microscopy of fluorescence in yeast cells containing RNA reporters that give a FRET signal when the gene promoter is activated. For each time frame, separate images are available for three spectral channels and the integrated intensity snapshot of the system. A large number of time-lapsed frames must be analyzed to identify each cell individually across time and space, as it is moving in and out of the focal plane of the microscope. This makes it a difficult image processing problem. We have proposed an algorithm here, based on scale-space technique, which solves the problem satisfactorily. The algorithm has multiple directions for even further improvement. The ability to rapidly measure changes in gene expression simultaneously in many cells in a population will open the opportunity for real-time studies of the heterogeneity of genetic response in a living cell population and the interactions between cells that occur in a mixed population, such as the ones found in the organs and tissues of multicellular organisms.

Non-invasive diagnosis and monitoring of actinic cheilitis with reflectance confocal microscopy.

PubMed

Ulrich, M; González, S; Lange-Asschenfeldt, B; Roewert-Huber, J; Sterry, W; Stockfleth, E; Astner, S

2011-03-01

Actinic cheilitis (AC) represents the equivalent of actinic keratosis on the lip. Various treatment modalities are available and the efficacy of diclofenac in hyaluronic acid has recently been described. Reflectance confocal microscopy (RCM) is a non-invasive imaging technique which has recently been applied for the diagnosis of actinic keratoses. Herein, we describe the applicability of RCM for the diagnosis of AC and for monitoring of treatment response of AC to diclofenac in hyaluronic acid. Ten Caucasian patients with clinical suspicion for AC were included in this study. To obtain a non-invasive diagnosis, RCM was performed at baseline, followed by biopsy and respective confocal-histopathological correlation. Six patients with a histological diagnosis of AC were treated with diclofenac in hyaluronic acid, whereby monitoring was performed by RCM. Reflectance confocal microscopy was able to correctly identify 6/7 cases of AC and 3/3 cases of benign lesions. The most important RCM criteria for diagnosis of AC were cellular atypia at the stratum spinosum and granulosum with atypical honeycomb pattern. One patient with AC was misclassified as inflammatory cheilitis by RCM as it showed marked inflammatory response and lacked clear signs of cellular atypia on RCM imaging. Following topical treatment with diclofenac gel, 5/6 patients (83%) showed a good treatment response with regression of dysplasia on consecutive RCM examination. Reflectance confocal microscopy is a promising tool for the non-invasive diagnosis and monitoring of actinic cheilitis. However, marked inflammation represents a potential diagnostic pitfall. In this regard, biopsy should be performed in doubtful cases. © 2010 The Authors. Journal of the European Academy of Dermatology and Venereology © 2010 European Academy of Dermatology and Venereology.

Revealing region-specific biofilm viscoelastic properties by means of a micro-rheological approach.

PubMed

Cao, Huayu; Habimana, Olivier; Safari, Ashkan; Heffernan, Rory; Dai, Yihong; Casey, Eoin

2016-01-01

Particle-tracking microrheology is an in situ technique that allows quantification of biofilm material properties. It overcomes the limitations of alternative techniques such as bulk rheology or force spectroscopy by providing data on region specific material properties at any required biofilm location and can be combined with confocal microscopy and associated structural analysis. This article describes single particle tracking microrheology combined with confocal laser scanning microscopy to resolve the biofilm structure in 3 dimensions and calculate the creep compliances locally. Samples were analysed from Pseudomonas fluorescens biofilms that were cultivated over two timescales (24 h and 48 h) and alternate ionic conditions (with and without calcium chloride supplementation). The region-based creep compliance analysis showed that the creep compliance of biofilm void zones is the primary contributor to biofilm mechanical properties, contributing to the overall viscoelastic character.

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

PubMed Central

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

2013-01-01

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

Confocal Raman studies in determining crystalline nature of PECVD grown Si nanowires

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

Ahmed, Nafis; Bhargav, P. Balaji; Ramasamy, P.

2015-06-24

Silicon nanowires of diameter ∼200 nm and length of 2-4 µm are grown in the plasma enhanced chemical vapour deposition technique using nanoclustered Au catalyst assisted vapour-liquid-solid process. The crystallinity in the as-grown and annealed samples is studied using confocal Raman spectroscopic studies. Amorphous phase is formed in the as-grown samples. Structural studies using high resolution transmission electron microscopy confirm the polycrystalline nature in the annealed sample.

Genetically encoded sensors and fluorescence microscopy for anticancer research

NASA Astrophysics Data System (ADS)

Zagaynova, Elena V.; Shirmanova, Marina V.; Sergeeva, Tatiana F.; Klementieva, Natalia V.; Mishin, Alexander S.; Gavrina, Alena I.; Zlobovskay, Olga A.; Furman, Olga E.; Dudenkova, Varvara V.; Perelman, Gregory S.; Lukina, Maria M.; Lukyanov, Konstantin A.

2017-02-01

Early response of cancer cells to chemical compounds and chemotherapeutic drugs were studied using novel fluorescence tools and microscopy techniques. We applied confocal microscopy, two-photon fluorescence lifetime imaging microscopy and super-resolution localization-based microscopy to assess structural and functional changes in cancer cells in vitro. The dynamics of energy metabolism, intracellular pH, caspase-3 activation during staurosporine-induced apoptosis as well as actin cytoskeleton rearrangements under chemotherapy were evaluated. We have showed that new genetically encoded sensors and advanced fluorescence microscopy methods provide an efficient way for multiparameter analysis of cell activities

Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy

PubMed Central

Siegel, Nisan; Brooker, Gary

2014-01-01

FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called “CINCH”. PMID:25321701

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.

Confocal Imaging of Confined Quiescent and Flowing Colloid-polymer Mixtures

PubMed Central

Conrad, Jacinta C.

2014-01-01

The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed assembly1-3, drug delivery4, improved hydrocarbon recovery5-7, and flowable electrodes for energy storage8. Suspensions containing fluorescent colloids and non-adsorbing polymers are appealing model systems, as the ratio of the polymer radius of gyration to the particle radius and concentration of polymer control the range and strength of the interparticle attraction, respectively. By tuning the polymer properties and the volume fraction of the colloids, colloid fluids, fluids of clusters, gels, crystals, and glasses can be obtained9. Confocal microscopy, a variant of fluorescence microscopy, allows an optically transparent and fluorescent sample to be imaged with high spatial and temporal resolution in three dimensions. In this technique, a small pinhole or slit blocks the emitted fluorescent light from regions of the sample that are outside the focal volume of the microscope optical system. As a result, only a thin section of the sample in the focal plane is imaged. This technique is particularly well suited to probe the structure and dynamics in dense colloidal suspensions at the single-particle scale: the particles are large enough to be resolved using visible light and diffuse slowly enough to be captured at typical scan speeds of commercial confocal systems10. Improvements in scan speeds and analysis algorithms have also enabled quantitative confocal imaging of flowing suspensions11-16,37. In this paper, we demonstrate confocal microscopy experiments to probe the confined phase behavior and flow properties of colloid-polymer mixtures. We first prepare colloid-polymer mixtures that are density- and refractive-index matched. Next, we report a standard protocol for imaging quiescent dense colloid-polymer mixtures under varying confinement in thin wedge-shaped cells. Finally, we demonstrate a protocol for imaging colloid-polymer mixtures during microchannel flow. PMID:24894062

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.

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.

High temporal resolution fluorescence measurements of a mitochondrial dye for detection of early stage apoptosis

PubMed Central

Iyer, Divya; Ray, Rachel D.; Pappas, Dimitri

2013-01-01

In the present study, early stage apoptosis is explored with high temporal resolution. In addition to monitoring early apoptosis induction in single cells by ultrasensitive confocal fluorescence microscopy (UCFM), the mitochondrial proteins release kinetics was explored. The current study shows development and optimization of a novel, rapid apoptosis assay to explore the earliest changes in cells by the intrinsic apoptosis pathway. We show that early apoptotic changes in the mitochondria begin nearly simultaneously with the addition of an apoptosis-inducing drug, such as staurosporine. With a temporal resolution of five minutes, this non-invasive analytical technique can elucidate the earliest apoptotic events in living cells. Moreover, our results show that the mitochondrial inter-membrane proteins are not involved in the extrinsic pathway of Ramos cells mediated by an anti-CD95 antibody. Additional techniques such as light microscopy and flow cytometry were employed to confirm the results obtained by ultrasensitive confocal fluorescence microscopy. The results of this study help to understand the earliest mechanisms of apoptosis induction in cells, enabling new methods of drug testing and dose-response analyses. PMID:23831722

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

Confocal Microscopy and Molecular-Specific Optical Contrast Agents for the Detection of Oral Neoplasia

PubMed Central

Carlson, Alicia L.; Gillenwater, Ann M.; Williams, Michelle D.; El-Naggar, Adel K.; Richards-Kortum, R. R.

2009-01-01

Using current clinical diagnostic techniques, it is difficult to visualize tumor morphology and architecture at the cellular level, which is necessary for diagnostic localization of pathologic lesions. Optical imaging techniques have the potential to address this clinical need by providing real-time, sub-cellular resolution images. This paper describes the use of dual mode confocal microscopy and optical molecular-specific contrast agents to image tissue architecture, cellular morphology, and sub-cellular molecular features of normal and neoplastic oral tissues. Fresh tissue slices were prepared from 33 biopsies of clinically normal and abnormal oral mucosa obtained from 14 patients. Reflectance confocal images were acquired after the application of 6% acetic acid, and fluorescence confocal images were acquired after the application of a fluorescence contrast agent targeting the epidermal growth factor receptor (EGFR). The dual imaging modes provided images similar to light microscopy of hematoxylin and eosin and immunohistochemistry staining, but from thick fresh tissue slices. Reflectance images provided information on the architecture of the tissue and the cellular morphology. The nuclear-to-cytoplasmic (N/C) ratio from the reflectance images was at least 7.5 times greater for the carcinoma than the corresponding normal samples, except for one case of highly keratinized carcinoma. Separation of carcinoma from normal and mild dysplasia was achieved using this ratio (p<0.01). Fluorescence images of EGFR expression yielded a mean fluorescence labeling intensity (FLI) that was at least 2.7 times higher for severe dysplasia and carcinoma samples than for the corresponding normal sample, and could be used to distinguish carcinoma from normal and mild dysplasia (p<0.01). Analyzed together, the N/C ratio and the mean FLI may improve the ability to distinguish carcinoma from normal squamous epithelium. PMID:17877424

Laser beam shaping for biomedical microscopy techniques

NASA Astrophysics Data System (ADS)

Laskin, Alexander; Kaiser, Peter; Laskin, Vadim; Ostrun, Aleksei

2016-04-01

Uniform illumination of a working field is very important in optical systems of confocal microscopy and various implementations of fluorescence microscopy like TIR, SSIM, STORM, PALM to enhance performance of these laser-based research techniques. Widely used TEM00 laser sources are characterized by essentially non-uniform Gaussian intensity profile which leads usually to non-uniform intensity distribution in a microscope working field or in a field of microlenses array of a confocal microscope optical system, this non-uniform illumination results in instability of measuring procedure and reducing precision of quantitative measurements. Therefore transformation of typical Gaussian distribution of a TEM00 laser to flat-top (top hat) profile is an actual technical task, it is solved by applying beam shaping optics. Due to high demands to optical image quality the mentioned techniques have specific requirements to a uniform laser beam: flatness of phase front and extended depth of field, - from this point of view the microscopy techniques are similar to holography and interferometry. There are different refractive and diffractive beam shaping approaches used in laser industrial and scientific applications, but only few of them are capable to fulfil the optimum conditions for beam quality required in discussed microscopy techniques. We suggest applying refractive field mapping beam shapers πShaper, which operational principle presumes almost lossless transformation of Gaussian to flat-top beam with flatness of output wavefront, conserving of beam consistency, providing collimated low divergent output beam, high transmittance, extended depth of field, negligible wave aberration, and achromatic design provides capability to work with several lasers with different wavelengths simultaneously. The main function of a beam shaper is transformation of laser intensity profile, further beam transformation to provide optimum for a particular technique spot size and shape has to be realized by an imaging optical system which can include microscope objectives and tube lenses. This paper will describe design basics of refractive beam shapers and optical layouts of their applying in microscopy systems. Examples of real implementations and experimental results will be presented as well.

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.

Simultaneous measurements of bulk moduli and particle dynamics in a sheared colloidal glass

NASA Astrophysics Data System (ADS)

Massa, Michael V.; Eisenmann, Christoph; Kim, Chanjoong; Weitz, David A.

2007-03-01

We present a novel study of glassy colloidal systems, using a stress-controlled rheometer in conjunction with a confocal microscope. This experimental setup combines the measurement of bulk moduli, using conventional rheology, with the ability to track the motion of individual particles, through confocal microscopy techniques. We explore the response of the system to applied shear, by simultaneously monitoring the macroscopic relaxation and microscopic particle dynamics, under conditions from the quiescent glass to a shear-melted liquid.

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.

Quantification of transendothelial migration using three-dimensional confocal microscopy.

PubMed

Cain, Robert J; d'Água, Bárbara Borda; Ridley, Anne J

2011-01-01

Migration of cells across endothelial barriers, termed transendothelial migration (TEM), is an important cellular process that underpins the pathology of many disease states including chronic inflammation and cancer metastasis. While this process can be modeled in vitro using cultured cells, many model systems are unable to provide detailed visual information of cell morphologies and distribution of proteins such as junctional markers, as well as quantitative data on the rate of TEM. Improvements in imaging techniques have made microscopy-based assays an invaluable tool for studying this type of detailed cell movement in physiological processes. In this chapter, we describe a confocal microscopy-based method that can be used to assess TEM of both leukocytes and cancer cells across endothelial barriers in response to a chemotactic gradient, as well as providing information on their migration into a subendothelial extracellular matrix, designed to mimic that found in vivo.

Basics of Confocal Microscopy and the Complexity of Diagnosing Skin Tumors: New Imaging Tools in Clinical Practice, Diagnostic Workflows, Cost-Estimate, and New Trends.

PubMed

Que, Syril Keena T; Grant-Kels, Jane M; Longo, Caterina; Pellacani, Giovanni

2016-10-01

The use of reflectance confocal microscopy (RCM) and other noninvasive imaging devices can potentially streamline clinical care, leading to more precise and efficient management of skin cancer. This article explores the potential role of RCM in cutaneous oncology, as an adjunct to more established techniques of detecting and monitoring for skin cancer, such as dermoscopy and total body photography. Discussed are current barriers to the adoption of RCM, diagnostic workflows and standards of care in the United States and Europe, and medicolegal issues. The potential role of RCM and other similar technological innovations in the enhancement of dermatologic care is evaluated. Copyright © 2016 Elsevier Inc. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

The potential role of in vivo reflectance confocal microscopy for evaluating oral cavity lesions: a systematic review.

PubMed

Lucchese, Alberta; Gentile, Enrica; Romano, Antonio; Maio, Claudio; Laino, Luigi; Serpico, Rosario

2016-11-01

Since the early 2000s, several studies have examined the application of reflectance confocal microscopy (RCM) to the oral cavity. This review gives an overview of the literature on reflectance confocal microscopy analysis of the oral cavity in vivo and identifies flaws in the studies, providing guidance to improve reflectance confocal microscopy applications and inform the design of future studies. The PubMed, ISI, Scopus, and Cochrane Library databases were searched for publications on RCM using the terms 'reflectance confocal microscopy' in combination with 'mouth' and other terms related to the topic of interest. The search gave 617 results. Seventeen studies were included in our final analysis. We decided to organize the selected articles according to four topics: healthy mucosa, autoimmune diseases, cancer and precancerous lesions, and hard dental tissues. Although reflectance confocal microscopy is promising for diagnosing and monitoring oral pathology, it has shortcomings and there are still too few publications on this topic. Further studies are needed to increase the quantity and quality of the results, to translate research into clinical practice. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

Chemical imaging of drug delivery systems with structured surfaces-a combined analytical approach of confocal raman microscopy and optical profilometry.

PubMed

Kann, Birthe; Windbergs, Maike

2013-04-01

Confocal Raman microscopy is an analytical technique with a steadily increasing impact in the field of pharmaceutics as the instrumental setup allows for nondestructive visualization of component distribution within drug delivery systems. Here, the attention is mainly focused on classic solid carrier systems like tablets, pellets, or extrudates. Due to the opacity of these systems, Raman analysis is restricted either to exterior surfaces or cross sections. As Raman spectra are only recorded from one focal plane at a time, the sample is usually altered to create a smooth and even surface. However, this manipulation can lead to misinterpretation of the analytical results. Here, we present a trendsetting approach to overcome these analytical pitfalls with a combination of confocal Raman microscopy and optical profilometry. By acquiring a topography profile of the sample area of interest prior to Raman spectroscopy, the profile height information allowed to level the focal plane to the sample surface for each spectrum acquisition. We first demonstrated the basic principle of this complementary approach in a case study using a tilted silica wafer. In a second step, we successfully adapted the two techniques to investigate an extrudate and a lyophilisate as two exemplary solid drug carrier systems. Component distribution analysis with the novel analytical approach was neither hampered by the curvature of the cylindrical extrudate nor the highly structured surface of the lyophilisate. Therefore, the combined analytical approach bears a great potential to be implemented in diversified fields of pharmaceutical sciences.

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.

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...

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.

Evaluation of the therapeutic results of actinic keratosis treated with topical 5% fluorouracil by reflectance confocal laser microscopy: preliminary study*

PubMed Central

Ishioka, Priscila; Maia, Marcus; Rodrigues, Sarita Bartholomei; Marta, Alessandra Cristina; Hirata, Sérgio Henrique

2015-01-01

Topical treatment for actinic keratosis with 5% fluorouracil has a recurrence rate of 54% in 12 months of follow-up. This study analyzed thirteen actinic keratoses on the upper limbs through confocal microscopy, at the time of clinical diagnosis and after 4 weeks of treatment with fluorouracil. After the treatment was established and evidence of clinical cure was achieved, in two of the nine actinic keratoses, confocal microscopy enabled visualization of focal areas of atypical honeycomb pattern in the epidermis indicating therapeutic failure. Preliminary data suggest the use of confocal microscopy as a tool for diagnosis and therapeutic control of actinic keratosis. PMID:26131881

Rapid detection of biofilms and adherent pathogens using scanning confocal laser microscopy and episcopic differential interference contrast microscopy.

PubMed

Keevil, C W

2003-01-01

Knowledge of biofilm structure and function has changed significantly in the last few years due to advances in light microscopy. One pertinent example is the use of scanning confocal laser microscopy (SCLM) to visualise corrosion pits caused by the biofilm mosaic footprint on corroding metal surfaces. Nevertheless, SCLM has some limitations as to its widespread use, including cost, inability to observe motile bacteria and eukaryotic grazers within biofilms, and difficulty to scan a curved surface. By contrast, episcopic differential interference contrast (EDIC) microscopy has provided a rapid, real time analysis of biofilms on opaque, curved, natural or man-made surfaces without the need for cover slips and oil. EDIC, coupled with epi-fluorescence (EDIC/EF), microscopy has been used successfully to visualise the 3-D biofilm structure, physiological niches, protozoal grazing and iron biomineralization, and the location of specific pathogens such as Legionella pneumophila, Campylobacter jejuni and Cryptosporidium parvum. These species were identified using gold nanoparticles or fluorophores coupled to monoclonal antibodies or 16S rRNA probes, respectively. Among its many potential uses, the EDIC technique will provide a rapid procedure to facilitate the calibration of the modern generation of biofilm-sensing electrodes.

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.

Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.

PubMed

Nyindodo-Ogari, Lilian; Schwartzbach, Steven D; Skalli, Omar; Estraño, Carlos E

2016-01-01

Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes.

Analysis of multi-channel microscopy: Spectral self-interference, multi-detector confocal and 4Pi systems

NASA Astrophysics Data System (ADS)

Davis, Brynmor J.

Fluorescence microscopy is an important and ubiquitous tool in biological imaging due to the high specificity with which fluorescent molecules can be attached to an organism and the subsequent nondestructive in-vivo imaging allowed. Focused-light microscopies allow three-dimensional fluorescence imaging but their resolution is restricted by diffraction. This effect is particularly limiting in the axial dimension as the diffraction-limited focal volume produced by a lens is more extensive along the optical axis than perpendicular to it. Approaches such as confocal microscopy and 4Pi microscopy have been developed to improve the axial resolution. Spectral Self-Interference Fluorescence Microscopy (SSFM) is another high-axial-resolution technique and is the principal subject of this dissertation. Nanometer-precision localization of a single fluorescent layer has been demonstrated using SSFM. This accuracy compares favorably with the axial resolutions given by confocal and 4Pi systems at similar operating parameters (these resolutions are approximately 350nm and 80nm respectively). This theoretical work analyzes the expected performance of the SSFM system when imaging a general object, i.e. an arbitrary fluorophore density function rather than a single layer. An existing model of SSFM is used in simulations to characterize the system's resolution. Several statistically-based reconstruction methods are applied to show that the expected resolution for SSFM is similar to 4Pi microscopy for a general object but does give very high localization accuracy when the object is known to consist of a limited number of layers. SSFM is then analyzed in a linear systems framework and shown to have strong connections, both physically and mathematically, to a multi-channel 4Pi microscope. Fourier-domain analysis confirms that SSFM cannot be expected to outperform this multi-channel 4Pi instrument. Differences between the channels in spatial-scanning, multi-channel microscopies are then exploited to show that such instruments can operate at a sub-Nyquist scanning rate but still produce images largely free of aliasing effects. Multi-channel analysis is also used to show how light typically discarded in confocal and 4Pi systems can be collected and usefully incorporated into the measured image.

Evaluating performance in three-dimensional fluorescence microscopy

PubMed Central

MURRAY, JOHN M; APPLETON, PAUL L; SWEDLOW, JASON R; WATERS, JENNIFER C

2007-01-01

In biological fluorescence microscopy, image contrast is often degraded by a high background arising from out of focus regions of the specimen. This background can be greatly reduced or eliminated by several modes of thick specimen microscopy, including techniques such as 3-D deconvolution and confocal. There has been a great deal of interest and some confusion about which of these methods is ‘better’, in principle or in practice. The motivation for the experiments reported here is to establish some rough guidelines for choosing the most appropriate method of microscopy for a given biological specimen. The approach is to compare the efficiency of photon collection, the image contrast and the signal-to-noise ratio achieved by the different methods at equivalent illumination, using a specimen in which the amount of out of focus background is adjustable over the range encountered with biological samples. We compared spot scanning confocal, spinning disk confocal and wide-field/deconvolution (WFD) microscopes and find that the ratio of out of focus background to in-focus signal can be used to predict which method of microscopy will provide the most useful image. We also find that the precision of measurements of net fluorescence yield is very much lower than expected for all modes of microscopy. Our analysis enabled a clear, quantitative delineation of the appropriate use of different imaging modes relative to the ratio of out-of-focus background to in-focus signal, and defines an upper limit to the useful range of the three most common modes of imaging. PMID:18045334

Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI

PubMed Central

Li, Yiwen; Song, Ying; Zhao, Lian; Gaidosh, Gabriel; Laties, Alan M; Wen, Rong

2009-01-01

We describe a protocol to rapidly and reliably visualize blood vessels in experimental animals. Blood vessels are directly labeled by cardiac perfusion using a specially formulated aqueous solution containing 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), a lipophilic carbocyanine dye, which incorporates into endothelial cell membranes upon contact. By lateral diffusion, DiI also stains membrane structures, including angiogenic sprouts and pseudopodial processes that are not in direct contact. Tissues can be immediately examined by conventional and confocal fluorescence microscopy. High-quality serial optical sections using confocal microscopy are obtainable from thick tissue sections, especially at low magnification, for three-dimensional reconstruction. It takes less than 1 h to stain the vasculature in a whole animal. Compared with alternative techniques to visualize blood vessels, including space-occupying materials such as India ink or fluorescent dye-conjugated dextran, the corrosion casting technique, endothelial cell-specific markers and lectins, the present method simplifies the visualization of blood vessels and data analysis. PMID:18846097

78 FR 33098 - Prospective Grant of Co-Exclusive Licenses: Multi-Focal Structured Illumination Microscopy...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-06-03

...-Exclusive Licenses: Multi-Focal Structured Illumination Microscopy Systems and Methods AGENCY: National... pertains to a system and method for digital confocal microscopy that rapidly processes enhanced images. In particular, the invention is a method for digital confocal microscopy that includes a digital mirror device...

Development of novel murine mammary imaging windows to examine wound healing effects on leukocyte trafficking in mammary tumors with intravital imaging

PubMed Central

Sobolik, Tammy; Su, Ying-Jun; Ashby, Will; Schaffer, David K.; Wells, Sam; Wikswo, John P.; Zijlstra, Andries; Richmond, Ann

2016-01-01

ABSTRACT We developed mammary imaging windows (MIWs) to evaluate leukocyte infiltration and cancer cell dissemination in mouse mammary tumors imaged by confocal microscopy. Previous techniques relied on surgical resection of a skin flap to image the tumor microenvironment restricting imaging time to a few hours. Utilization of mammary imaging windows offers extension of intravital imaging of the tumor microenvironment. We have characterized strengths and identified some previously undescribed potential weaknesses of MIW techniques. Through iterative enhancements of a transdermal portal we defined conditions for improved quality and extended confocal imaging time for imaging key cell-cell interactions in the tumor microenvironment. PMID:28243517

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.

Development of novel murine mammary imaging windows to examine wound healing effects on leukocyte trafficking in mammary tumors with intravital imaging.

PubMed

Sobolik, Tammy; Su, Ying-Jun; Ashby, Will; Schaffer, David K; Wells, Sam; Wikswo, John P; Zijlstra, Andries; Richmond, Ann

2016-01-01

We developed mammary imaging windows (MIWs) to evaluate leukocyte infiltration and cancer cell dissemination in mouse mammary tumors imaged by confocal microscopy. Previous techniques relied on surgical resection of a skin flap to image the tumor microenvironment restricting imaging time to a few hours. Utilization of mammary imaging windows offers extension of intravital imaging of the tumor microenvironment. We have characterized strengths and identified some previously undescribed potential weaknesses of MIW techniques. Through iterative enhancements of a transdermal portal we defined conditions for improved quality and extended confocal imaging time for imaging key cell-cell interactions in the tumor microenvironment.

Visualizing and quantifying the in vivo structure and dynamics of the Arabidopsis cortical cytoskeleton using CLSM and VAEM.

PubMed

Rosero, Amparo; Zárský, Viktor; Cvrčková, Fatima

2014-01-01

The cortical microtubules, and to some extent also the actin meshwork, play a central role in the shaping of plant cells. Transgenic plants expressing fluorescent protein markers specifically tagging the two main cytoskeletal systems are available, allowing noninvasive in vivo studies. Advanced microscopy techniques, in particular confocal laser scanning microscopy (CLSM) and variable angle epifluorescence microscopy (VAEM), can be nowadays used for imaging the cortical cytoskeleton of living cells with unprecedented spatial and temporal resolution. With the aid of suitable computing techniques, quantitative information can be extracted from microscopic images and video sequences, providing insight into both architecture and dynamics of the cortical cytoskeleton.

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.

Elucidation of the internal physical and chemical microstructure of pharmaceutical granules using X-ray micro-computed tomography, Raman microscopy and infrared spectroscopy.

PubMed

Crean, Barry; Parker, Andrew; Roux, Delphine Le; Perkins, Mark; Luk, Shen Y; Banks, Simon R; Melia, Colin D; Roberts, Clive J

2010-11-01

X-ray micro-computed tomography (XMCT) was used in conjunction with confocal Raman mapping to measure the intra-granular pore size, binder volumes and to provide spatial and chemical maps of internal granular components in α-lactose monohydrate granules formulated with different molecular weights of polyvinyl pyrrolidone (PVP). Infrared spectroscopy was used to understand the molecular association of binder domains. Granules were prepared by high-shear aqueous granulation from α-lactose monohydrate and PVP K29/32 or K90. XMCT was used to visualise the granule microstructure, intra-granular binder distribution and measure intra-granular porosity, which was subsequently related to intrusion porosimetry measurements. Confocal Raman microscopy and infrared microscopy were employed to investigate the distribution of components within the granule and explore the nature of binder substrate interactions. XMCT data sets of internal granule microstructure provided values of residual porosity in the lactose:PVP K29/32 and lactose:PVP K90 granules of 32.41 ± 4.60% and 22.40 ± 0.03%, respectively. The binder volumes of the lactose:PVP K29/32 and lactose:PVP K90 granules were 2.98 ± 0.10% and 3.38 ± 0.07%, respectively, and were attributed to PVP-rich binder domains within the granule. Confocal Raman microscopy revealed anisotropic domains of PVP between 2 μm and 20 μm in size surrounded by larger particles of lactose, in both granule types. Raman data showed that PVP domains contained various amounts of lactose, whilst IR microscopy determined that the PVP was molecularly associated with lactose, rather than residual water. The work shows that XMCT can be applied to investigate granular microstructure and resolve the porosity and the excipient and binder volumes. Combining this technique with vibrational techniques provides further structural information and aids the interpretations of the XMCT images. When used complementarily, these techniques highlighted that porosity and binder volume were the most significant microstructural differences between the α-lactose monohydrate granules formulated with the different grades of PVP. Copyright © 2010 Elsevier B.V. All rights reserved.

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

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 Light Absorption and Scattering Spectroscopic (CLASS) imaging: From cancer detection to sub-cellular function

NASA Astrophysics Data System (ADS)

Qiu, Le

Light scattering spectroscopy (LSS), an optical technique that relates the spectroscopic properties of light elastically scattered by small particles to their size, refractive index and shape, has been recently successfully employed for sensing morphological and biochemical properties of epithelial tissues and cells in vivo. LSS does not require exogenous markers, is non-invasive, and, due to its multispectral nature, can sense biological structures well beyond the diffraction limit. All that makes LSS be a very good candidate to be used both in clinical medicine for in vivo detection of disease and in cell biology to monitor cell function on the organelle scale. Recently we developed two LSS-based imaging modalities: clinical Polarized LSS (PLSS) Endoscopic Technique for locating early pre-cancerous changes in GI tract and Confocal Light Absorption and Scattering Spectroscopic (CLASS) Microscopy for studying cells in vivo without exogenous markers. One important application of the clinical PLSS endoscopic instrument, a noncontact scanning imaging device compatible with the standard clinical endoscopes and capable of detecting dysplastic changes, is to serve as a guide for biopsy in Barrett's esophagus (BE). The instrument detects parallel and perpendicular components of the polarized light, backscattered from epithelial tissues, and determines characteristics of epithelial nuclei from the residual spectra. It also can find tissue oxygenation, hemoglobin content and other properties from the diffuse light component. By rapidly scanning esophagus the PLSS endoscopic instrument makes sure the entire BE portion is scanned and examined for the presence of dysplasia. CLASS microscopy, on the other hand, combines principles of light scattering spectroscopy (LSS) with confocal microscopy. Its main purpose is to image cells on organelle scale in vivo without the use of exogenous labels which may affect the cell function. The confocal geometry selects specific region and images are obtained by scanning the confocal volume across the sample. The new beam scanning CLASS microscope is a significant improvement over the previous proof-of-principle device. With this new device we have already performed experiments to monitor morphological changes in cells during apoptosis, differentiated fetal from maternal nucleated red blood cells, and detected plasmon scattering spectra of single gold nanorod.

Deep Tissue Fluorescent Imaging in Scattering Specimens Using Confocal Microscopy

PubMed Central

Clendenon, Sherry G.; Young, Pamela A.; Ferkowicz, Michael; Phillips, Carrie; Dunn, Kenneth W.

2015-01-01

In scattering specimens, multiphoton excitation and nondescanned detection improve imaging depth by a factor of 2 or more over confocal microscopy; however, imaging depth is still limited by scattering. We applied the concept of clearing to deep tissue imaging of highly scattering specimens. Clearing is a remarkably effective approach to improving image quality at depth using either confocal or multiphoton microscopy. Tissue clearing appears to eliminate the need for multiphoton excitation for deep tissue imaging. PMID:21729357

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.

Reflectance confocal microscopy of tinea nigra: comparing images with dermoscopy and mycological examination results.

PubMed

Veasey, John Verrinder; Avila, Ricardo Bertozzi de; Ferreira, Marcus Antônio Maia de Olivas; Lazzarini, Rosana

2017-01-01

Tinea nigra is a superficial mycosis whose diagnosis is confirmed by isolating the infectious agent Hortae werneckii through mycological examinations. In vivo reflectance confocal microscopy, initially used in melanocytic dermatosis, has been used with skin infectious diseases to identify the parasite at the cellular level. We report, for the first time in the scientific literature, the use of reflectance confocal microscopy in a case of tinea nigra and compare its findings to dermoscopy and mycological examination results.

Reflectance confocal microscopy of tinea nigra: comparing images with dermoscopy and mycological examination results*

PubMed Central

Veasey, John Verrinder; de Avila, Ricardo Bertozzi; Ferreira, Marcus Antônio Maia de Olivas; Lazzarini, Rosana

2017-01-01

Tinea nigra is a superficial mycosis whose diagnosis is confirmed by isolating the infectious agent Hortae werneckii through mycological examinations. In vivo reflectance confocal microscopy, initially used in melanocytic dermatosis, has been used with skin infectious diseases to identify the parasite at the cellular level. We report, for the first time in the scientific literature, the use of reflectance confocal microscopy in a case of tinea nigra and compare its findings to dermoscopy and mycological examination results. PMID:28954116

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.

Validation of nonlinear interferometric vibrational imaging as a molecular OCT technique by the use of Raman microscopy

NASA Astrophysics Data System (ADS)

Benalcazar, Wladimir A.; Jiang, Zhi; Marks, Daniel L.; Geddes, Joseph B.; Boppart, Stephen A.

2009-02-01

We validate a molecular imaging technique called Nonlinear Interferometric Vibrational Imaging (NIVI) by comparing vibrational spectra with those acquired from Raman microscopy. This broadband coherent anti-Stokes Raman scattering (CARS) technique uses heterodyne detection and OCT acquisition and design principles to interfere a CARS signal generated by a sample with a local oscillator signal generated separately by a four-wave mixing process. These are mixed and demodulated by spectral interferometry. Its confocal configuration allows the acquisition of 3D images based on endogenous molecular signatures. Images from both phantom and mammary tissues have been acquired by this instrument and its spectrum is compared with its spontaneous Raman signatures.

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.

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.

Comparison of Confocal and Super-Resolution Reflectance Imaging of Metal Oxide Nanoparticles

PubMed Central

Guggenheim, Emily J.; Khan, Abdullah; Pike, Jeremy; Chang, Lynne; Lynch, Iseult; Rappoport, Joshua Z.

2016-01-01

The potential for human exposure to manufactured nanoparticles (NPs) has increased in recent years, in part through the incorporation of engineered particles into a wide range of commercial goods and medical applications. NP are ideal candidates for use as therapeutic and diagnostic tools within biomedicine, however concern exists regarding their efficacy and safety. Thus, developing techniques for the investigation of NP uptake into cells is critically important. Current intracellular NP investigations rely on the use of either Transmission Electron Microscopy (TEM), which provides ultrahigh resolution, but involves cumbersome sample preparation rendering the technique incompatible with live cell imaging, or fluorescent labelling, which suffers from photobleaching, poor bioconjugation and, often, alteration of NP surface properties. Reflected light imaging provides an alternative non-destructive label free technique well suited, but not limited to, the visualisation of NP uptake within model systems, such as cells. Confocal reflectance microscopy provides optical sectioning and live imaging capabilities, with little sample preparation. However confocal microscopy is diffraction limited, thus the X-Y resolution is restricted to ~250 nm, substantially larger than the <100 nm size of NPs. Techniques such as super-resolution light microscopy overcome this fundamental limitation, providing increased X-Y resolution. The use of Reflectance SIM (R-SIM) for NP imaging has previously only been demonstrated on custom built microscopes, restricting the widespread use and limiting NP investigations. This paper demonstrates the use of a commercial SIM microscope for the acquisition of super-resolution reflectance data with X-Y resolution of 115 nm, a greater than two-fold increase compared to that attainable with RCM. This increase in resolution is advantageous for visualising small closely spaced structures, such as NP clusters, previously unresolvable by RCM. This is advantageous when investigating the subcellular trafficking of NP within fluorescently labelled cellular compartments. NP signal can be observed using RCM, R-SIM and TEM and a direct comparison is presented. Each of these techniques has its own benefits and limitations; RCM and R-SIM provide novel complementary information while the combination of modalities provides a unique opportunity to gain additional information regarding NP uptake. The use of multiple imaging methods therefore greatly enhances the range of NPs that can be studied under label-free conditions. PMID:27695038

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

Cardiovascular Imaging Using Two-Photon Microscopy

PubMed Central

Scherschel, John A.; Rubart, Michael

2008-01-01

Two-photon excitation microscopy has become the standard technique for high resolution deep tissue and intravital imaging. It provides intrinsic three-dimensional resolution in combination with increased penetration depth compared to single-photon confocal microscopy. This article will describe the basic physical principles of two-photon excitation and will review its multiple applications to cardiovascular imaging, including second harmonic generation and fluorescence laser scanning microscopy. In particular, the capability and limitations of multiphoton microscopy to assess functional heterogeneity on a cellular scale deep within intact, Langendorff-perfused hearts are demonstrated. It will also discuss the use of two-photon excitation-induced release of caged compounds for the study of intracellular calcium signaling and intercellular dye transfer. PMID:18986603

Confocal fluorescence microscopy in a murine model of microdissection testicular sperm extraction to improve sperm retrieval.

PubMed

Smith, Ryan P; Lowe, Greg J; Kavoussi, Parviz K; Steers, William D; Costabile, Raymond A; Herr, John C; Shetty, Jagathpala; Lysiak, Jeffrey J

2012-05-01

Microdissection testicular sperm extraction markedly improves the sperm retrieval rates in men with nonobstructive azoospermia. However, localizing sperm foci can be time-consuming and it is not always successful. Fiberoptic confocal fluorescent microscopy offers the advantage of rapid in vivo detection of fluorescently labeled sperm in the seminiferous tubules. After establishing the feasibility of fiberoptic confocal fluorescent microscopy to identify antibody labeled sperm in vivo C57/B6 mice underwent intraperitoneal injection of busulfan to induce azoospermia. During spermatogenesis reestablishment at approximately 16 weeks the mice were anesthetized and the testes were delivered through a low midline incision. Fluorescein isothiocyanate labeled antibody to intra-acrosomal protein Hs-14 was injected retrograde into a single murine rete testis. The testes were imaged in vivo with fiberoptic confocal fluorescent microscopy and sperm foci were detected. The respective seminiferous tubules were excised and squash prepared for immunofluorescence microscopy. Sperm foci were identified in the testis injected with fluorescently tagged antibody by in vivo fiberoptic confocal fluorescence microscopy. The contralateral control testis of each mouse showed no specific signal. Immunofluorescence microscopy of the excised tubules provided morphological confirmation of the presence of labeled sperm with an absence in controls. Findings were consistent in the feasibility portion of the study and in the busulfan model of nonobstructive azoospermia. Fiberoptic confocal fluorescent microscopy was feasible during microdissection testicular sperm extraction in an azoospermic mouse model to identify fluorescently labeled sperm in vivo. Translation to the clinical setting could decrease operative time and improve the sperm harvest rate. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Lensless microscopy technique for static and dynamic colloidal systems.

PubMed

Alvarez-Palacio, D C; Garcia-Sucerquia, J

2010-09-15

We present the application of a lensless microscopy technique known as digital in-line holographic microscopy (DIHM) to image dynamic and static colloidal systems of microspheres. DIHM has been perfected up to the point that submicrometer lateral resolution with several hundreds of micrometers depth of field is achieved with visible light; it is shown that the lateral resolution of DIHM is enough to resolve self-assembled colloidal monolayers built up from polystyrene spheres with submicrometer diameters. The time resolution of DIHM is of the order of 4 frames/s at 2048 x 2048 pixels, which represents an overall improvement of 16 times the time resolution of confocal scanning microscopy. This feature is applied to the visualization of the migration of dewetting fronts in dynamic colloidal systems and the formation of front-like arrangements of particles. Copyright 2010 Elsevier Inc. All rights reserved.

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.

Enhancing microparticle internalization by nonphagocytic cells through the use of noncovalently conjugated polyethyleneimine

PubMed Central

Patiño, Tania; Nogués, Carme; Ibáñez, Elena; Barrios, Leonardo

2012-01-01

Development of micro- and nanotechnology for the study of living cells, especially in the field of drug delivery, has gained interest in recent years. Although several studies have reported successful results in the internalization of micro- and nanoparticles in phagocytic cells, when nonphagocytic cells are used, the low internalization efficiency represents a limitation that needs to be overcome. It has been reported that covalent surface modification of micro- and nanoparticles increases their internalization rate. However, this surface modification represents an obstacle for their use as drug-delivery carriers. For this reason, the aim of the present study was to increase the capability for microparticle internalization of HeLa cells through the use of noncovalently bound transfection reagents: polyethyleneimine (PEI) Lipofectamine™ 2000 and FuGENE 6®. Both confocal microscopy and flow cytometry techniques allowed us to precisely quantify the efficiency of microparticle internalization by HeLa cells, yielding similar results. In addition, intracellular location of microparticles was analyzed through transmission electron microscopy and confocal microscopy procedures. Our results showed that free PEI at a concentration of 0.05 mM significantly increased microparticle uptake by cells, with a low cytotoxic effect. As determined by transmission electron and confocal microscopy analyses, microparticles were engulfed by plasma-membrane projections during internalization, and 24 hours later they were trapped in a lysosomal compartment. These results show the potential use of noncovalently conjugated PEI in microparticle internalization assays. PMID:23152683

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.

Laboratory-based characterization of plutonium in soil particles using micro-XRF and 3D confocal XRF

DOE PAGES

McIntosh, Kathryn Gallagher; Cordes, Nikolaus Lynn; Patterson, Brian M.; ...

2015-03-29

The investigation of plutonium (Pu) in a soil matrix is of interest in safeguards, nuclear forensics, and environmental remediation activities. The elemental composition of two plutonium contaminated soil particles was characterized nondestructively using a pair of micro X-ray fluorescence spectrometry (micro-XRF) techniques including high resolution X-ray (hiRX) and 3D confocal XRF. The three dimensional elemental imaging capability of confocal XRF permitted the identification two distinct Pu particles within the samples: one external to the Ferich soil matrix and another co-located with Cu within the soil matrix. The size and morphology of the particles was assessed with X-ray transmission microscopy andmore » micro X-ray computed tomography (micro-CT) providing complementary morphological information. Limits of detection for a 30 μm Pu particle are <10 ng for each of the XRF techniques. Ultimately, this study highlights the capability for lab-based, nondestructive, spatially resolved characterization of heterogeneous matrices on the micrometer scale with nanogram sensitivity.« less

Structure and chemical composition of the dentin-enamel junction analyzed by Confocal Raman Microscopy

NASA Astrophysics Data System (ADS)

Desoutter, A.; Salehi, H.; Slimani, A.; Marquet, P.; Jacquot, B.; Tassery, H.; Cuisinier, F. J. G.

2014-02-01

The structure and chemical composition of the human dentin-enamel junction (DEJ) was studied using confocal Raman microscopy - a chemical imaging technique. Slices of non-fixed, sound teeth were prepared with an Isomet diamond saw and scanned with Witec Alpha300R system. The combination of different characteristics peaks of phosphate, carbonate and organic matrix (respectively 960, 1072 and 1545 cm-1), generates images representing the chemical composition of the DEJ area. Images are also calculated using peak ratios enabling precise determination of the chemical composition across the DEJ. Then, with two characterized peaks, different pictures are calculated to show the ratio of two components. The images of the spatial distribution of mineral phosphate (960cm-1) to organic matrix (1545 cm-1) ratios, mineral carbonates (1072cm-1) to mineral phosphate ratios; and mineral carbonates to organic matrix ratios were reconstructed. Cross sectional and calculated graphic profile show the variations of the different chemical component ratios through the enamel and the dentin. Phosphate to organic ratio shows an accumulation of organic material under the enamel surface. The cross sectional profile of these pictures shows a high phosphate content compared to enamel in the vicinity of the DEJ. The Confocal Raman imaging technique can be used to further provide full chemical imaging of tooth, particularly of the whole DEJ and to study enamel and dentin decay.

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.

High Definition Confocal Imaging Modalities for the Characterization of Tissue-Engineered Substitutes.

PubMed

Mayrand, Dominique; Fradette, Julie

2018-01-01

Optimal imaging methods are necessary in order to perform a detailed characterization of thick tissue samples from either native or engineered tissues. Tissue-engineered substitutes are featuring increasing complexity including multiple cell types and capillary-like networks. Therefore, technical approaches allowing the visualization of the inner structural organization and cellular composition of tissues are needed. This chapter describes an optical clearing technique which facilitates the detailed characterization of whole-mount samples from skin and adipose tissues (ex vivo tissues and in vitro tissue-engineered substitutes) when combined with spectral confocal microscopy and quantitative analysis on image renderings.

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.

Using confocal laser scanning microscopy to probe the milk fat globule membrane and associated proteins.

PubMed

Gallier, Sophie; Gragson, Derek; Jiménez-Flores, Rafael; Everett, David

2010-04-14

The bovine milk fat globule membrane (MFGM) is an important, biologically relevant membrane due to its functional and health properties. Its composition has been thoroughly studied, but its structure, especially the lateral organization of its components, still remains unclear. We have used confocal laser scanning microscopy (CLSM) to investigate the surface structure of the MFGM in globules with different degrees of processing using two types of fluorescently labeled phospholipid probes and a protein dye. Using this technique, we have observed heterogeneities in the distribution of MFGM lipids and proteins relating to the processing and size of the globules. The effect of pretreating the milk (centrifugation, pasteurization-homogenization and churning) was studied by double-staining the surface of the milk fat globules, followed by observation using CLSM, and by determining the phospholipid profile of raw milk, raw cream, processed milk and buttermilk powder. Our findings agree with other techniques by showing that the composition of the MFGM changes with processing through the loss of phospholipids and the adsorption of caseins and whey proteins onto the surface.

Confocal observation of hydrophilic and lipophilic photosensitizers in endothelial cells, lumen of the vessels, interstitium, and tumor cells using the chicken chorioallantoic membrane

NASA Astrophysics Data System (ADS)

Rueck, Angelika C.; Akguen, Nermin; Heckelsmiller, K.; Beck, Gerd C.; Genze, Felicitas; Steiner, Rudolf W.

1998-05-01

The dynamic behavior of lipophilic and hydrophilic sensitizers in cell cultures and non animal in vivo systems with varying incubation but also during the photodynamic therapy will be summarized within the presentation. As an appropriate in vivo system we used the chorioallantoic membrane (CAM) of fertilized eggs, which served as a substrate for tumor cells. Because the CAM is a transparent membrane it is possible to view individual blood vessels and to examine tumor cells as well as structural changes of the supplying vasculature. To adapt this system to high magnification microscopy, we established a new technique for in vivo observation of the CAM tissue. This technique enables online investigations of alterations at cellular level induced by drugs with confocal laser scanning microscopy. The localization of the drugs with clinical importance was observed after different application times in the lumen of the vessels, the endothelial cells and the tumor cells. In addition light induced subcellular Ca2+-changes were observed and correlated with the photodynamic process.

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: AXIAL RESOLUTION

EPA Science Inventory

Abstract

Confocal Microscopy System Performance: Axial resolution.
Robert M. Zucker, PhD

Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Re...

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 raman microscopy as a non-invasive tool to investigate the phase composition of frozen complex cryopreservation media.

PubMed

Kreiner-Møller, A; Stracke, F; Zimmermann, H

2013-01-01

Various cryoprotective agents (CPA) are added to cell media in order to avoid cell injury during cryo preservation. The resulting complex environment of the preserved cell, consisting of crystalline and liquid phases can however not be investigated non-invasively by established methods in cryobiology. This study shows how scanning confocal Raman microscopy can non-invasively extract information on chemical composition, phase domain and distribution at cryogenic temperatures. The formation of the salt hydrate, hydrohalite NaCl∙H2O, in solutions comprised of phosphate buffered saline (PBS) and dimethyl sulphoxide (DMSO) is studied in particular. Scanning confocal Raman microscopy can be used to unambiguously identify hydrohalite in a medium containing DMSO and saline. The confocal Raman microscopy imaging along with differential scanning calorimetric measurements further show that the hydrohalite is formed without eutectic formation. This method also allows for discrimination between closely packed hydrohalite crystals that are oriented differently.

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.

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.

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

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.

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.

The Use of Confocal Photoluminescence Microscopy for Determination of Defect Densities in Various 2-6 Semiconductors

DTIC Science & Technology

2014-03-11

optical configuration that significantly enhances both lateral and depth resolution and returns crisp PL images 1. REPORT DATE (DD-MM-YYYY) 4. TITLE AND...that significantly enhances both lateral and depth resolution and returns crisp PL images with high contrast. This technique revolutionized fluorescent...resolution and returns crisp PL images with high contrast. This technique revolutionized fluorescent imaging in biology, and has the potential to

Frequency domain phase-shifted confocal microscopy (FDPCM) with array detection

NASA Astrophysics Data System (ADS)

Ge, Baoliang; Huang, Yujia; Fang, Yue; Kuang, Cuifang; Xiu, Peng; Liu, Xu

2017-09-01

We proposed a novel method to reconstruct images taken by array detected confocal microscopy without prior knowledge about its detector distribution. The proposed frequency domain phase-shifted confocal microscopy (FDPCM) shifts the image from each detection channel to its corresponding place by substituting the phase information in Fourier domain. Theoretical analysis shows that our method could approach the resolution nearly twofold of wide-field microscopy. Simulation and experiment results are also shown to verify the applicability and effectiveness of our method. Compared to Airyscan, our method holds the advantage of simplicity and convenience to be applied to array detectors with different structure, which makes FDPCM have great potential in the application of biomedical observation in the future.

CONFOCAL LASER SCANNING MICROSCOPY OF APOPTOSIS IN WHOLE MOUSE OVARIES

EPA Science Inventory

Confocal Laser Scanning Microscopy of Apoptosis in Whole Mouse Ovaries. Robert M. Zucker Susan C. Jeffay and Sally D. Perreault Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle...

Unwarping confocal microscopy images of bee brains by nonrigid registration to a magnetic resonance microscopy image.

PubMed

Rohlfing, Torsten; Schaupp, Frank; Haddad, Daniel; Brandt, Robert; Haase, Axel; Menzel, Randolf; Maurer, Calvin R

2005-01-01

Confocal microscopy (CM) is a powerful image acquisition technique that is well established in many biological applications. It provides 3-D acquisition with high spatial resolution and can acquire several different channels of complementary image information. Due to the specimen extraction and preparation process, however, the shapes of imaged objects may differ considerably from their in vivo appearance. Magnetic resonance microscopy (MRM) is an evolving variant of magnetic resonance imaging, which achieves microscopic resolutions using a high magnetic field and strong magnetic gradients. Compared to CM imaging, MRM allows for in situ imaging and is virtually free of geometrical distortions. We propose to combine the advantages of both methods by unwarping CM images using a MRM reference image. Our method incorporates a sequence of image processing operators applied to the MRM image, followed by a two-stage intensity-based registration to compute a nonrigid coordinate transformation between the CM images and the MRM image. We present results obtained using CM images from the brains of 20 honey bees and a MRM image of an in situ bee brain. Copyright 2005 Society of Photo-Optical Instrumentation Engineers.

Combined use of confocal laser scanning microscopy (CLSM) and Raman microscopy (RM): investigations on EPS-Matrix.

PubMed

Wagner, Michael; Ivleva, Natalia P; Haisch, Christoph; Niessner, Reinhard; Horn, Harald

2009-01-01

Confocal laser scanning microscopy (CLSM) was applied in combination with Raman microscopy (RM) for the characterization of heterotrophic biofilms. Compared to CLSM, RM allows for a deeper insight into the chemical structure of extracellular polymeric substances (EPS) of the biofilm matrix. A low load of glucose (2 g m(-2)d(-1)) was applied as substrate to ensure small growth rates of the heterotrophic biofilm. To investigate the influence of hydrodynamic conditions on the chemical composition of EPS, a three funnel flow system was used, wherein biofilms were grown at Reynolds numbers of 1000, 2500 and 4000, respectively. 31 and 92 days after inoculation with activated sludge supernatant RM was applied as an additional technique to standard CLSM measurements for a more detailed analysis of the biofilm matrix. Polysaccharide-related Raman bands are in good agreement with the lectin binding analysis from CLSM. For the older biofilm, lectin binding analysis showed no change in the composition of EPS, whereas Raman spectra pointed out a change of EPS composition from predominantly polysaccharides to predominantly (glyco) proteins. For the applied substrate condition no significant influence of the Reynolds number on the chemical properties was observed.

Evaluating ex vivo fluorescence confocal microscopy images of basal cell carcinomas in Mohs excised tissue.

PubMed

Longo, C; Rajadhyaksha, M; Ragazzi, M; Nehal, K; Gardini, S; Moscarella, E; Lallas, A; Zalaudek, I; Piana, S; Argenziano, G; Pellacani, G

2014-09-01

Fluorescence confocal microscopy (FCM) is an emerging technology for rapid imaging of excised tissue, without the need for frozen- or fixed-section processing. Basal cell carcinomas (BCCs) can be detected in Mohs excisions although few studies have described the major BCC findings as seen on FCM. To describe the major BCC findings of excised tissue during Mohs surgery and to correlate them with histopathology. Freshly excised tumours and frozen-thawed discarded tissue of BCC during Mohs surgery were analysed by means of FCM. A side-by-side correlation between FCM images and histological sections was performed. The FCM features of overlying skin and adnexal structures were also described. Sixty-four BCC cases were analysed. Distinct BCC types appeared unique in terms of shape and size of tumour islands [bigger in nodular (18/25), smaller and rounded in micronodular (7/7) and tiny cords for infiltrative ones (24/30)] and for the presence of clefting, palisading and increased nucleus/cytoplasm ratio. An excellent correlation was found between FCM and histological findings (Cohen's κ statistics = 0·9). In six cases, the presence of sebaceous glands and intense stroma reaction represented possible confounders. Fluorescence confocal microscopy is a fast and new imaging technique that allows an excellent visualization of skin structures and BCC findings during Mohs surgery. © 2014 British Association of Dermatologists.

Three-dimensional textures and defects of soft material layering revealed by thermal sublimation.

PubMed

Yoon, Dong Ki; Kim, Yun Ho; Kim, Dae Seok; Oh, Seong Dae; Smalyukh, Ivan I; Clark, Noel A; Jung, Hee-Tae

2013-11-26

Layering is found and exploited in a variety of soft material systems, ranging from complex macromolecular self-assemblies to block copolymer and small-molecule liquid crystals. Because the control of layer structure is required for applications and characterization, and because defects reveal key features of the symmetries of layered phases, a variety of techniques have been developed for the study of soft-layer structure and defects, including X-ray diffraction and visualization using optical transmission and fluorescence confocal polarizing microscopy, atomic force microscopy, and SEM and transmission electron microscopy, including freeze-fracture transmission electron microscopy. Here, it is shown that thermal sublimation can be usefully combined with such techniques to enable visualization of the 3D structure of soft materials. Sequential sublimation removes material in a stepwise fashion, leaving a remnant layer structure largely unchanged and viewable using SEM, as demonstrated here using a lamellar smectic liquid crystal.

Classifying distinct basal cell carcinoma subtype by means of dermatoscopy and reflectance confocal microscopy.

PubMed

Longo, Caterina; Lallas, Aimilios; Kyrgidis, Athanassios; Rabinovitz, Harold; Moscarella, Elvira; Ciardo, Silvana; Zalaudek, Iris; Oliviero, Margaret; Losi, Amanda; Gonzalez, Salvador; Guitera, Pascale; Piana, Simonetta; Argenziano, Giuseppe; Pellacani, Giovanni

2014-10-01

The current guidelines for the management of basal cell carcinoma (BCC) suggest a different therapeutic approach according to histopathologic subtype. Although dermatoscopic and confocal criteria of BCC have been investigated, no specific studies were performed to evaluate the distinct reflectance confocal microscopy (RCM) aspects of BCC subtypes. To define the specific dermatoscopic and confocal criteria for delineating different BCC subtypes. Dermatoscopic and confocal images of histopathologically confirmed BCCs were retrospectively evaluated for the presence of predefined criteria. Frequencies of dermatoscopic and confocal parameters are provided. Univariate and adjusted odds ratios were calculated. Discriminant analyses were performed to define the independent confocal criteria for distinct BCC subtypes. Eighty-eight BCCs were included. Dermatoscopically, superficial BCCs (n=44) were primarily typified by the presence of fine telangiectasia, multiple erosions, leaf-like structures, and revealed cords connected to the epidermis and epidermal streaming upon RCM. Nodular BCCs (n=22) featured the classic dermatoscopic features and well outlined large basaloid islands upon RCM. Infiltrative BCCs (n=22) featured structureless, shiny red areas, fine telangiectasia, and arborizing vessels on dermatoscopy and dark silhouettes upon RCM. The retrospective design. Dermatoscopy and confocal microscopy can reliably classify different BCC subtypes. Copyright © 2014 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

Confocal fluorescence microscopy for rapid evaluation of invasive tumor cellularity of inflammatory breast carcinoma core needle biopsies.

PubMed

Dobbs, Jessica; Krishnamurthy, Savitri; Kyrish, Matthew; Benveniste, Ana Paula; Yang, Wei; Richards-Kortum, Rebecca

2015-01-01

Tissue sampling is a problematic issue for inflammatory breast carcinoma, and immediate evaluation following core needle biopsy is needed to evaluate specimen adequacy. We sought to determine if confocal fluorescence microscopy provides sufficient resolution to evaluate specimen adequacy by comparing invasive tumor cellularity estimated from standard histologic images to invasive tumor cellularity estimated from confocal images of breast core needle biopsy specimens. Grayscale confocal fluorescence images of breast core needle biopsy specimens were acquired following proflavine application. A breast-dedicated pathologist evaluated invasive tumor cellularity in histologic images with hematoxylin and eosin staining and in grayscale and false-colored confocal images of cores. Agreement between cellularity estimates was quantified using a kappa coefficient. 23 cores from 23 patients with suspected inflammatory breast carcinoma were imaged. Confocal images were acquired in an average of less than 2 min per core. Invasive tumor cellularity estimated from histologic and grayscale confocal images showed moderate agreement by kappa coefficient: κ = 0.48 ± 0.09 (p < 0.001). Grayscale confocal images require less than 2 min for acquisition and allow for evaluation of invasive tumor cellularity in breast core needle biopsy specimens with moderate agreement to histologic images. We show that confocal fluorescence microscopy can be performed immediately following specimen acquisition and could indicate the need for additional biopsies at the initial visit.

CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: QA TESTS, QUANTITATION AND SPECTROSCOPY

EPA Science Inventory

Confocal Microscopy System Performance: QA tests, Quantitation and Spectroscopy.

Robert M. Zucker 1 and Jeremy M. Lerner 2,
1Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research Development, U.S. Environmen...

Multispectral confocal microscopy images and artificial neural nets to monitor the photosensitizer uptake and degradation in Candida albicans cells

NASA Astrophysics Data System (ADS)

Romano, Renan A.; Pratavieira, Sebastião.; da Silva, Ana P.; Kurachi, Cristina; Guimarães, Francisco E. G.

2017-07-01

This study clearly demonstrates that multispectral confocal microscopy images analyzed by artificial neural networks provides a powerful tool to real-time monitoring photosensitizer uptake, as well as photochemical transformations occurred.

Mapping of dendritic lesions in patients with herpes simplex keratitis using in vivo confocal microscopy

PubMed Central

Yokogawa, Hideaki; Kobayashi, Akira; Mori, Natsuko; Sugiyama, Kazuhisa

2015-01-01

Purpose To produce a two-dimensional reconstruction map of dendritic lesions in patients with herpes simplex keratitis (HSK) using in vivo confocal microscopy. Methods Four eyes of four patients (mean 65.8 years) with HSK presenting with a dendritic lesion were enrolled. Slit-lamp biomicroscopy and in vivo laser confocal microscopy were performed. Acquired confocal images at the level of the epithelium were arranged and mapped into subconfluent montages. Changes in the shape and degree of light reflection of abnormal cells and deposits around dendritic lesions as well as other corneal layers were qualitatively evaluated. Results Mapping of dendritic lesion was successful in all cases, and the subconfluent montages clearly showed the larger image of dendritic lesion. In all cases, the dendritic lesion consisted of hyperreflective irregular epithelial cells, and was surrounded by distorted and elongated epithelial cells. In three cases, hyperreflective deposits were noted at the midline of the lesion. The corneal stroma showed a hyperreflective honeycomb pattern. In two cases, inflammatory cells were observed at the level of endothelial cell layer. Conclusion Mapping of dendritic lesions in patients with HSK was successful in all patients using in vivo confocal microscopy. Cellular level observation of dendritic lesion at a relatively larger magnification may help understand the in vivo morphological change of HSK. Further study in more patients with HSK and nonherpetic dendritic lesion is needed to utilize confocal microscopy images in differential diagnosis and follow-up of the epithelial lesions with dendrite. PMID:26445524

Mapping of dendritic lesions in patients with herpes simplex keratitis using in vivo confocal microscopy.

PubMed

Yokogawa, Hideaki; Kobayashi, Akira; Mori, Natsuko; Sugiyama, Kazuhisa

2015-01-01

To produce a two-dimensional reconstruction map of dendritic lesions in patients with herpes simplex keratitis (HSK) using in vivo confocal microscopy. Four eyes of four patients (mean 65.8 years) with HSK presenting with a dendritic lesion were enrolled. Slit-lamp biomicroscopy and in vivo laser confocal microscopy were performed. Acquired confocal images at the level of the epithelium were arranged and mapped into subconfluent montages. Changes in the shape and degree of light reflection of abnormal cells and deposits around dendritic lesions as well as other corneal layers were qualitatively evaluated. Mapping of dendritic lesion was successful in all cases, and the subconfluent montages clearly showed the larger image of dendritic lesion. In all cases, the dendritic lesion consisted of hyperreflective irregular epithelial cells, and was surrounded by distorted and elongated epithelial cells. In three cases, hyperreflective deposits were noted at the midline of the lesion. The corneal stroma showed a hyperreflective honeycomb pattern. In two cases, inflammatory cells were observed at the level of endothelial cell layer. Mapping of dendritic lesions in patients with HSK was successful in all patients using in vivo confocal microscopy. Cellular level observation of dendritic lesion at a relatively larger magnification may help understand the in vivo morphological change of HSK. Further study in more patients with HSK and nonherpetic dendritic lesion is needed to utilize confocal microscopy images in differential diagnosis and follow-up of the epithelial lesions with dendrite.

Saturated virtual fluorescence emission difference microscopy based on detector array

NASA Astrophysics Data System (ADS)

Liu, Shaocong; Sun, Shiyi; Kuang, Cuifang; Ge, Baoliang; Wang, Wensheng; Liu, Xu

2017-07-01

Virtual fluorescence emission difference microscopy (vFED) has been proposed recently to enhance the lateral resolution of confocal microscopy with a detector array, implemented by scanning a doughnut-shaped pattern. Theoretically, the resolution can be enhanced by around 1.3-fold compared with that in confocal microscopy. For further improvement of the resolving ability of vFED, a novel method is presented utilizing fluorescence saturation for super-resolution imaging, which we called saturated virtual fluorescence emission difference microscopy (svFED). With a point detector array, matched solid and hollow point spread functions (PSF) can be obtained by photon reassignment, and the difference results between them can be used to boost the transverse resolution. Results show that the diffraction barrier can be surpassed by at least 34% compared with that in vFED and the resolution is around 2-fold higher than that in confocal microscopy.

Actinic Keratosis and Non-Invasive Diagnostic Techniques: An Update

PubMed Central

Casari, Alice; Chester, Johanna; Pellacani, Giovanni

2018-01-01

Actinic keratosis represents the earliest manifestation of non-melanoma skin cancer. Because of their risk of progression to invasive squamous cell carcinoma, an earlier diagnosis and treatment are mandatory. Their diagnosis sometimes could represent a challenge even for expert dermatologists. Dermoscopy, confocal laser microscopy and optical coherence tomography could help clinicians in diagnosis. PMID:29316678

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

PubMed

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

2018-01-01

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

New telemedicine techniques in dermatology - evaluation with reflectance confocal microscopy via cloud-based platform.

PubMed

Łudzik, Joanna; Witkowski, Alexander Michael; Roterman-Konieczna, Irena

Dermoscopically equivocal skin lesions may present a diagnostic challenge in daily clinical practice and are regularly sent for second expert opinion. We present a new approach to handling these cases in a consultation referral system that enables communication between the initial doctor at the image upload site and dermatology experts at a distance via cloud-based telemedicine. In our study we retrospectively evaluated 100 equivocal cases with complete digital dermoscopy-reflectance confocal microscopy image sets and compared suggested management of the initial doctor to a second expert confocal reader. We evaluated the effect of reader concordance on final management of these lesions resulting in a single reader overall sensitivity of 89% and specificity of 66% and double reader concordance method sensitivity of 98% and specificity of 54%. In conclusion, we found that application of double reader evaluation of these image sets with automatic referral of lesions for removal in the case of discordant diagnosis between two doctors improved the sensitivity of diagnosis in this subset of lesions and may increase the safety threshold of management choice reducing potential misdiagnosis in telemedicine settings. This paper concerns the application of telemedicine in practical medicine.

Clinical significance of owl eye morphologic features by in vivo laser confocal microscopy in patients with cytomegalovirus corneal endotheliitis.

PubMed

Kobayashi, Akira; Yokogawa, Hideaki; Higashide, Tomomi; Nitta, Koji; Sugiyama, Kazuhisa

2012-03-01

To demonstrate the clinical significance of owl eye morphologic features observed by in vivo laser confocal microscopy in patients with cytomegalovirus (CMV) corneal endotheliitis. Observational case series. participants: Six eyes of 6 patients (6 men; mean age, 73.3 years) with cytomegalovirus corneal endotheliitis diagnosed by clinical manifestations together with polymerase chain reaction from aqueous humor samples. intervention: All patients were examined by slit-lamp biomicroscopy and in vivo laser confocal microscopy. main outcome measures: Clinical manifestations were summarized by reviewing medical records. Selected confocal images of corneal layers were evaluated qualitatively for shape and degree of light reflection of abnormal cells and deposits. All patients had long histories of anterior uveitis with intraocular pressure elevation, corneal edema with keratic precipitates, and decrease of endothelial cell densities. Coin-shaped lesions were observed by slit lamp only in 1 patient at the first visit and in 2 additional patients at subsequent follow-up. In all patients, confocal microscopy demonstrated reduced subepithelial nerves, subepithelial opacity, increased reflectivity of keratocytes, highly reflective dots, and needle-shaped bodies. Owl eye morphologic features were observed consistently in all patients at the initial visit, and highly reflective round bodies were detected in 5 patients; most notably, these confocal features were reversible after resolution of endotheliitis. Owl eye morphologic features and highly reflective round bodies observed by confocal microscopy may be useful as an adjunct for the noninvasive diagnosis of cytomegalovirus corneal endotheliitis. Reversibility of these features after resolution of endotheliitis may be useful for monitoring the therapeutic effects without multiple anterior chamber tap. Copyright © 2012 Elsevier Inc. All rights reserved.

Optical Diagnostics in Medicine

NASA Astrophysics Data System (ADS)

Iftimia, Nicusor

2003-03-01

Light has a unique potential for non-invasive tissue diagnosis. The relatively short wavelength of light allows imaging of tissue at the resolution of histopathology. While strong multiple scattering of light in tissue makes attainment of this resolution difficult for thick tissues, most pathology emanates from epithelial surfaces. Therefore, high-resolution diagnosis of many important diseases may be achieved by transmitting light to the surface of interest. The recent fiber-optic implementation of technologies that reject multiple scattering, such as confocal microscopy and optical low coherence interferometry, have brought us one step closer to realizing non-invasive imaging of architectural and cellular features of tissue. Optical coherence tomography (OCT) can produce high-resolution cross-sectional images of biological structures. Clinical OCT studies conducted in the gastrointestinal tract and cardiovascular system have shown that OCT is capable of providing images of the architectural (> 20 µm) microanatomy of a variety of epithelial tissues, including the layered structure of squamous epithelium and arterial vessels. Fine Needle Aspiration- Low Coherence Interferometry (FNA-LCI) is another optical diagnostics technique, which is a suitable solution to increase the effectiveness of the FNA procedures. LCI is capable of measuring depth resolved (axial, z) tissue structure, birefringence, flow (Doppler shift), and spectra at a resolution of several microns. Since LCI systems are fiber-optic based, LCI probes may easily fit within the bore of a fine gauge needle, allowing diagnostic information to be obtained directly from the FNA biopsy site. Fiber optic spectrally encoded confocal microscopy (SECM) is a new confocal microscopy method, which eliminates the need for rapid beam scanning within the optical probe. This advance enables confocal microscopy to be performed through small diameter probes and will allow assessment of internal human tissues in vivo at the cellular level. A detailed description of several fiber optics based systems for early diseases diagnosis, as well as preliminary clinic results, will be presented.

MAMMALIAN APOPTOSIS IN WHOLE NEONATAL OVARIES USING CONFOCAL LASER SCANNING MICROSCOPY

EPA Science Inventory

MAMMALIAN APOPTOSIS IN WHOLE NEONATAL OVARIES USING CONFOCAL LASER SCANNING MICROSCOPY

Robert M. Zucker Susan C. Jeffery and Sally D. Perreault

Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Prot...

CONFOCAL LASER SCANNING MICROSCOPY OF APOPTOSIS IN WHOLE MOUSE AND RAT OVARIES

EPA Science Inventory

Confocal Laser Scanning Microscopy of Apoptosis in Whole Mouse and Rat Ovaries. Robert M. Zucker Susan C. Jeffay and Sally D. Perreault Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research ...

WHOLE INSECT AND MAMMALIAN EMBRYO IMAGING WITH CONFOCAL MICROSCOPY: MORPHOLOGY AND APOPTOSIS

EPA Science Inventory

Background: After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and indicates...

Analysis of Orientations of Collagen Fibers by Novel Fiber-Tracking Software

NASA Astrophysics Data System (ADS)

Wu, Jun; Rajwa, Bartlomiej; Filmer, David L.; Hoffmann, Christoph M.; Yuan, Bo; Chiang, Ching-Shoei; Sturgis, Jennie; Robinson, J. Paul

2003-12-01

Recent evidence supports the notion that biological functions of extracellular matrix (ECM) are highly correlated to not only its composition but also its structure. This article integrates confocal microscopy imaging and image-processing techniques to analyze the microstructural properties of ECM. This report describes a two- and three-dimensional fiber middle-line tracing algorithm that may be used to quantify collagen fibril organization. We utilized computer simulation and statistical analysis to validate the developed algorithm. These algorithms were applied to confocal images of collagen gels made with reconstituted bovine collagen type I, to demonstrate the computation of orientations of individual fibers.

Fluorescence Imaging of Posterior Spiracles from Second and Third Instars of Forensically-important Chrysomya rufifacies (Diptera: Calliphoridae)*

PubMed Central

Flores, Danielle; Miller, Amy L.; Showman, Angelique; Tobita, Caitlyn; Shimoda, Lori M.N.; Sung, Carl; Stokes, Alexander J.; Tomberlin, Jeffrey K.; Carter, David O.; Turner, Helen

2016-01-01

Entomological protocols for aging blow fly (Diptera: Calliphoridae) larvae to estimate the time of colonization (TOC) are commonly used to assist in death investigations. While the methodologies for analysing fly larvae differ, most rely on light microscopy, genetic analysis or, more rarely, electron microscopy. This pilot study sought to improve resolution of larval stage in the forensically-important blow fly Chrysomya rufifacies using high-content fluorescence microscopy and biochemical measures of developmental marker proteins. We established fixation and mounting protocols, defined a set of measurable morphometric criteria and captured developmental transitions of 2nd instar to 3rd instar using both fluorescence microscopy and anti-ecdysone receptor Western blot analysis. The data show that these instars can be distinguished on the basis of robust, non-bleaching, autofluorescence of larval posterior spiracles. High content imaging techniques using confocal microscopy, combined with morphometric and biochemical techniques, may therefore aid forensic entomologists in estimating TOC. PMID:27706817

Using corneal confocal microscopy to track changes in the corneal layers of dry eye patients after autologous serum treatment.

PubMed

Mahelkova, Gabriela; Jirsova, Katerina; Seidler Stangova, Petra; Palos, Michalis; Vesela, Viera; Fales, Ivan; Jiraskova, Nada; Dotrelova, Dagmar

2017-05-01

In vivo corneal confocal microscopy allows the examination of each layer of the cornea in detail and the identification of pathological changes at the cellular level. The purpose of this study was to identify the possible effects of a three-month treatment with autologous serum eye-drops in different corneal layers of patients with severe dry eye disease using corneal confocal microscopy. Twenty-six patients with dry eye disease were included in the study. Corneal fluorescein staining was performed. The corneas of the right eyes were examined using in vivo corneal confocal microscopy before and after a three-month treatment with autologous serum drops. The densities of superficial and basal epithelial cells, Langerhans cells, the keratocytes and activated keratocytes, the density of endothelial cells and the status of the sub-basal nerve plexus fibres were evaluated. A significant decrease in corneal fluorescein staining was found after the three-month autologous serum treatment (p = 0.0006). The basal epithelial cell density decreased significantly (p = 0.001), while the density of superficial epithelial cells did not change significantly (p = 0.473) nor did the number of Langerhans cells or activated keratocytes (p = 0.223; p = 0.307, respectively). There were no differences in the other corneal cell layers or in the status of the nerve fibres. The results demonstrate the ability of corneal confocal microscopy to evaluate an improvement in the basal epithelial cell layer of the cornea after autologous serum treatment in patients with dry eye disease. More studies with longer follow-up periods are needed to elucidate the suitability of corneal confocal microscopy to follow the effect of autologous serum treatment on nerve fibres or other corneal layers in dry eye disease patients. © 2016 Optometry Australia.

Evaluation of the Surface Characteristics of Various Implant Abutment Materials Using Confocal Microscopy and White Light Interferometry.

PubMed

Park, Jun-Beom; Yang, Seung-Min; Ko, Youngkyung

2015-12-01

The purpose of this study was to evaluate the surface characteristics of various implant abutment materials, such as of titanium alloy (Ti6Al4V; Ma), machined cobalt-chrome-molybdenum alloy (CCM), titanium nitride coating on a titanium alloy disc (TiN), anodic oxidized titanium alloy disc (AO), composite resin coating on a titanium alloy disc (Res), and zirconia disc (Zr), using confocal microscopy and white light interferometry. Measurements from the 2 methods were evaluated to see if these methods would give equivalent results. The precision of measurements were evaluated by the coefficient of variation. Five discs each of Ma, CCM, TiN, AO, Res, and Zr were used. The surface roughness was evaluated by confocal laser microscopy and white light interferometry. Confocal microscopy showed that the Res group showed significantly greater Ra, Rq, Rz, Sa, Sq, and Sz values compared with those of the Ma group (P < 0.05). The white light interferometry results showed that the Res group had significantly higher Ra, Rq, Rz, Rt, Sa, Sq, Sz, and Sdr values compared with the Ma group (P < 0.05). All the roughness parameters obtained from the 2 methods differed, and the Sa values of the Zr group from confocal microscopy were greater by 0.163 μm than those obtained by white light interferometry. Least difference was seen in the TiN group where the difference was 0.058 μm. Roughness parameters of different abutment materials varied significantly. Precision of measurement differed according to the characteristics of the material used. White light interferometry could be recommended for measurement of TiN and AO. Confocal microscopy gave more precise measurements for Ma and CCM groups. The optical characteristics of the surface should be considered before choosing the examination method.

Scatter sensitive microscopic techniques to identify contrasting mucosal structures in ultrahigh-resolution optical coherence tomograms of mouse colon

NASA Astrophysics Data System (ADS)

Tumlinson, Alexandre R.; Hariri, Lida P.; Drexler, Wolfgang; Barton, Jennifer K.

2008-02-01

Optical coherence tomography, optical coherence microscopy, reflectance confocal microscopy, and darkfield microscopy all derive contrast from the intensity of endogenous tissue scatter. We have imaged excised mouse colon tissue with these complimentary technologies to make conclusions about structural origins of scatter in the mouse colonic mucosa observed with endoscopic OCT. We find hyperintense scattering both from the cytoplasm of epithelial cells and from the boundary between epithelia and the lamina propria. We find almost no scatter from the portion of epithelial cells containing the nucleus. These observations substantiate explanations for the appearance of colonic crypts and the luminal surface.

Probing effects of pressure release on virus capture during virus filtration using confocal microscopy.

PubMed

Dishari, Shudipto K; Venkiteshwaran, Adith; Zydney, Andrew L

2015-10-01

Virus filtration is used to ensure drug safety in the production of biotherapeutics. Several recent studies have shown a dramatic decrease in virus retention as a result of a process disruption, e.g., a transient pressure release. In this work, a novel two-label fluorescence technique was developed to probe virus capture within virus filtration membranes using confocal microscopy. Experiments were performed with Ultipor® DV20, Viresolve® Pro, and Viresolve® NFP membranes using bacteriophage φx174 as a model virus. The filters were challenged with two batches of fluorescently labeled phage: one labeled with red dye (Cy5) and one with green dye (SYBR Gold) to visualize captured phage from before and after the pressure release. The capture patterns seen in the confocal images were a strong function of the underlying membrane morphology and pore structure. The DV20 and Viresolve® NFP showed migration of previously captured phage further into the filter, consistent with the observed loss of virus retention after the pressure release. In contrast, there was no migration of captured virus in the Viresolve® Pro membranes, and these filters were also the only ones to show stable virus retention after a pressure release. The direct visualization of virus capture using the two-label fluorescence technique provides unique insights into the factors controlling the retention characteristics of virus filters with different pore structure. © 2015 Wiley Periodicals, Inc.

Cell Invasion in Collagen Scaffold Architectures Characterized by Percolation Theory.

PubMed

Ashworth, Jennifer C; Mehr, Marco; Buxton, Paul G; Best, Serena M; Cameron, Ruth E

2015-06-24

The relationship between biological scaffold interconnectivity and cell migration is an important but poorly understood factor in tissue regeneration. Here a scale-independent technique for characterization of collagen scaffold interconnectivity is presented, using a combination of X-ray microcomputed tomography and percolation theory. Confocal microscopy of connective tissue cells reveals this technique as highly relevant for determining the extent of cell invasion. © 2015 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An optimized protocol for handling and processing fragile acini cultured with the hanging drop technique.

PubMed

Snyman, Celia; Elliott, Edith

2011-12-15

The hanging drop three-dimensional culture technique allows cultivation of functional three-dimensional mammary constructs without exogenous extracellular matrix. The fragile acini are, however, difficult to preserve during processing steps for advanced microscopic investigation. We describe adaptations to the protocol for handling of hanging drop cultures to include investigation using confocal, scanning, and electron microscopy, with minimal loss of cell culture components. Copyright © 2011 Elsevier Inc. All rights reserved.

New Diagnostic Aides for Melanoma

PubMed Central

Ferris, Laura K.; Harris, Ryan J.

2012-01-01

Synopsis Detection of melanoma at an early stage is crucial to improving survival rates in melanoma. Accurate diagnosis by current techniques including dermatoscopy remains difficult, and new tools are needed to improve our diagnostic abilities. This article discusses recent advances in diagnostic techniques including confocal scanning laser microscopy, MelaFind, Siascopy, noninvasive genomic detection, as well as other future possibilities to aid in diagnosing melanoma. Advantages and barriers to implementation of the various technologies are discussed as well. PMID:22800557

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

EPA Science Inventory

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

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

Preparation of HIV monoclonal antibody-conjugated pulchellin in order to study its intracellular trafficking pathway in HIV-infected cells by confocal microscopy

NASA Astrophysics Data System (ADS)

Sadraeian, M.; Tsutae, F. M.; Moreira, H. H. T.; Araujo, A. P. U.; Guimarães, F. E. G.; Pincus, S. H.

2015-06-01

Pulchellin is a type 2 of ribosome-inactivating proteins isolated from some seeds significantly growing in Brazil. It is a potent agent to inhibit the protein synthesis in cancer cells and also HIV-infected cells. Pulchellin can be conjugated to HIV monoclonal antibodies to specifically target the HIV-infected cells. To analyze the protein synthesis inhibition by Pulchellin, the intracellular localization of the immunoconjugate should be compared to Pulchellin. In this case, the intracellular trafficking of this protein in cells can be determined by confocal microscopy. In our study, we utilized Pulchellin to construct HIV monoclonal antibody-conjugated Pulchellin A chain in order to target HIV-infected lymphocyte cells. Afterward the conjugation was labeled with the superior Alexa Fluor 488 dye. As a subsequent step, we are interested in studying the intracellular trafficking pathway of this novel conjugation in HIV-infected cells by confocal microscopy. Moreover, possible quantitative methods for fluorescent labeling of the immunoconjugate during confocal microscopy will be investigated.

A novel method for enhancing the lateral resolution and image SNR in confocal microscopy

NASA Astrophysics Data System (ADS)

Chen, Youhua; Zhu, Dazhao; Fang, Yue; Kuang, Cuifang; Liu, Xu

2017-12-01

There is always a tradeoff between the resolution and the signal-to-noise ratio (SNR) in confocal microscopy. In particular, the pinhole size is very important for maintaining a balance between them. In this paper, we propose a method for improving the lateral resolution and image SNR in confocal microscopy without making any changes to the hardware. By using the fluorescence emission difference (FED) approach, we divide the images acquired by different pinhole sizes into one image acquired by the central pinhole and several images acquired by ring-shaped pinholes. Then, they are added together with the deconvolution method. Simulation and experimental results for fluorescent particles and cells show that our method can achieve a far better resolution than a large pinhole and a higher SNR than a small pinhole. Moreover, our method can improve the performance of classic confocal laser scanning microscopy (CLSM) to a certain extent, especially CLSM with a continuously variable pinhole.

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection

PubMed Central

Brown, Koshonna; Thurn, Ted; Xin, Lun; Liu, William; Bazak, Remon; Chen, Si; Lai, Barry; Vogt, Stefan; Jacobsen, Chris; Paunesku, Tatjana; Woloschak, Gayle E.

2018-01-01

Titanium dioxide (TiO2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here. PMID:29541425

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection.

PubMed

Brown, Koshonna; Thurn, Ted; Xin, Lun; Liu, William; Bazak, Remon; Chen, Si; Lai, Barry; Vogt, Stefan; Jacobsen, Chris; Paunesku, Tatjana; Woloschak, Gayle E

2018-01-01

Titanium dioxide (TiO 2 ) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.

Confocal microscopy to guide laser ablation of basal cell carinoma: a preliminary feasibility study

NASA Astrophysics Data System (ADS)

Larson, Bjorg A.; Sierra, Heidy; Chen, Jason; Rajadhyaksha, Milind

2013-03-01

Laser ablation may be a promising method for removal of skin lesions, with the potential for better cosmetic outcomes and reduced scarring and infection. An obstacle to implementing laser ablation is that the treatment leaves no tissue for histopathological analysis. Pre-operative and intra-operative mapping of BCCs using confocal microscopy may guide the ablation of the tumor until all tumor is removed. We demonstrate preliminary feasibility of confocal microscopy to guide laser ablation of BCCs in freshly excised tissue from Mohs surgery. A 2940 nm Er:YAG laser provides efficient ablation of tumor with reduced thermal damage to the surrounding tissue.

Single-Molecule Three-Color FRET with Both Negligible Spectral Overlap and Long Observation Time

PubMed Central

Hohng, Sungchul

2010-01-01

Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET) experiments. Existing single-molecule three-color FRET techniques, however, suffer from severe photobleaching of Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a single-molecule three-color FRET technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time and improved data quality. Because the absorption spectra of three fluorophores are well separated, real-time monitoring of three FRET efficiencies was possible by incorporating the alternating laser excitation (ALEX) technique both in confocal microscopy and in total-internal-reflection fluorescence (TIRF) microscopy. PMID:20808851

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.

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

NASA Astrophysics Data System (ADS)

Upputuri, Paul Kumar; Pramanik, Manojit

2017-12-01

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

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

EPA Science Inventory

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

Confocal laser scanning microscopy of apoptosis in organogenesis-stage mouse embryos

EPA Science Inventory

Confocal laser scanning microscopy combined with a vital stain has been used to study apoptosis in organogenesis-stage mouse embryos. In order to achieve optical sectioning through embryos, it was necessary to use low power objectives and to prepare the sample appropriately. Mous...

Applicability of confocal laser scanning microscopy for evaluation and monitoring of cutaneous wound healing

NASA Astrophysics Data System (ADS)

Lange-Asschenfeldt, Susanne; Bob, Adrienne; Terhorst, Dorothea; Ulrich, Martina; Fluhr, Joachim; Mendez, Gil; Roewert-Huber, Hans-Joachim; Stockfleth, Eggert; Lange-Asschenfeldt, Bernhard

2012-07-01

There is a high demand for noninvasive imaging techniques for wound assessment. In vivo reflectance confocal laser scanning microscopy (CLSM) represents an innovative optical technique for noninvasive evaluation of normal and diseased skin in vivo at near cellular resolution. This study was designed to test the feasibility of CLSM for noninvasive analysis of cutaneous wound healing in 15 patients (7 male/8 female), including acute and chronic, superficial and deep dermal skin wounds. A commercially available CLSM system was used for the assessment of wound bed and wound margins in order to obtain descriptive cellular and morphological parameters of cutaneous wound repair noninvasively and over time. CLSM was able to visualize features of cutaneous wound repair in epidermal and superficial dermal wounds, including aspects of inflammation, neovascularisation, and tissue remodelling in vivo. Limitations include the lack of mechanic fixation of the optical system on moist surfaces restricting the analysis of chronic skin wounds to the wound margins, as well as a limited optical resolution in areas of significant slough formation. By describing CLSM features of cutaneous inflammation, vascularisation, and epithelialisation, the findings of this study support the role of CLSM in modern wound research and management.

Two-dimensional confocal laser scanning microscopy image correlation for nanoparticle flow velocimetry

NASA Astrophysics Data System (ADS)

Jun, Brian; Giarra, Matthew; Golz, Brian; Main, Russell; Vlachos, Pavlos

2016-11-01

We present a methodology to mitigate the major sources of error associated with two-dimensional confocal laser scanning microscopy (CLSM) images of nanoparticles flowing through a microfluidic channel. The correlation-based velocity measurements from CLSM images are subject to random error due to the Brownian motion of nanometer-sized tracer particles, and a bias error due to the formation of images by raster scanning. Here, we develop a novel ensemble phase correlation with dynamic optimal filter that maximizes the correlation strength, which diminishes the random error. In addition, we introduce an analytical model of CLSM measurement bias error correction due to two-dimensional image scanning of tracer particles. We tested our technique using both synthetic and experimental images of nanoparticles flowing through a microfluidic channel. We observed that our technique reduced the error by up to a factor of ten compared to ensemble standard cross correlation (SCC) for the images tested in the present work. Subsequently, we will assess our framework further, by interrogating nanoscale flow in the cell culture environment (transport within the lacunar-canalicular system) to demonstrate our ability to accurately resolve flow measurements in a biological system.

Integral refractive index imaging of flowing cell nuclei using quantitative phase microscopy combined with fluorescence microscopy.

PubMed

Dardikman, Gili; Nygate, Yoav N; Barnea, Itay; Turko, Nir A; Singh, Gyanendra; Javidi, Barham; Shaked, Natan T

2018-03-01

We suggest a new multimodal imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells in suspension. For this aim, we combined quantitative phase microscopy with simultaneous 2-D fluorescence microscopy. We used 2-D fluorescence microscopy to localize the nucleus inside the quantitative phase map of the cell, as well as for measuring the nucleus radii. As verified offline by both 3-D confocal fluorescence microscopy and 2-D fluorescence microscopy while rotating the cells during flow, the nucleus of cells in suspension that are not during division can be assumed to be an ellipsoid. The entire shape of a cell in suspension can be assumed to be a sphere. Then, the cell and nucleus 3-D shapes can be evaluated based on their in-plain radii available from the 2-D phase and fluorescent measurements, respectively. Finally, the nucleus integral refractive index profile is calculated. We demonstrate the new technique on cancer cells, obtaining nucleus refractive index values that are lower than those of the cytoplasm, coinciding with recent findings. We believe that the proposed technique has the potential to be used for flow cytometry, where full 3-D refractive index tomography is too slow to be implemented during flow.

An instrumental approach to combining confocal microspectroscopy and 3D scanning probe nanotomography.

PubMed

Mochalov, Konstantin E; Chistyakov, Anton A; Solovyeva, Daria O; Mezin, Alexey V; Oleinikov, Vladimir A; Vaskan, Ivan S; Molinari, Michael; Agapov, Igor I; Nabiev, Igor; Efimov, Anton E

2017-11-01

In the past decade correlative microscopy, which combines the potentials of different types of high-resolution microscopies with a variety of optical microspectroscopy techniques, has been attracting increasing attention in material science and biological research. One of outstanding solutions in this area is the combination of scanning probe microscopy (SPM), which provides data on not only the topography, but also the spatial distribution of a wide range of physical properties (elasticity, conductivity, etc.), with ultramicrotomy, allowing 3D multiparametric examination of materials. The combination of SPM and ultramicrotomy (scanning probe nanotomography) is very appropriate for characterization of soft multicompound nanostructurized materials, such as polymer matrices and microstructures doped with different types of nanoparticles (magnetic nanoparticles, quantum dots, nanotubes, etc.), and biological materials. A serious problem of this technique is a lack of chemical and optical characterization tools, which may be solved by using optical microspectroscopy. Here, we report the development of an instrumental approach to combining confocal microspectroscopy and 3D scanning probe nanotomography in a single apparatus. This approach retains all the advantages of SPM and upright optical microspectroscopy and allows 3D multiparametric characterization using both techniques. As the first test of the system developed, we have performed correlative characterization of the morphology and the magnetic and fluorescent properties of fluorescent magnetic microspheres doped with a fluorescent dye and magnetic nanoparticles. The results of this study can be used to obtain 3D volume images of a specimen for most high-resolution near-field scanning probe microscopies: SNOM, TERS, AFM-IR, etc. This approach will result in development of unique techniques combining the advantages of SPM (nanoscale morphology and a wide range of physical parameters) and high-resolution optical microspectroscopy (nanoscale chemical mapping and optical properties) and allowing simultaneous 3D measurements. Copyright © 2017 Elsevier B.V. All rights reserved.

Analyzing Structure and Function of Vascularization in Engineered Bone Tissue by Video-Rate Intravital Microscopy and 3D Image Processing.

PubMed

Pang, Yonggang; Tsigkou, Olga; Spencer, Joel A; Lin, Charles P; Neville, Craig; Grottkau, Brian

2015-10-01

Vascularization is a key challenge in tissue engineering. Three-dimensional structure and microcirculation are two fundamental parameters for evaluating vascularization. Microscopic techniques with cellular level resolution, fast continuous observation, and robust 3D postimage processing are essential for evaluation, but have not been applied previously because of technical difficulties. In this study, we report novel video-rate confocal microscopy and 3D postimage processing techniques to accomplish this goal. In an immune-deficient mouse model, vascularized bone tissue was successfully engineered using human bone marrow mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in a poly (D,L-lactide-co-glycolide) (PLGA) scaffold. Video-rate (30 FPS) intravital confocal microscopy was applied in vitro and in vivo to visualize the vascular structure in the engineered bone and the microcirculation of the blood cells. Postimage processing was applied to perform 3D image reconstruction, by analyzing microvascular networks and calculating blood cell viscosity. The 3D volume reconstructed images show that the hMSCs served as pericytes stabilizing the microvascular network formed by HUVECs. Using orthogonal imaging reconstruction and transparency adjustment, both the vessel structure and blood cells within the vessel lumen were visualized. Network length, network intersections, and intersection densities were successfully computed using our custom-developed software. Viscosity analysis of the blood cells provided functional evaluation of the microcirculation. These results show that by 8 weeks, the blood vessels in peripheral areas function quite similarly to the host vessels. However, the viscosity drops about fourfold where it is only 0.8 mm away from the host. In summary, we developed novel techniques combining intravital microscopy and 3D image processing to analyze the vascularization in engineered bone. These techniques have broad applicability for evaluating vascularization in other engineered tissues as well.

Association between dermoscopic and reflectance confocal microscopy features of cutaneous melanoma with BRAF mutational status.

PubMed

Bombonato, C; Ribero, S; Pozzobon, F C; Puig-Butille, J A; Badenas, C; Carrera, C; Malvehy, J; Moscarella, E; Lallas, A; Piana, S; Puig, S; Argenziano, G; Longo, C

2017-04-01

Melanomas harbouring common genetic mutations might share certain morphological features detectable with dermoscopy and reflectance confocal microscopy. BRAF mutational status is crucial for the management of metastatic melanoma. To correlate the dermoscopic characteristics of primary cutaneous melanomas with BRAF mutational status. Furthermore, a subset of tumours has also been analysed for the presence of possible confocal features that might be linked with BRAF status. Retrospectively acquired dermoscopic and confocal images of patients with melanoma in tertiary referral academic centres: Skin Cancer Unit in Reggio Emilia and at the Melanoma Unit in Barcelona. Kruskal-Wallis test, logistic regressions, univariate and multivariate analyses have been performed to find dermoscopic and confocal features significantly correlated with BRAF mutational status. Dermoscopically, the presence of irregular peripheral streaks and ulceration were positive predictors of BRAF-mutated melanomas with a statistically significance value, while dotted vessels were more represented in wild-type melanomas. None of the evaluated reflectance confocal microscopy features were correlated with genetic profiling. Ulceration and irregular peripheral streaks represent dermoscopic feature indicative for BRAF-mutated melanoma, while dotted vessels are suggestive for wild-type melanoma. © 2016 European Academy of Dermatology and Venereology.

Sensitivity and specificity for detecting basal cell carcinomas in Mohs excisions with confocal fluorescence mosaicing microscopy

NASA Astrophysics Data System (ADS)

Gareau, Daniel S.; Karen, Julie K.; Dusza, Stephen W.; Tudisco, Marie; Nehal, Kishwer S.; Rajadhyaksha, Milind

2009-05-01

Recent studies have demonstrated the ability of confocal fluorescence mosaicing microscopy to rapidly detect basal cell carcinomas (BCCs) directly in thick and fresh Mohs surgical excisions. Mosaics of confocal images display large areas of tissue with high resolution and magnification equivalent to 2×, which is the standard magnification when examining pathology. Comparison of mosaics to Mohs frozen histopathology was shown to be excellent for all types of BCCs. However, comparisons in the previous studies were visual and qualitative. In this work, we report the results of a semiquantitative preclinical study in which 45 confocal mosaics are blindly evaluated for the presence (or absence) of BCC tumor. The evaluations are made by two clinicians: a senior Mohs surgeon with prior expertise in interpreting confocal images, and a novice Mohs fellow with limited experience. The blinded evaluation is compared to the gold standard of frozen histopathology. BCCs are detected with an overall sensitivity of 96.6%, specificity of 89.2%, positive predictive value of 93.0%, and negative predictive value of 94.7%. The results demonstrate the potential clinical utility of confocal mosaicing microscopy toward rapid surgical pathology at the bedside to expedite and guide surgery.

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

NASA Astrophysics Data System (ADS)

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

2016-03-01

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

Development of confocal immunofluorescence FRET microscopy to Investigate eNOS and GSNOR localization and interaction in pulmonary endothelial cells

NASA Astrophysics Data System (ADS)

Rehman, Shagufta; Brown-Steinke, Kathleen; Palmer, Lisa; Periasamy, Ammasi

2015-03-01

Confocal FRET microscopy is a widely used technique for studying protein-protein interactions in live or fixed cells. Endothelial nitric oxide synthase (eNOS) and S-nitrosoglutathione reductase (GSNOR) are enzymes involved in regulating the bioavailability of S-nitrosothiols (SNOs) in the pulmonary endothelium and have roles in the development of pulmonary arterial hypertension. Labeling of endogenous proteins to better understand a disease process can be challenging. We have used immunofluorescence to detect endogenous eNOS and GSNOR in primary pulmonary endothelial cells to co-localize these proteins as well as to study their interaction by FRET. The challenge has been in selecting the right immunofluorescence labeling condition, right antibody, the right blocking reagent, the right FRET pair and eliminating cross-reactivity of secondary antibodies. We have used Alexa488 and Alexa568 as a FRET pair. After a series of optimizations, the data from Confocal Laser Scanning Microscopy (CLSM) demonstrate co-localization of eNOS and GSNOR in the perinuclear region of the pulmonary endothelial cell primarily within the cis-Golgi with lower levels of co-localization seen within the trans-Golgi. FRET studies demonstrate, for the first time, interaction between eNOS and GSNOR in both murine and bovine pulmonary endothelial cells. Further characterization of eNOSGSNOR interaction and the subcellular location of this interaction will provide mechanistic insight into the importance of S-nitrosothiol signaling in pulmonary biology, physiology and pathology.

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

Correlated Light and Electron Microscopy/Electron Tomography of Mitochondria In Situ

PubMed Central

Perkins, Guy A.; Sun, Mei G.; Frey, Terrence G.

2009-01-01

Three-dimensional light microscopy and three-dimensional electron microscopy (electron tomography) separately provide very powerful tools to study cellular structure and physiology, including the structure and physiology of mitochondria. Fluorescence microscopy allows one to study processes in live cells with specific labels and stains that follow the movement of labeled proteins and changes within cellular compartments but does not have sufficient resolution to define the ultrastructure of intracellular organelles such as mitochondria. Electron microscopy and electron tomography provide the highest resolution currently available to study mitochondrial ultrastructure but cannot follow processes in living cells. We describe the combination of these two techniques in which fluorescence confocal microscopy is used to study structural and physiologic changes in mitochondria within apoptotic HeLa cells to define the apoptotic timeframe. Cells can then be selected at various stages of the apoptotic timeframe for examination at higher resolution by electron microscopy and electron tomography. This is a form of “virtual” 4-dimensional electron microscopy that has revealed interesting structural changes in the mitochondria of HeLa cells during apoptosis. The same techniques can be applied, with modification, to study other dynamic processes within cells in other experimental contexts. PMID:19348881

Microbiologically influenced corrosion: looking to the future.

PubMed

Videla, Héctor A; Herrera, Liz K

2005-09-01

This review discusses the state-of-the-art of research into biocorrosion and the biofouling of metals and alloys of industrial usage. The key concepts needed to understand the main effects of microorganisms on metal decay, and current trends in monitoring and control strategies to mitigate the deleterious effects of biocorrosion and biofouling are also described. Several relevant cases of biocorrosion studied by our research group are provided as examples: (i) biocorrosion of aluminum and its alloys by fungal contaminants of jet fuels; (ii) sulfate-reducing bacteria (SRB)-induced corrosion of steel; (iii) biocorrosion and biofouling interactions in the marine environment; (iv) monitoring strategies for assessing biocorrosion in industrial water systems; (v) microbial inhibition of corrosion; (vi) use and limitations of electrochemical techniques for evaluating biocorrosion effects. Future prospects in the field are described with respect to the potential of innovative techniques in microscopy (environmental scanning electron microscopy, confocal scanning laser microscopy, atomic force microscopy), new spectroscopic techniques for the study of corrosion products and biofilms (energy dispersion X-ray analysis, X-ray photoelectron spectroscopy, electron microprobe analysis) and electrochemistry (electrochemical impedance spectroscopy, electrochemical noise analysis).

Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

PubMed Central

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

2012-01-01

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass. PMID:23083712

Comparison between reflectance confocal microscopy and two-photon microscopy in early detection of cutaneous radiation injury in a mouse model in-vivo.

PubMed

Jang, Won Hyuk; Kwon, Soonjae; Shim, Sehwan; Jang, Won-Suk; Myung, Jae Kyung; Yang, Sejung; Park, Sunhoo; Kim, Ki Hean

2018-05-12

Cutaneous radiation injury (CRI) is a skin injury caused by high dose exposure of ionizing radiation (IR). For proper treatment, early detection of CRI before clinical symptoms is important. Optical microscopic techniques such as reflectance confocal microscopy (RCM) and two-photon microscopy (TPM) have been tested as the early diagnosis method by detecting cellular changes. In this study, RCM and TPM were compared in the detection of cellular changes caused by CRI in an in-vivo mouse model. CRI was induced on the mouse hindlimb skin with various IR doses and the injured skin regions were imaged longitudinally by both modalities until the onset of clinical symptoms. Both RCM and TPM detected the changes of epidermal cells and sebaceous glands before clinical symptoms in different optical contrasts. RCM detected changes of cell morphology and scattering property based on light reflection. TPM detected detail changes of cellular structures based on autofluorescence of cells. Since both RCM and TPM were sensitive to the early-stage CRI by using different contrasts, the optimal method for clinical CRI diagnosis could be either individual methods or their combination. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Confocal laser scanning microscopy coupled to a spectrofluorometric detector as a rapid tool for determining the in vivo effect of metals on phototrophic bacteria.

PubMed

Burnat, Mireia; Diestra, Elia; Esteve, Isabel; Solé, Antonio

2010-01-01

In this paper, we determine for the first time the in vivo effect of heavy metals in a phototrophic bacterium. We used Confocal Laser Scanning Microscopy coupled to a spectrofluorometric detector as a rapid technique to measure pigment response to heavy-metal exposure. To this end, we selected lead and copper (toxic and essential metals) and Microcoleus sp. as the phototrophic bacterium because it would be feasible to see this cyanobacterium as a good biomarker, since it covers large extensions of coastal sediments. The results obtained demonstrate that, while cells are still viable, pigment peak decreases whereas metal concentration increases (from 0.1 to 1 mM Pb). Pigments are totally degraded when cultures were polluted with lead and copper at the maximum doses used (25 mM Pb(NO(3))(2) and 10 mM CuSO(4)). The aim of this study was also to identify the place of metal accumulation in Microcoleus cells. Element analysis of this cyanobacterium in the above mentioned conditions determined by Energy Dispersive X-ray microanalysis (EDX) coupled to Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), shows that Pb (but not Cu) accumulates externally and internally in cells.

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

Multi-scale Observation of Biological Interactions of Nanocarriers: from Nano to Macro

PubMed Central

Jin, Su-Eon; Bae, Jin Woo; Hong, Seungpyo

2010-01-01

Microscopic observations have played a key role in recent advancements in nanotechnology-based biomedical sciences. In particular, multi-scale observation is necessary to fully understand the nano-bio interfaces where a large amount of unprecedented phenomena have been reported. This review describes how to address the physicochemical and biological interactions of nanocarriers within the biological environments using microscopic tools. The imaging techniques are categorized based on the size scale of detection. For observation of the nano-scale biological interactions of nanocarriers, we discuss atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). For the micro to macro-scale (in vitro and in vivo) observation, we focus on confocal laser scanning microscopy (CLSM) as well as in vivo imaging systems such as magnetic resonance imaging (MRI), superconducting quantum interference devices (SQUIDs), and IVIS®. Additionally, recently developed combined techniques such as AFM-CLSM, correlative Light and Electron Microscopy (CLEM), and SEM-spectroscopy are also discussed. In this review, we describe how each technique helps elucidate certain physicochemical and biological activities of nanocarriers such as dendrimers, polymers, liposomes, and polymeric/inorganic nanoparticles, thus providing a toolbox for bioengineers, pharmaceutical scientists, biologists, and research clinicians. PMID:20232368

In vivo assessment of cytological changes by means of reflectance confocal microscopy - demonstration of the effect of topical vitamin E on skin irritation caused by sodium lauryl sulfate.

PubMed

Casari, Alice; Farnetani, Francesca; De Pace, Barbara; Losi, Amanda; Pittet, Jean-Christophe; Pellacani, Giovanni; Longo, Caterina

2017-03-01

Irritant contact dermatitis is caused by skin barrier damage. Vitamin E is an antioxidant that is commonly used in cosmetics to prevent photo-damage. To show the usefulness of reflectance confocal microscopy in the assessment of irritant skin damage caused by sodium lauryl sulfate (SLS) and of the protective action of vitamin E applied prior to skin irritation. Ten healthy volunteers were enrolled. Irritation was induced by the application of a patch test containing SLS 5% aq. for 24 h. Three sites were compared: one site on which a product with vitamin E was applied before SLS treatment, one site on which the same product was applied after SLS treatment, and one control site (SLS only). Each site was evaluated with reflectance confocal microscopy, providing in vivo tissue images at nearly histological resolution. We also performed a computerized analysis of the VivaStack® images. Reflectance confocal microscopy is able to identify signs of skin irritation and the preventive effect of vitamin E application. Reflectance confocal microscopy is useful in the objective assessment of irritative skin damage. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

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...

New techniques for fluorescence background rejection in microscopy and endoscopy

NASA Astrophysics Data System (ADS)

Ventalon, Cathie

2009-03-01

Confocal microscopy is a popular technique in the bioimaging community, mainly because it provides optical sectioning. However, its standard implementation requires 3-dimensional scanning of focused illumination throughout the sample. Efficient non-scanning alternatives have been implemented, among which the simple and well-established incoherent structured illumination microscopy (SIM) [1]. We recently proposed a similar technique, called Dynamic Speckle Illumination (DSI) microscopy, wherein the incoherent grid illumination pattern is replaced with a coherent speckle illumination pattern from a laser, taking advantage of the fact that speckle contrast is highly maintained in a scattering media, making the technique well adapted to tissue imaging [2]. DSI microscopy relies on the illumination of a sample with a sequence of dynamic speckle patterns and an image processing algorithm based only on an a priori knowledge of speckle statistics. The choice of this post-processing algorithm is crucial to obtain a good sectioning strength: in particular, we developed a novel post-processing algorithm based one wavelet pre-filtering of the raw images and obtained near-confocal fluorescence sectioning in a mouse brain labeled with GFP, with a good image quality maintained throughout a depth of ˜100 μm [3]. In the purpose of imaging fluorescent tissue at higher depth, we recently applied structured illumination to endoscopy. We used a similar set-up wherein the illumination pattern (a one-dimensional grid) is transported to the sample with an imaging fiber bundle with miniaturized objective and the fluorescence image is collected through the same bundle. Using a post-processing algorithm similar to the one previously described [3], we obtained high-quality images of a fluorescein-labeled rat colonic mucosa [4], establishing the potential of our endomicroscope for bioimaging applications. [4pt] Ref: [0pt] [1] M. A. A. Neil et al, Opt. Lett. 22, 1905 (1997) [0pt] [2] C. Ventalon et al, Opt. Lett. 30, 3350 (2005) [0pt] [3] C. Ventalon et al, Opt. Lett. 32, 1417 (2007) [0pt] [4] N. Bozinovic et al, Opt. Express 16, 8016 (2008)

Annexin-V/quantum dot probes for multimodal apoptosis monitoring in living cells: improving bioanalysis using electrochemistry

NASA Astrophysics Data System (ADS)

Montón, Helena; Parolo, Claudio; Aranda-Ramos, Antonio; Merkoçi, Arben; Nogués, Carme

2015-02-01

There is a great demand to develop novel techniques that allow useful and complete monitoring of apoptosis, which is a key factor of several diseases and a target for drug development. Here, we present the use of a novel dual electrochemical/optical label for the detection and study of apoptosis. We combined the specificity of Annexin-V for phosphatidylserine, a phospholipid expressed in the outer membrane of apoptotic cells, with the optical and electrochemical properties of quantum dots to create a more efficient label. Using this conjugate we addressed three important issues: (i) we made the labeling of apoptotic cells faster (30 min) and easier; (ii) we fully characterized the samples by common cell biological techniques (confocal laser scanning microscopy, scanning electron microscopy and flow cytometry); and (iii) we developed a fast, cheap and quantitative electrochemical detection method for apoptotic cells with results in full agreement with those obtained by flow cytometry.There is a great demand to develop novel techniques that allow useful and complete monitoring of apoptosis, which is a key factor of several diseases and a target for drug development. Here, we present the use of a novel dual electrochemical/optical label for the detection and study of apoptosis. We combined the specificity of Annexin-V for phosphatidylserine, a phospholipid expressed in the outer membrane of apoptotic cells, with the optical and electrochemical properties of quantum dots to create a more efficient label. Using this conjugate we addressed three important issues: (i) we made the labeling of apoptotic cells faster (30 min) and easier; (ii) we fully characterized the samples by common cell biological techniques (confocal laser scanning microscopy, scanning electron microscopy and flow cytometry); and (iii) we developed a fast, cheap and quantitative electrochemical detection method for apoptotic cells with results in full agreement with those obtained by flow cytometry. Electronic supplementary information (ESI) available: Optical microscopy images of apoptotic induced cell cultures at different times and negative control of flow cytometry. See DOI: 10.1039/c4nr07191c

Time-resolved wide-field optically sectioned fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dupuis, Guillaume; Benabdallah, Nadia; Chopinaud, Aurélien; Mayet, Céline; Lévêque-Fort, Sandrine

2013-02-01

We present the implementation of a fast wide-field optical sectioning technique called HiLo microscopy on a fluorescence lifetime imaging microscope. HiLo microscopy is based on the fusion of two images, one with structured illumination and another with uniform illumination. Optically sectioned images are then digitally generated thanks to a fusion algorithm. HiLo images are comparable in quality with confocal images but they can be acquired faster over larger fields of view. We obtain 4D imaging by combining HiLo optical sectioning, time-gated detection, and z-displacement. We characterize the performances of this set-up in terms of 3D spatial resolution and time-resolved capabilities in both fixed- and live-cell imaging modes.

Effect of Autologous Serum Eye Drops in Patients with Sjögren Syndrome-related Dry Eye: Clinical and In Vivo Confocal Microscopy Evaluation of the Ocular Surface.

PubMed

Semeraro, Francesco; Forbice, Eliana; Nascimbeni, Giuseppe; Taglietti, Marco; Romano, Vito; Guerra, Germano; Costagliola, Ciro

To evaluate in vivo changes after therapy using autologous serum (AS) eye drops in Sjögren's syndrome (SS)-related dry eyes by confocal microscopy. In this study, 24 patients with SS-related dry eyes [12 in AS eye drop therapy and 12 in artificial tear (AT) therapy] and 24 healthy volunteers were recruited. Ocular Surface Disease Index (OSDI), central corneal thickness, tear film, break-up time, corneal and conjunctival staining, Schirmer's test and corneal confocal microscopy were investigated. Tear production, tear stability, corneal staining, inflammation, and central corneal thickness, Langherans cells, activated keratocytes, intermediate epithelial cell density, nerve tortuosity, number of sub-basal nerve branches, and number of bead-like formations differed between patients and controls (p<0.0001). The AT and AS groups differed in the OSDI, number of branches, and number of beadings (p<0.0001). AS eye drops improve symptoms and confocal microscopy findings in SS-related dry eyes. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer

NASA Astrophysics Data System (ADS)

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

2013-04-01

Optical imaging techniques using a variety of contrast mechanisms are under evaluation for early detection of epithelial precancer; however, tradeoffs in field of view (FOV) and resolution may limit their application. Therefore, we present a multiscale multimodal optical imaging system combining macroscopic biochemical imaging of fluorescence lifetime imaging (FLIM) with subcellular morphologic imaging of reflectance confocal microscopy (RCM). The FLIM module images a 16×16 mm2 tissue area with 62.5 μm lateral and 320 ps temporal resolution to guide cellular imaging of suspicious regions. Subsequently, coregistered RCM images are acquired at 7 Hz with 400 μm diameter FOV, <1 μm lateral and 3.5 μm axial resolution. FLIM-RCM imaging was performed on a tissue phantom, normal porcine buccal mucosa, and a hamster cheek pouch model of oral carcinogenesis. While FLIM is sensitive to biochemical and macroscopic architectural changes in tissue, RCM provides images of cell nuclear morphology, all key indicators of precancer progression.

Characterization of Protein-Excipient Microheterogeneity in Biopharmaceutical Solid-State Formulations by Confocal Fluorescence Microscopy.

PubMed

Koshari, Stijn H S; Ross, Jean L; Nayak, Purnendu K; Zarraga, Isidro E; Rajagopal, Karthikan; Wagner, Norman J; Lenhoff, Abraham M

2017-02-06

Protein-stabilizer microheterogeneity is believed to influence long-term protein stability in solid-state biopharmaceutical formulations and its characterization is therefore essential for the rational design of stable formulations. However, the spatial distribution of the protein and the stabilizer in a solid-state formulation is, in general, difficult to characterize because of the lack of a functional, simple, and reliable characterization technique. We demonstrate the use of confocal fluorescence microscopy with fluorescently labeled monoclonal antibodies (mAbs) and antibody fragments (Fabs) to directly visualize three-dimensional particle morphologies and protein distributions in dried biopharmaceutical formulations, without restrictions on processing conditions or the need for extensive data analysis. While industrially relevant lyophilization procedures of a model IgG1 mAb generally lead to uniform protein-excipient distribution, the method shows that specific spray-drying conditions lead to distinct protein-excipient segregation. Therefore, this method can enable more definitive optimization of formulation conditions than has previously been possible.

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

NASA Astrophysics Data System (ADS)

Boichenko, Stepan

2018-04-01

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

Nanoparticle uptake and their co-localization with cell compartments - a confocal Raman microscopy study at single cell level

NASA Astrophysics Data System (ADS)

Estrela-Lopis, I.; Romero, G.; Rojas, E.; Moya, S. E.; Donath, E.

2011-07-01

Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al2O3 NPs were found in the cytoplasm. CeO2 NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.

Growth and development of the barnacle Amphibalanus amphitrite: time and spatially resolved structure and chemistry of the base plate

PubMed Central

Burden, Daniel K.; Spillmann, Christopher M.; Everett, Richard K.; Barlow, Daniel E.; Orihuela, Beatriz; Deschamps, Jeffrey R.; Fears, Kenan P.; Rittschof, Daniel; Wahl, Kathryn J.

2014-01-01

The radial growth and advancement of the adhesive interface to the substratum of many species of acorn barnacles occurs underwater and beneath an opaque, calcified shell. Here, the time-dependent growth processes involving various autofluorescent materials within the interface of live barnacles are imaged for the first time using 3D time-lapse confocal microscopy. Key features of the interface development in the striped barnacle, Amphibalanus (= Balanus) amphitrite were resolved in situ and include advancement of the barnacle/substratum interface, epicuticle membrane development, protein secretion, and calcification. Microscopic and spectroscopic techniques provide ex situ material identification of regions imaged by confocal microscopy. In situ and ex situ analysis of the interface support the hypothesis that barnacle interface development is a complex process coupling sequential, timed secretory events and morphological changes. This results in a multi-layered interface that concomitantly fulfills the roles of strongly adhering to a substratum while permitting continuous molting and radial growth at the periphery. PMID:25115515

Growth and development of the barnacle Amphibalanus amphitrite: time and spatially resolved structure and chemistry of the base plate.

PubMed

Burden, Daniel K; Spillmann, Christopher M; Everett, Richard K; Barlow, Daniel E; Orihuela, Beatriz; Deschamps, Jeffrey R; Fears, Kenan P; Rittschof, Daniel; Wahl, Kathryn J

2014-01-01

The radial growth and advancement of the adhesive interface to the substratum of many species of acorn barnacles occurs underwater and beneath an opaque, calcified shell. Here, the time-dependent growth processes involving various autofluorescent materials within the interface of live barnacles are imaged for the first time using 3D time-lapse confocal microscopy. Key features of the interface development in the striped barnacle, Amphibalanus (= Balanus) amphitrite were resolved in situ and include advancement of the barnacle/substratum interface, epicuticle membrane development, protein secretion, and calcification. Microscopic and spectroscopic techniques provide ex situ material identification of regions imaged by confocal microscopy. In situ and ex situ analysis of the interface support the hypothesis that barnacle interface development is a complex process coupling sequential, timed secretory events and morphological changes. This results in a multi-layered interface that concomitantly fulfills the roles of strongly adhering to a substratum while permitting continuous molting and radial growth at the periphery.

Fast imaging of live organisms with sculpted light sheets

NASA Astrophysics Data System (ADS)

Chmielewski, Aleksander K.; Kyrsting, Anders; Mahou, Pierre; Wayland, Matthew T.; Muresan, Leila; Evers, Jan Felix; Kaminski, Clemens F.

2015-04-01

Light-sheet microscopy is an increasingly popular technique in the life sciences due to its fast 3D imaging capability of fluorescent samples with low photo toxicity compared to confocal methods. In this work we present a new, fast, flexible and simple to implement method to optimize the illumination light-sheet to the requirement at hand. A telescope composed of two electrically tuneable lenses enables us to define thickness and position of the light-sheet independently but accurately within milliseconds, and therefore optimize image quality of the features of interest interactively. We demonstrated the practical benefit of this technique by 1) assembling large field of views from tiled single exposure each with individually optimized illumination settings; 2) sculpting the light-sheet to trace complex sample shapes within single exposures. This technique proved compatible with confocal line scanning detection, further improving image contrast and resolution. Finally, we determined the effect of light-sheet optimization in the context of scattering tissue, devising procedures for balancing image quality, field of view and acquisition speed.

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.

Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues

PubMed Central

Benninger, Richard K.P.; Piston, David W.

2013-01-01

Two-photon excitation microscopy is an alternative to confocal microscopy that provides advantages for three-dimensional and deep tissue imaging. This unit will describe the basic physical principles behind two-photon excitation and discuss the advantages and limitations of its use in laser-scanning microscopy. The principal advantages of two-photon microscopy are reduced phototoxicity, increased imaging depth, and the ability to initiate highly localized photochemistry in thick samples. Practical considerations for the application of two-photon microscopy will then be discussed, including recent technological advances. This unit will conclude with some recent applications of two-photon microscopy that highlight the key advantages over confocal microscopy and the types of experiments which would benefit most from its application. PMID:23728746

Multimodality optical imaging of embryonic heart microstructure

PubMed Central

Yelin, Ronit; Yelin, Dvir; Oh, Wang-Yuhl; Yun, Seok H.; Boudoux, Caroline; Vakoc, Benjamin J.; Bouma, Brett E.; Tearney, Guillermo J.

2009-01-01

Study of developmental heart defects requires the visualization of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xenopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence microscopy with subcellular (1 to 2 µm) resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed (up to 900 frames per second) that enables real-time 4-D high resolution imaging in vivo. These imaging modalities can provide a comprehensive picture of the morphologic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investigation of the teratogenic effects of ethanol. Microstructural abnormalities associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecular mass. PMID:18163837

Multimodality optical imaging of embryonic heart microstructure.

PubMed

Yelin, Ronit; Yelin, Dvir; Oh, Wang-Yuhl; Yun, Seok H; Boudoux, Caroline; Vakoc, Benjamin J; Bouma, Brett E; Tearney, Guillermo J

2007-01-01

Study of developmental heart defects requires the visualization of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xenopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence microscopy with subcellular (1 to 2 microm) resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed (up to 900 frames per second) that enables real-time 4-D high resolution imaging in vivo. These imaging modalities can provide a comprehensive picture of the morphologic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investigation of the teratogenic effects of ethanol. Microstructural abnormalities associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecular mass.

Multiphoton imaging with high peak power VECSELs

NASA Astrophysics Data System (ADS)

Mirkhanov, Shamil; Quarterman, Adrian H.; Swift, Samuel; Praveen, Bavishna B.; Smyth, Conor J. C.; Wilcox, Keith G.

2016-03-01

Multiphoton imaging (MMPI) has become one of thee key non-invasive light microscopy techniques. This technique allows deep tissue imaging with high resolution and less photo-damage than conventional confocal microscopy. MPI is type of laser-scanning microscopy that employs localized nonlinear excitation, so that fluorescence is excited only with is scanned focal volume. For many years, Ti: sapphire femtosecond lasers have been the leading light sources for MPI applications. However, recent developments in laser sources and new types of fluorophores indicate that longer wavelength excitation could be a good alternative for these applications. Mode-locked VECSEELs have the potential to be low cost, compact light sources for MPI systems, with the additional advantage of broad wavelength coverage through use of different semiconductor material systems. Here, we use a femtosecond fibber laser to investigate the effect average power and repetition rate has on MPI image quality, to allow us to optimize our mode-locked VVECSELs for MPI.

In situ mechanical characterization of the cell nucleus by atomic force microscopy.

PubMed

Liu, Haijiao; Wen, Jun; Xiao, Yun; Liu, Jun; Hopyan, Sevan; Radisic, Milica; Simmons, Craig A; Sun, Yu

2014-04-22

The study of nuclear mechanical properties can provide insights into nuclear dynamics and its role in cellular mechanotransduction. While several methods have been developed to characterize nuclear mechanical properties, direct intracellular probing of the nucleus in situ is challenging. Here, a modified AFM (atomic force microscopy) needle penetration technique is demonstrated to mechanically characterize cell nuclei in situ. Cytoplasmic and nuclear stiffness were determined based on two different segments on the AFM indentation curves and were correlated with simultaneous confocal Z-stack microscopy reconstructions. On the basis of direct intracellular measurement, we show that the isolated nuclei from fibroblast-like cells exhibited significantly lower Young's moduli than intact nuclei in situ. We also show that there is in situ nucleus softening in the highly metastatic bladder cancer cell line T24 when compared to its less metastatic counterpart RT4. This technique has potential to become a reliable quantitative measurement tool for intracellular mechanics studies.

Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy.

PubMed

Nooh, Mohammed M; Bahouth, Suleiman W

2017-01-01

G protein-coupled receptors (GPCRs) are recognized as one of the most fruitful group of therapeutic targets, accounting for more than 40% of all approved pharmaceuticals on the market. Therefore, the search for selective agents that affect GPCR function is of major interest to the pharmaceutical industry. This chapter describes methods for measuring agonist-promoted GPCR trafficking, which involves the internalization of the GPCR and its subsequent recycling back to the plasma membrane or retention and eventual degradation. These pathways will be analyzed by confocal cellular imaging, using the β 1 -adrenergic receptor (β 1 -AR) as a primary model. A major problem encountered in studying GPCR trafficking is the unavailability of antibodies that would recognize the native receptor in cells or tissues. Therefore, wild-type, point mutants, and β 1 -AR chimeras are generated as epitope-tagged proteins, which are stably- or transiently expressed in mammalian cells. GPCR are labeled with a fluorophore-conjugated antibody directed against the N-terminal epitope tag. The trafficking of the fluorophore-tagged GPCR between divergent trafficking pathways that result in retention and eventual degradation or recycling and reinsertion into the plasma membrane can be followed by confocal immunofluorescence microscopy techniques outlined in this review. © 2017 Elsevier Inc. All rights reserved.

IMART software for correction of motion artifacts in images collected in intravital microscopy

PubMed Central

Dunn, Kenneth W; Lorenz, Kevin S; Salama, Paul; Delp, Edward J

2014-01-01

Intravital microscopy is a uniquely powerful tool, providing the ability to characterize cell and organ physiology in the natural context of the intact, living animal. With the recent development of high-resolution microscopy techniques such as confocal and multiphoton microscopy, intravital microscopy can now characterize structures at subcellular resolution and capture events at sub-second temporal resolution. However, realizing the potential for high resolution requires remarkable stability in the tissue. Whereas the rigid structure of the skull facilitates high-resolution imaging of the brain, organs of the viscera are free to move with respiration and heartbeat, requiring additional apparatus for immobilization. In our experience, these methods are variably effective, so that many studies are compromised by residual motion artifacts. Here we demonstrate the use of IMART, a software tool for removing motion artifacts from intravital microscopy images collected in time series or in three dimensions. PMID:26090271

Doppler optical coherence microscopy and tomography applied to inner ear mechanics

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

Page, Scott; Freeman, Dennis M.; Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts

While it is clear that cochlear traveling waves underlie the extraordinary sensitivity, frequency selectivity, and dynamic range of mammalian hearing, the underlying micromechanical mechanisms remain unresolved. Recent advances in low coherence measurement techniques show promise over traditional laser Doppler vibrometry and video microscopy, which are limited by low reflectivities of cochlear structures and restricted optical access. Doppler optical coherence tomography (DOCT) and Doppler optical coherence microscopy (DOCM) both utilize a broadband source to limit constructive interference of scattered light to a small axial depth called a coherence gate. The coherence gate can be swept axially to image and measure sub-nanometermore » motions of cochlear structures throughout the cochlear partition. The coherence gate of DOCT is generally narrower than the confocal gate of the focusing optics, enabling increased axial resolution (typically 15 μm) within optical sections of the cochlear partition. DOCM, frequently implemented in the time domain, centers the coherence gate on the focal plane, achieving enhanced lateral and axial resolution when the confocal gate is narrower than the coherence gate. We compare these two complementary systems and demonstrate their utility in studying cellular and micromechanical mechanisms involved in mammalian hearing.« less

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.

Intracellular in situ labeling of TiO 2 nanoparticles for fluorescence microscopy detection

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

Brown, Koshonna; Thurn, Ted; Xin, Lun

Titanium dioxide (TiO 2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. In this paper, we describe two in situ posttreatmentmore » labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyneconjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Finally and therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.« less

Intracellular in situ labeling of TiO 2 nanoparticles for fluorescence microscopy detection

DOE PAGES

Brown, Koshonna; Thurn, Ted; Xin, Lun; ...

2017-07-19

Titanium dioxide (TiO 2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. In this paper, we describe two in situ posttreatmentmore » labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyneconjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Finally and therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.« less

A multimodal microcharacterisation of trace-element zonation and crystallographic orientation in natural cassiterite by combining cathodoluminescence, EBSD, EPMA and contribution of confocal Raman-in-SEM imaging.

PubMed

Wille, G; Lerouge, C; Schmidt, U

2018-01-16

In cassiterite, tin is associated with metals (titanium, niobium, tantalum, indium, tungsten, iron, manganese, mercury). Knowledge of mineral chemistry and trace-element distribution is essential for: the understanding of ore formation, the exploration phase, the feasibility of ore treatment, and disposal/treatment of tailings after the exploitation phase. However, the availability of analytical methods make these characterisations difficult. We present a multitechnical approach to chemical and structural data that includes scanning electron microscopy (SEM)-based imaging and microanalysis techniques such as: secondary and backscattered electrons, cathodoluminescence (CL), electron probe microanalyser (EPMA), electron backscattered diffraction (EBSD) and confocal Raman-imaging integrated in a SEM (RISE). The presented results show the complementarity of the used analytical techniques. SEM, CL, EBSD, EPMA provide information from the interaction of an electron beam with minerals, leading to atomistic information about their composition, whereas RISE, Raman spectroscopy and imaging completes the studies with information about molecular vibrations, which are sensitive to structural modifications of the minerals. The correlation of Raman bands with the presence/absence of Nb, Ta, Fe (heterovalent substitution) and Ti (homovalent substitution) is established at a submicrometric scale. Combination of the different techniques makes it possible to establish a direct link between chemical and crystallographic data of cassiterite. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Antioxidant-based topical formulations influence on the inflammatory response of Japanese skin: A clinical study using non-invasive techniques.

PubMed

Wagemaker, Tais A L; Maia Campos, Patrícia M B G; Shimizu, Kenji; Kyotani, Daiki; Yoshida, Daisuke

2017-08-01

Cutaneous irritants exposure induces an excess of ROS in the skin and can ensue an inflammatory response. Topical antioxidant-based formulations can help to counteract ROS generation. This study evaluated the influence of antioxidant-based topical formulations on the inflammatory response of skin, using a combination of in vivo real-time non-invasive techniques. Nine test areas were defined on each volar forearm of the 25 Japanese volunteers. Measurements were performed before and after treatment with 15μL of a 5% sodium dodecyl sulfate solution and 15μL of the same based formulation or the vehicle with 1% of the antioxidants. Volunteers without antioxidant treatment showed more pronounced erythematous areas. Transepidermal water loss of areas treated with green tea polyphenol (GTP)-based formulation showed fully recovered skin. Skin barrier damage caused by repeated applications of SDS showed characteristic alterations, detectable by in vivo confocal microscopy such as desquamation, spongiosis and inflammatory infiltrates. The majority of confocal microscopy inflammation signs were found in skin without treatment followed by the vehicle. Ascorbyl tetraisopalmitate, Coenzyme Q 10 , GTP- and Resveratrol-based formulations reduced the anti-inflammatory cytokines release and attenuated inflammatory signs. The combination of techniques provides results that highlight the importance of antioxidant-based formulations for rapid skin recovery. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

4Pi microscopy of the nuclear pore complex.

PubMed

Kahms, Martin; Hüve, Jana; Peters, Reiner

2015-01-01

4Pi microscopy is a far-field fluorescence microscopy technique, in which the wave fronts of two opposing illuminating beams are adjusted to constructively interfere in a common focus. This yields a diffraction pattern in the direction of the optical axis, which essentially consists of a main focal spot accompanied by two smaller side lobes. At optimal conditions, the main peak of this so-called point spread function has a full width at half maximum: fixed phrase of 100 nm in the direction of the optical axis, and thus is 6-7-fold smaller than that of a confocal microscope. In this chapter, we describe the basic features of 4Pi microscopy and its application to cell biology using the example of the nuclear pore complex, a large protein assembly spanning the nuclear envelope.

Multi-imaging analysis of nascent surface structures generated during femtosecond laser irradiation of silicon in high vacuum

NASA Astrophysics Data System (ADS)

Gesuele, F.; JJ Nivas, J.; Fittipaldi, R.; Altucci, C.; Bruzzese, R.; Maddalena, P.; Amoruso, S.

2018-02-01

We report a correlative imaging analysis of a crystalline silicon target after irradiation with a low number of 1055 nm, 850 fs laser pulses with several microscopy techniques (e.g., scanning electron microscopy, atomic force microscopy, Raman micro-imaging and confocal optical microscopy). The analysis is carried out on samples irradiated both in high vacuum and at atmospheric pressure conditions, evidencing interesting differences induced by the ambient environment. In high-vacuum conditions, the results evidence the formation of a halo, which is constituted by alternate stripes of amorphous and crystalline silicon, around the nascent ablation crater. In air, such an effect is drastically reduced, due to the significant back-deposition of nanoparticulate material induced by the larger ambient pressure.

Evaluation of drug delivery to intact and porated skin by coherent Raman scattering and fluorescence microscopies.

PubMed

Belsey, Natalie A; Garrett, Natalie L; Contreras-Rojas, L Rodrigo; Pickup-Gerlaugh, Adam J; Price, Gareth J; Moger, Julian; Guy, Richard H

2014-01-28

Stimulated Raman scattering microscopy was used to assess the permeation of topically applied drugs and formulation excipients into porcine skin. This chemically selective technique generates high-resolution 3D images, from which semi-quantitative information may be elucidated. Ibuprofen, applied as a close-to-saturated solution in propylene glycol, was directly observed to crystallise in/on the skin, as the co-solvent permeated more rapidly, resulting in precipitation of the drug. Coherent Raman scattering microscopy is also an excellent tool, in conjunction with more conventional confocal fluorescence microscopy, with which to image micro/nanoparticle-based formulations. Specifically, the uptake of particles into thermal ablation transport pathways in the skin has been examined. Copyright © 2013 Elsevier B.V. All rights reserved.

Real-time high dynamic range laser scanning microscopy

NASA Astrophysics Data System (ADS)

Vinegoni, C.; Leon Swisher, C.; Fumene Feruglio, P.; Giedt, R. J.; Rousso, D. L.; Stapleton, S.; Weissleder, R.

2016-04-01

In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

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.

Reflection imaging of China ink-perfused brain vasculature using confocal laser-scanning microscopy after clarification of brain tissue by the Spalteholz method.

PubMed

Gutierre, R C; Vannucci Campos, D; Mortara, R A; Coppi, A A; Arida, R M

2017-04-01

Confocal laser-scanning microscopy is a useful tool for visualizing neurons and glia in transparent preparations of brain tissue from laboratory animals. Currently, imaging capillaries and venules in transparent brain tissues requires the use of fluorescent proteins. Here, we show that vessels can be imaged by confocal laser-scanning microscopy in transparent cortical, hippocampal and cerebellar preparations after clarification of China ink-injected specimens by the Spalteholz method. This method may be suitable for global, three-dimensional, quantitative analyses of vessels, including stereological estimations of total volume and length and of surface area of vessels, which constitute indirect approaches to investigate angiogenesis. © 2017 Anatomical Society.

Application of Multiphoton Microscopy in Dermatological Studies: a Mini-Review

PubMed Central

Yew, Elijah; Rowlands, Christopher

2014-01-01

This review summarizes the historical and more recent developments of multiphoton microscopy, as applied to dermatology. Multiphoton microscopy offers several advantages over competing microscopy techniques: there is an inherent axial sectioning, penetration depths that compete well with confocal microscopy on account of the use of near-infrared light, and many two-photon contrast mechanisms, such as second-harmonic generation, have no analogue in one-photon microscopy. While the penetration depths of photons into tissue are typically limited on the order of hundreds of microns, this is of less concern in dermatology, as the skin is thin and readily accessible. As a result, multiphoton microscopy in dermatology has generated a great deal of interest, much of which is summarized here. The review covers the interaction of light and tissue, as well as the various considerations that must be made when designing an instrument. The state of multiphoton microscopy in imaging skin cancer and various other diseases is also discussed, along with the investigation of aging and regeneration phenomena, and finally, the use of multiphoton microscopy to analyze the transdermal transport of drugs, cosmetics and other agents is summarized. The review concludes with a look at potential future research directions, especially those that are necessary to push these techniques into widespread clinical acceptance. PMID:25075226

Water-induced phase separation of miconazole-poly (vinylpyrrolidone-co-vinyl acetate) amorphous solid dispersions: Insights with confocal fluorescence microscopy.

PubMed

Saboo, Sugandha; Taylor, Lynne S

2017-08-30

The aim of this study was to evaluate the utility of confocal fluorescence microscopy (CFM) to study the water-induced phase separation of miconazole-poly (vinylpyrrolidone-co-vinyl acetate) (mico-PVPVA) amorphous solid dispersions (ASDs), induced during preparation, upon storage at high relative humidity (RH) and during dissolution. Different fluorescent dyes were added to drug-polymer films and the location of the dyes was evaluated using CFM. Orthogonal techniques, in particular atomic force microscopy (AFM) coupled with nanoscale infrared spectroscopy (AFM-nanoIR), were used to provide additional analysis of the drug-polymer blends. The initial miscibility of mico-PVPVA ASDs prepared under low humidity conditions was confirmed by AFM-nanoIR. CFM enabled rapid identification of drug-rich and polymer-rich phases in phase separated films prepared under high humidity conditions. The identity of drug- and polymer-rich domains was confirmed using AFM-nanoIR imaging and localized IR spectroscopy, together with Lorentz contact resonance (LCR) measurements. The CFM technique was then utilized successfully to further investigate phase separation in mico-PVPVA films exposed to high RH storage and to visualize phase separation dynamics following film immersion in buffer. CFM is thus a promising new approach to study the phase behavior of ASDs, utilizing drug and polymer specific dyes to visualize the evolution of heterogeneity in films exposed to water. Copyright © 2017 Elsevier B.V. All rights reserved.

Liposuction Preserves the Morphological Integrity of the Microvascular Network: Flow Cytometry and Confocal Microscopy Evidence in a Controlled Study.

PubMed

Bertheuil, Nicolas; Chaput, Benoit; Berger-Müller, Sandra; Ménard, Cédric; Mourcin, Frédéric; Watier, Eric; Grolleau, Jean-Louis; Garrido, Ignacio; Tarte, Karin; Sensébé, Luc; Varin, Audrey

2016-05-01

Liposuction is a very popular technique in plastic surgery that allows for the taking adipose tissue (AT) on large surfaces with little risk of morbidity. Although liposuction was previously shown to preserve large perforator vessels, little is known about the effects of liposuction on the microvasculature network. The aim of this study was to analyze the effect of liposuction on the preservation of microvessels at tissue and cellular levels by flow cytometry and confocal microscopy following abdominoplasty procedure. Percentage of endothelial cells in AT from liposuction and en bloc AT was determined by multicolor flow cytometry. Moreover, vessel density and adipocyte content were analyzed in situ in 3 different types of AT (en bloc, from liposuction, and residual AT after liposuction) by confocal microscopy. Flow cytometric analysis showed that en bloc AT contained 30.6% ± 12.9% and AT from liposuction 21.6% ± 9.9% of endothelial cells (CD31(pos)/CD45(neg)/CD235a(neg)/CD11b(neg)) (P = .009). Moreover, analysis of paired AT from the same patients (n = 5) confirmed a lower percentage of endothelial cells in AT from liposuction compared to en bloc AT (17.7% ± 4.5% vs 21.9% ± 3.3%, P = .031). Likewise, confocal microscopy showed that en bloc AT contained 8.2% ± 6.3%, AT from liposuction only 1.6% ± 1.0% (P < .0001), and AT after liposuction 8.9% ± 4.1% (P = .111) of CD31(pos) vessels. Conversely, adipocyte content was 39.5% ± 14.5% in the en bloc AT, 45% ± 18.4% in AT from liposuction (P = .390), and 18.8 ± 14.8% in AT after liposuction (P = .011). For the first time, we demonstrate that liposuction preserves the microvascular network. Indeed, a low percentage of endothelial cells was found in AT from liposuction and we confirm the persistence of microvessels in the tissue after liposuction. © 2015 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com.

A comparison of image restoration approaches applied to three-dimensional confocal and wide-field fluorescence microscopy.

PubMed

Verveer, P. J; Gemkow, M. J; Jovin, T. M

1999-01-01

We have compared different image restoration approaches for fluorescence microscopy. The most widely used algorithms were classified with a Bayesian theory according to the assumed noise model and the type of regularization imposed. We considered both Gaussian and Poisson models for the noise in combination with Tikhonov regularization, entropy regularization, Good's roughness and without regularization (maximum likelihood estimation). Simulations of fluorescence confocal imaging were used to examine the different noise models and regularization approaches using the mean squared error criterion. The assumption of a Gaussian noise model yielded only slightly higher errors than the Poisson model. Good's roughness was the best choice for the regularization. Furthermore, we compared simulated confocal and wide-field data. In general, restored confocal data are superior to restored wide-field data, but given sufficient higher signal level for the wide-field data the restoration result may rival confocal data in quality. Finally, a visual comparison of experimental confocal and wide-field data is presented.

In vivo laser confocal microscopy findings of Thygeson superficial punctate keratitis.

PubMed

Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2011-06-01

We looked for microstructural corneal characteristics of Thygeson superficial punctate keratitis (TSPK) in an in vivo investigation using laser scanning confocal microscopy. Five patients (3 men and 2 women; mean age, 51.8 years) with clinically diagnosed TSPK were enrolled in this study. All patients were examined by slit-lamp biomicroscopy and in vivo laser confocal microscopy. Deposits in selected confocal images of all corneal layers were evaluated qualitatively for shape and degree of light reflection. The most characteristic finding was aggregates of highly reflective deposits with a starburst-like appearance that corresponded with epithelial punctate lesions identified by slit-lamp biomicroscopy; the aggregates were sporadically observed in all cases at the superficial and basal epithelial cell layers. Subepithelial haze was observed in all cases. Langerhans cells were also sporadically observed in all cases at the basal epithelial layer. Bowman layer abnormalities were observed in 3 of 5 cases; all these patients had a long history of TSPK (eg, more than 1 year). In addition, the 3 patients had highly reflective, tiny, needle-shaped materials in the corneal stroma. In vivo laser confocal microscopy is capable of identifying characteristic corneal microstructural changes related to TSPK with a higher resolution than is available with slit-lamp biomicroscopy. It may also be a valuable tool for further research to elucidate both pathogenesis and the natural course of TSPK.

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.

Confocal microscopy evaluation of stromal fluorescence intensity after standard and accelerated iontophoresis-assisted corneal cross-linking.

PubMed

Lanzini, Manuela; Curcio, Claudia; Spoerl, Eberhard; Calienno, Roberta; Mastropasqua, Alessandra; Colasante, Martina; Mastropasqua, Rodolfo; Nubile, Mario; Mastropasqua, Leonardo

2017-02-01

The aim of this study is to determine modifications in stromal fluorescence intensity after different corneal cross-linking (CXL) procedures and to correlate stromal fluorescence to corneal biomechanical resistance. For confocal microscopy study, 15 human cadaver corneas were examined. Three served as control (group 1), three were just soaked with iontophoresis procedure (group 2), three were treated with standard epi-off technique (group 3), and six underwent iontophoresis imbibition. Three of later six were irradiated for 30 min with 3 mW/cm 2 UVA (group 4) and three for 9 min at 10 mW/cm 2 UVA (group 5). Confocal microscopy was performed to quantify the fluorescence intensity in the cornea at different stromal depths. For biomechanical study, 30 human cadaver corneas were randomly divided into five groups and treated as previously described. Static stress-strain measurements of the corneas were performed. Iontophoresis imbibition followed by 10mW/cm 2 irradiation proved to increase stromal fluorescence into the corneal stroma and significant differences were revealed between group 3 and 5 both at 100 (p = 0.0171) and 250 µm (p = 0.0024), respectively. Biomechanical analysis showed an improvement of corneal resistance in group 5. Iontophoresis imbibition followed by accelerated irradiation increased the stromal fluorescence and is related to an improvement of biomechanical resistance. This approach may represent a new strategy to achieve greater concentrations of riboflavin without removing corneal epithelium and improve clinical results while reducing the side effects of CXL.

Demonstration of prominent actin filaments in the root columella

NASA Technical Reports Server (NTRS)

Collings, D. A.; Zsuppan, G.; Allen, N. S.; Blancaflor, E. B.; Brown, C. S. (Principal Investigator)

2001-01-01

The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling.

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

Parallel detection experiment of fluorescence confocal microscopy using DMD.

PubMed

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

2016-05-01

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

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.

Adaptive optics in spinning disk microscopy: improved contrast and brightness by a simple and fast method.

PubMed

Fraisier, V; Clouvel, G; Jasaitis, A; Dimitrov, A; Piolot, T; Salamero, J

2015-09-01

Multiconfocal microscopy gives a good compromise between fast imaging and reasonable resolution. However, the low intensity of live fluorescent emitters is a major limitation to this technique. Aberrations induced by the optical setup, especially the mismatch of the refractive index and the biological sample itself, distort the point spread function and further reduce the amount of detected photons. Altogether, this leads to impaired image quality, preventing accurate analysis of molecular processes in biological samples and imaging deep in the sample. The amount of detected fluorescence can be improved with adaptive optics. Here, we used a compact adaptive optics module (adaptive optics box for sectioning optical microscopy), which was specifically designed for spinning disk confocal microscopy. The module overcomes undesired anomalies by correcting for most of the aberrations in confocal imaging. Existing aberration detection methods require prior illumination, which bleaches the sample. To avoid multiple exposures of the sample, we established an experimental model describing the depth dependence of major aberrations. This model allows us to correct for those aberrations when performing a z-stack, gradually increasing the amplitude of the correction with depth. It does not require illumination of the sample for aberration detection, thus minimizing photobleaching and phototoxicity. With this model, we improved both signal-to-background ratio and image contrast. Here, we present comparative studies on a variety of biological samples. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

In Vivo Confocal Microscopy of Corneal Nerves in Health and Disease

PubMed Central

Cruzat, Andrea; Qazi, Yureeda; Hamrah, Pedram

2016-01-01

In vivo confocal microscopy (IVCM) is becoming an indispensable tool for studying corneal physiology and disease. Enabling the dissection of corneal architecture at a cellular level, this technique offers fast and noninvasive in vivo imaging of the cornea with images comparable to that of ex vivo histochemical techniques. Corneal nerves bear substantial relevance to clinicians and scientists alike, given their pivotal roles in regulation of corneal sensation, maintenance of epithelial integrity, and proliferation and promotion of wound healing. Thus, IVCM offers a unique method to study corneal nerve alterations in a myriad of conditions, such as ocular and systemic diseases and following corneal surgery, without altering the tissue microenvironment. Of particular interest has been the correlation of corneal subbasal nerves to their function, which has been studied in normal eyes, contact lens wearers, and patients with keratoconus, infectious keratitis, corneal dystrophies, and neurotrophic keratopathy. Longitudinal studies have applied IVCM to investigate the effects of corneal surgery on nerves, demonstrating their regenerative capacity. IVCM is increasingly important in the diagnosis and management of systemic conditions such as peripheral diabetic neuropathy and, more recently, in ocular diseases. In this review, we outline the principles and applications of IVCM in the study of corneal nerves in various ocular and systemic diseases. PMID:27771327

A model system using confocal fluorescence microscopy for examining real-time intracellular sodium ion regulation.

PubMed

Lee, Jacqueline A; Collings, David A; Glover, Chris N

2016-08-15

The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid-base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na(+)]i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na(+)]i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye-ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na(+)]i, whereas no significant differences in branchial [Na(+)]i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na(+)]i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms. Copyright © 2016 Elsevier Inc. All rights reserved.

High-resolution confocal imaging of wall ingrowth deposition in plant transfer cells: Semi-quantitative analysis of phloem parenchyma transfer cell development in leaf minor veins of Arabidopsis.

PubMed

Nguyen, Suong T T; McCurdy, David W

2015-04-23

Transfer cells (TCs) are trans-differentiated versions of existing cell types designed to facilitate enhanced membrane transport of nutrients at symplasmic/apoplasmic interfaces. This transport capacity is conferred by intricate wall ingrowths deposited secondarily on the inner face of the primary cell wall, hence promoting the potential trans-membrane flux of solutes and consequently assigning TCs as having key roles in plant growth and productivity. However, TCs are typically positioned deep within tissues and have been studied mostly by electron microscopy. Recent advances in fluorophore labelling of plant cell walls using a modified pseudo-Schiff-propidium iodide (mPS-PI) staining procedure in combination with high-resolution confocal microscopy have allowed visualization of cellular details of individual tissue layers in whole mounts, hence enabling study of tissue and cellular architecture without the need for tissue sectioning. Here we apply a simplified version of the mPS-PI procedure for confocal imaging of cellulose-enriched wall ingrowths in vascular TCs at the whole tissue level. The simplified mPS-PI staining procedure produced high-resolution three-dimensional images of individual cell types in vascular bundles and, importantly, wall ingrowths in phloem parenchyma (PP) TCs in minor veins of Arabidopsis leaves and companion cell TCs in pea. More efficient staining of tissues was obtained by replacing complex clearing procedures with a simple post-fixation bleaching step. We used this modified procedure to survey the presence of PP TCs in other tissues of Arabidopsis including cotyledons, cauline leaves and sepals. This high-resolution imaging enabled us to classify different stages of wall ingrowth development in Arabidopsis leaves, hence enabling semi-quantitative assessment of the extent of wall ingrowth deposition in PP TCs at the whole leaf level. Finally, we conducted a defoliation experiment as an example of using this approach to statistically analyze responses of PP TC development to leaf ablation. Use of a modified mPS-PI staining technique resulted in high-resolution confocal imaging of polarized wall ingrowth deposition in TCs. This technique can be used in place of conventional electron microscopy and opens new possibilities to study mechanisms determining polarized deposition of wall ingrowths and use reverse genetics to identify regulatory genes controlling TC trans-differentiation.

Confocal Raman microscopy of morphological changes in poly(ethylene terephthalate) film induced by supercritical CO(2).

PubMed

Fleming, Oliver S; Kazarian, Sergei G

2004-04-01

Poly(ethylene terephthalate) (PET) film was exposed to supercritical (sc) CO(2) and confocal Raman microscopy was used to investigate the morphological changes induced. The study evaluates the use of oil and dry objectives in confocal mode to obtain depth profiles of PET film. These results were compared with the data obtained by mapping of the film cross-section. A significant gradient of degree of crystallinity normal to the surface of PET film down to 60 microm has been observed. The gradient of the degree of morphological changes are functions of exposure time and pressure.

Evaluation of agave fiber delignification by means of microscopy techniques and image analysis.

PubMed

Hernández-Hernández, Hilda M; Chanona-Pérez, Jorge J; Calderón-Domínguez, Georgina; Perea-Flores, María J; Mendoza-Pérez, Jorge A; Vega, Alberto; Ligero, Pablo; Palacios-González, Eduardo; Farrera-Rebollo, Reynold R

2014-10-01

Recently, the use of different types of natural fibers to produce paper and textiles from agave plants has been proposed. Agave atrovirens can be a good source of cellulose and lignin; nevertheless, the microstructural changes that happen during delignification have scarcely been studied. The aim of this work was to study the microstructural changes that occur during the delignification of agave fibers by means of microscopy techniques and image analysis. The fibers of A. atrovirens were obtained from leaves using convective drying, milling, and sieving. Fibers were processed using the Acetosolv pulping method at different concentrations of acetic acid; increasing acid concentration promoted higher levels of delignification, structural damage, and the breakdown of fiber clumps. Delignification followed by spectrometric analysis and microstructural studies were carried out by light, confocal laser scanning and scanning electron microscopy and showed that the delignification process follows three stages: initial, bulk, and residual. Microscopy techniques and image analysis were efficient tools for microstructural characterization during delignification of agave fibers, allowing quantitative evaluation of the process and the development of linear prediction models. The data obtained integrated numerical and microstructural information that could be valuable for the study of pulping of lignocellulosic materials.

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

Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.

PubMed

Kaufmann, Anna; Mickoleit, Michaela; Weber, Michael; Huisken, Jan

2012-09-01

Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging optical imaging techniques, such as optical projection tomography (OPT).

Real-time high dynamic range laser scanning microscopy

PubMed Central

Vinegoni, C.; Leon Swisher, C.; Fumene Feruglio, P.; Giedt, R. J.; Rousso, D. L.; Stapleton, S.; Weissleder, R.

2016-01-01

In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging. PMID:27032979

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.

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

PubMed

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

2012-10-17

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0-85 μm from the surface of a coverglass. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Development of a viability standard curve for microencapsulated probiotic bacteria using confocal microscopy and image analysis software.

PubMed

Moore, Sarah; Kailasapathy, Kasipathy; Phillips, Michael; Jones, Mark R

2015-07-01

Microencapsulation is proposed to protect probiotic strains from food processing procedures and to maintain probiotic viability. Little research has described the in situ viability of microencapsulated probiotics. This study successfully developed a real-time viability standard curve for microencapsulated bacteria using confocal microscopy, fluorescent dyes and image analysis software. Copyright © 2015 Elsevier B.V. All rights reserved.

Confocal laser scanning microscopy and area-scale analysis used to quantify enamel surface textural changes from citric acid demineralization and salivary remineralization in vitro.

PubMed

Austin, R S; Giusca, C L; Macaulay, G; Moazzez, R; Bartlett, D W

2016-02-01

This paper investigates the application of confocal laser scanning microscopy to determine the effect of acid-mediated erosive enamel wear on the micro-texture of polished human enamel in vitro. Twenty polished enamel samples were prepared and subjected to a citric acid erosion and pooled human saliva remineralization model. Enamel surface microhardness was measured using a Knoop hardness tester, which confirmed that an early enamel erosion lesion was formed which was then subsequently completely remineralized. A confocal laser scanning microscope was used to capture high-resolution images of the enamel surfaces undergoing demineralization and remineralization. Area-scale analysis was used to identify the optimal feature size following which the surface texture was determined using the 3D (areal) texture parameter Sa. The Sa successfully characterized the enamel erosion and remineralization for the polished enamel samples (P<0.001). Areal surface texture characterization of the surface events occurring during enamel demineralization and remineralization requires optical imaging instrumentation with lateral resolution <2.5 μm, applied in combination with appropriate filtering in order to remove unwanted waviness and roughness. These techniques will facilitate the development of novel methods for measuring early enamel erosion lesions in natural enamel surfaces in vivo. Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Comparison of divided and full pupil configurations for line-scanning confocal microscopy in human skin and oral mucosa

NASA Astrophysics Data System (ADS)

Larson, Bjorg; Abeytunge, Sanjeewa; Glazowski, Chris; Rajadhyaksha, Milind

2012-02-01

Confocal point-scanning microscopy has been showing promise in the detection, diagnosing and mapping of skin lesions in clinical settings. The noninvasive technique allows provides optical sectioning and cellular resolution for in vivo diagnosis of melanoma and basal cell carcinoma and pre-operative and intra-operative mapping of margins. The imaging has also enabled more accurate "guided" biopsies while minimizing the otherwise large number of "blind" biopsies. Despite these translational advances, however, point-scanning technology remains relatively complex and expensive. Line-scanning technology may offer an alternative approach to accelerate translation to the clinic. Line-scanning, using fewer optical components, inexpensive linear-array detectors and custom electronics, may enable smaller, simpler and lower-cost confocal microscopes. A line is formed using a cylindrical lens and scanned through the back focal plane of the objective with a galvanometric scanner. A linear CCD is used for detection. Two pupil configurations were compared for performance in imaging human tissue. In the full-pupil configuration, illumination and detection is made through the full objective pupil. In the divided pupil approach, half the pupil is illuminated and the other half is used for detection. The divided pupil configuration loses spatial and axial resolution due to a diminished NA, but the sectioning capability and rejection of background is improved. Imaging in skin and oral mucosa illustrate the performance of the two configurations.

In vivo laser confocal microscopy after non-Descemet's stripping automated endothelial keratoplasty.

PubMed

Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2009-07-01

To investigate in vivo corneal changes in patients with bullous keratopathy who underwent non-Descemet's stripping automated endothelial keratoplasty (nDSAEK) with the use of laser confocal microscopy. Single-center, prospective clinical study. Ten eyes (10 patients; 3 men and 7 women; mean age, 73.5+/-6.6 years [mean+/-standard deviation]) with bullous keratopathy were evaluated in this study. In vivo laser confocal microscopy was performed before and 1, 3, and 6 months after nDSAEK. Selected confocal images of corneal layers were evaluated qualitatively and quantitatively for degree of haze and density of deposits. Before surgery, the following were observed in all patients: corneal epithelial edema, subepithelial haze, keratocytes in a honeycomb pattern, and tiny needle-shaped materials in the stroma. After nDSAEK, subepithelial haze, donor-recipient interface haze, and interface particles were observed in all measurable cases; postoperative haze, interface particles, and needle-shaped materials decreased statistically significantly (P<0.05) over the course of follow-up. In addition, hyperreflective giant interface particles were observed after nDSAEK in all patients. In vivo laser confocal microscopy can identify subclinical corneal abnormalities after nDSAEK such as subepithelial haze, host-recipient interface haze, host stromal needle-shaped materials, and host-recipient interface particles with characteristic giant particles. Further studies with this technology in a large number of patients and long-term follow-up are needed to understand fully the long-term corneal stromal changes after nDSAEK.

Confocal Imaging of porous media

NASA Astrophysics Data System (ADS)

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

2012-12-01

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

Molecular and Cellular Quantitative Microscopy: theoretical investigations, technological developments and applications to neurobiology

NASA Astrophysics Data System (ADS)

Esposito, Alessandro

2006-05-01

This PhD project aims at the development and evaluation of microscopy techniques for the quantitative detection of molecular interactions and cellular features. The primarily investigated techniques are Fαrster Resonance Energy Transfer imaging and Fluorescence Lifetime Imaging Microscopy. These techniques have the capability to quantitatively probe the biochemical environment of fluorophores. An automated microscope capable of unsupervised operation has been developed that enables the investigation of molecular and cellular properties at high throughput levels and the analysis of cellular heterogeneity. State-of-the-art Förster Resonance Energy Transfer imaging, Fluorescence Lifetime Imaging Microscopy, Confocal Laser Scanning Microscopy and the newly developed tools have been combined with cellular and molecular biology techniques for the investigation of protein-protein interactions, oligomerization and post-translational modifications of α-Synuclein and Tau, two proteins involved in Parkinson’s and Alzheimer’s disease, respectively. The high inter-disciplinarity of this project required the merging of the expertise of both the Molecular Biophysics Group at the Debye Institute - Utrecht University and the Cell Biophysics Group at the European Neuroscience Institute - Gαttingen University. This project was conducted also with the support and the collaboration of the Center for the Molecular Physiology of the Brain (Göttingen), particularly with the groups associated with the Molecular Quantitative Microscopy and Parkinson’s Disease and Aggregopathies areas. This work demonstrates that molecular and cellular quantitative microscopy can be used in combination with high-throughput screening as a powerful tool for the investigation of the molecular mechanisms of complex biological phenomena like those occurring in neurodegenerative diseases.

Image scanning fluorescence emission difference microscopy based on a detector array.

PubMed

Li, Y; Liu, S; Liu, D; Sun, S; Kuang, C; Ding, Z; Liu, X

2017-06-01

We propose a novel imaging method that enables the enhancement of three-dimensional resolution of confocal microscopy significantly and achieve experimentally a new fluorescence emission difference method for the first time, based on the parallel detection with a detector array. Following the principles of photon reassignment in image scanning microscopy, images captured by the detector array were arranged. And by selecting appropriate reassign patterns, the imaging result with enhanced resolution can be achieved with the method of fluorescence emission difference. Two specific methods are proposed in this paper, showing that the difference between an image scanning microscopy image and a confocal image will achieve an improvement of transverse resolution by approximately 43% compared with that in confocal microscopy, and the axial resolution can also be enhanced by at least 22% experimentally and 35% theoretically. Moreover, the methods presented in this paper can improve the lateral resolution by around 10% than fluorescence emission difference and 15% than Airyscan. The mechanism of our methods is verified by numerical simulations and experimental results, and it has significant potential in biomedical applications. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Scanning transmission X-ray, laser scanning, and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms.

PubMed

Lawrence, J R; Swerhone, G D W; Leppard, G G; Araki, T; Zhang, X; West, M M; Hitchcock, A P

2003-09-01

Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.

Analysis of a nanocrystalline polymer dispersion of ebselen using solid-state NMR, Raman microscopy, and powder X-ray diffraction.

PubMed

Vogt, Frederick G; Williams, Glenn R

2012-07-01

Nanocrystalline drug-polymer dispersions are of significant interest in pharmaceutical delivery. The purpose of this work is to demonstrate the applicability of methods based on two-dimensional (2D) and multinuclear solid-state NMR (SSNMR) to a novel nanocrystalline pharmaceutical dispersion of ebselen with polyvinylpyrrolidone-vinyl acetate (PVP-VA), after initial characterization with other techniques. A nanocrystalline dispersion of ebselen with PVP-VA was prepared and characterized by powder X-ray diffraction (PXRD), confocal Raman microscopy and mapping, and differential scanning calorimetry (DSC), and then subjected to detailed 1D and 2D SSNMR analysis involving ¹H, ¹³C, and ⁷⁷Se isotopes and ¹H spin diffusion. PXRD was used to show that dispersion contains nanocrystalline ebselen in the 35-60 nm size range. Confocal Raman microscopy and spectral mapping were able to detect regions where short-range interactions may occur between ebselen and PVP-VA. Spin diffusion effects were analyzed using 2D SSNMR experiments and are able to directly detect interactions between ebselen and the surrounding PVP-VA. The methods used here, particularly the 2D SSNMR methods based on spin diffusion, provided detailed structural information about a nanocrystalline polymer dispersion of ebselen, and should be useful in other studies of these types of materials.

Real-time dynamic range and signal to noise enhancement in beam-scanning microscopy by integration of sensor characteristics, data acquisition hardware, and statistical methods

NASA Astrophysics Data System (ADS)

Kissick, David J.; Muir, Ryan D.; Sullivan, Shane Z.; Oglesbee, Robert A.; Simpson, Garth J.

2013-02-01

Despite the ubiquitous use of multi-photon and confocal microscopy measurements in biology, the core techniques typically suffer from fundamental compromises between signal to noise (S/N) and linear dynamic range (LDR). In this study, direct synchronous digitization of voltage transients coupled with statistical analysis is shown to allow S/N approaching the theoretical maximum throughout an LDR spanning more than 8 decades, limited only by the dark counts of the detector on the low end and by the intrinsic nonlinearities of the photomultiplier tube (PMT) detector on the high end. Synchronous digitization of each voltage transient represents a fundamental departure from established methods in confocal/multi-photon imaging, which are currently based on either photon counting or signal averaging. High information-density data acquisition (up to 3.2 GB/s of raw data) enables the smooth transition between the two modalities on a pixel-by-pixel basis and the ultimate writing of much smaller files (few kB/s). Modeling of the PMT response allows extraction of key sensor parameters from the histogram of voltage peak-heights. Applications in second harmonic generation (SHG) microscopy are described demonstrating S/N approaching the shot-noise limit of the detector over large dynamic ranges.

Comparison of Cornea Module and DermaInspect for noninvasive imaging of ocular surface pathologies

NASA Astrophysics Data System (ADS)

Steven, Philipp; Müller, Maya; Koop, Norbert; Rose, Christian; Hüttmann, Gereon

2009-11-01

Minimally invasive imaging of ocular surface pathologies aims at securing clinical diagnosis without actual tissue probing. For this matter, confocal microscopy (Cornea Module) is in daily use in ophthalmic practice. Multiphoton microscopy is a new optical technique that enables high-resolution imaging and functional analysis of living tissues based on tissue autofluorescence. This study was set up to compare the potential of a multiphoton microscope (DermaInspect) to the Cornea Module. Ocular surface pathologies such as pterygia, papillomae, and nevi were investigated in vivo using the Cornea Module and imaged immediately after excision by DermaInspect. Two excitation wavelengths, fluorescence lifetime imaging and second-harmonic generation (SHG), were used to discriminate different tissue structures. Images were compared with the histopathological assessment of the samples. At wavelengths of 730 nm, multiphoton microscopy exclusively revealed cellular structures. Collagen fibrils were specifically demonstrated by second-harmonic generation. Measurements of fluorescent lifetimes enabled the highly specific detection of goblet cells, erythrocytes, and nevus-cell clusters. At the settings used, DermaInspect reaches higher resolutions than the Cornea Module and obtains additional structural information. The parallel detection of multiphoton excited autofluorescence and confocal imaging could expand the possibilities of minimally invasive investigation of the ocular surface toward functional analysis at higher resolutions.

Visualization and identification of the structures formed during early stages of fibrin polymerization

PubMed Central

Chernysh, Irina N.; Nagaswami, Chandrasekaran

2011-01-01

We determined the sequence of events and identified and quantitatively characterized the mobility of moving structures present during the early stages of fibrin-clot formation from the beginning of polymerization to the gel point. Three complementary techniques were used in parallel: spinning-disk confocal microscopy, transmission electron microscopy, and turbidity measurements. At the beginning of polymerization the major structures were monomers, whereas at the middle of the lag period there were monomers, oligomers, protofibrils (defined as structures that consisted of more than 8 monomers), and fibers. At the end of the lag period, there were primarily monomers and fibers, giving way to mainly fibers at the gel point. Diffusion rates were calculated from 2 different results, one based on sizes and another on the velocity of the observed structures, with similar results in the range of 3.8-0.1 μm2/s. At the gel point, the diffusion coefficients corresponded to very large, slow-moving structures and individual protofibrils. The smallest moving structures visible by confocal microscopy during fibrin polymerization were identified as protofibrils with a length of approximately 0.5 μm. The sequence of early events of clotting and the structures present are important for understanding hemostasis and thrombosis. PMID:21248064

Confocal arthroscopy-based patient-specific constitutive models of cartilaginous tissues - II: prediction of reaction force history of meniscal cartilage specimens.

PubMed

Taylor, Zeike A; Kirk, Thomas B; Miller, Karol

2007-10-01

The theoretical framework developed in a companion paper (Part I) is used to derive estimates of mechanical response of two meniscal cartilage specimens. The previously developed framework consisted of a constitutive model capable of incorporating confocal image-derived tissue microstructural data. In the present paper (Part II) fibre and matrix constitutive parameters are first estimated from mechanical testing of a batch of specimens similar to, but independent from those under consideration. Image analysis techniques which allow estimation of tissue microstructural parameters form confocal images are presented. The constitutive model and image-derived structural parameters are then used to predict the reaction force history of the two meniscal specimens subjected to partially confined compression. The predictions are made on the basis of the specimens' individual structural condition as assessed by confocal microscopy and involve no tuning of material parameters. Although the model does not reproduce all features of the experimental curves, as an unfitted estimate of mechanical response the prediction is quite accurate. In light of the obtained results it is judged that more general non-invasive estimation of tissue mechanical properties is possible using the developed framework.

Confocal endomicroscopy: Is it time to move on?

PubMed

Robles-Medranda, Carlos

2016-01-10

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

X-ray microscopy of live biological micro-organisms

NASA Astrophysics Data System (ADS)

Raja Al-Ani, Ma'an Nassar

Real-time, compact x-ray microscopy has the potential to benefit many scientific fields, including microbiology, pharmacology, organic chemistry, and physics. Single frame x-ray micro-radiography, produced by a compact, solid-state laser plasma source, allows scientists to use x-ray emission for elemental analysis, and to observe biological specimens in their natural state. In this study, x-ray images of mouse kidney tissue, live bacteria, Pseudomonas aeruginosa and Burkholderia cepacia, and the bacteria's interaction with the antibiotic gentamicin, are examined using x-ray microscopy. For the purposes of comparing between confocal microscopy and x-ray microscopy, we introduced to our work the technique of gold labeling. Indirect immunofluorescence staining and immuno-gold labeling were applied on human lymphocytes and human tumor cells. Differential interference contrast microscopy (DIC) showed the lymphocyte body and nucleus, as did x-ray microscopy. However, the high resolution of x-ray microscopy allows us to differentiate between the gold particles bound to the antibodies and the free gold. A compact, tabletop Nd: glass laser is used in this study to produce x-rays from an Yttrium target. An atomic force microscope is used to scan the x-ray images from the developed photo-resist. The use of compact, tabletop laser plasma sources, in conjunction with x-ray microscopy, is a new technique that has great potential as a flexible, user-friendly scientific research tool.

Intracellular photoinduced oxidative stress by zinc phthalocyanine photosensitization: a study of the early events in real time using confocal microscopy

NASA Astrophysics Data System (ADS)

Alexandratou, Eleni; Yova, Dido; Handris, Panagiotis; Kletsas, Dimitris; Loukas, Spyros

2003-10-01

Oxidative stress has been implicated in several biological and pathological aspects. Reactive oxygen species (ROS) have been proposed to act as signal transduction molecules activating reactions leading to cell rescue or to cell apoptosis/necrosis. In the present study, oxidative stress was induced by photosensitization of zinc phthalocyanine (ZnPc) in human fibroblasts using a photodynamic dose that did not lead to apoptosis or necrosis. The induction of oxidative stress was performed at the microscope stage in preassigned time. The cascade of phenomena evoked was studied in real time and at the single cell level using confocal laser scanning microscopy. Using specific vital fluorescent probes, alterations induced by oxidative stress in mitochondria membrane potential, in intracellular pH and in calcium concentration were recorded. Image processing and analysis techniques were used to quantify the observed changes. Subcellular localization of the photosensitizer was studied in order to determine the primary and immediate ROS target. It was found that ZnPc is mainly localized in the mitochondria region.

Classification of M1/M2-polarized human macrophages by label-free hyperspectral reflectance confocal microscopy and multivariate analysis.

PubMed

Bertani, Francesca R; Mozetic, Pamela; Fioramonti, Marco; Iuliani, Michele; Ribelli, Giulia; Pantano, Francesco; Santini, Daniele; Tonini, Giuseppe; Trombetta, Marcella; Businaro, Luca; Selci, Stefano; Rainer, Alberto

2017-08-21

The possibility of detecting and classifying living cells in a label-free and non-invasive manner holds significant theranostic potential. In this work, Hyperspectral Imaging (HSI) has been successfully applied to the analysis of macrophagic polarization, given its central role in several pathological settings, including the regulation of tumour microenvironment. Human monocyte derived macrophages have been investigated using hyperspectral reflectance confocal microscopy, and hyperspectral datasets have been analysed in terms of M1 vs. M2 polarization by Principal Components Analysis (PCA). Following PCA, Linear Discriminant Analysis has been implemented for semi-automatic classification of macrophagic polarization from HSI data. Our results confirm the possibility to perform single-cell-level in vitro classification of M1 vs. M2 macrophages in a non-invasive and label-free manner with a high accuracy (above 98% for cells deriving from the same donor), supporting the idea of applying the technique to the study of complex interacting cellular systems, such in the case of tumour-immunity in vitro models.

High-definition optical coherence tomography and reflectance confocal microscopy in the in vivo visualization of a reaction to permanent make-up.

PubMed

Maier, T; Flaig, M J; Ruzicka, T; Berking, C; Pavicic, T

2015-03-01

After permanent make-up treatments, eczematous and granulomatous reactions may occur which need anti-inflammatory treatment. For the definite diagnosis oftentimes biopsies are recommended. In vivo imaging such as reflectance confocal microscopy (RCM) and high-definition optical coherence tomography (HD-OCT) has been successfully used in the non-invasive diagnosis of various dermatoses before. Here, we report on non-invasive imaging of a reaction towards permanent make-up in a 40-year-old woman by using HD-OCT and RCM. Both in HD-OCT and in RCM subepidermal pigment and granulomatous changes could be visualized and correlated with the histopathological findings. Regression of the lesions in response to topical steroids and intralesional injections of steroids and 5-fluorouracil is reported and treatment options are discussed. Non-invasive imaging techniques such as HD-OCT and RCM allow the visualization and localization of exogenous pigment and help in the evaluation of adverse reactions due to permanent make-up tattooing. © 2014 European Academy of Dermatology and Venereology.

Quantitative Confocal Microscopy Analysis as a Basis for Search and Study of Potassium Kv1.x Channel Blockers

NASA Astrophysics Data System (ADS)

Feofanov, Alexey V.; Kudryashova, Kseniya S.; Nekrasova, Oksana V.; Vassilevski, Alexander A.; Kuzmenkov, Alexey I.; Korolkova, Yuliya V.; Grishin, Eugene V.; Kirpichnikov, Mikhail P.

Artificial KcsA-Kv1.x (x = 1, 3) receptors were recently designed by transferring the ligand-binding site from human Kv1.x voltage-gated potassium channels into corresponding domain of the bacterial KscA channel. We found that KcsA-Kv1.x receptors expressed in E. coli cells are embedded into cell membrane and bind ligands when the cells are transformed to spheroplasts. We supposed that E. coli spheroplasts with membrane-embedded KcsA-Kv1.x and fluorescently labeled ligand agitoxin-2 (R-AgTx2) can be used as elements of an advanced analytical system for search and study of Kv1-channel blockers. To realize this idea, special procedures were developed for measurement and quantitative treatment of fluorescence signals obtained from spheroplast membrane using confocal laser scanning microscopy (CLSM). The worked out analytical "mix and read" systems supported by quantitative CLSM analysis were demonstrated to be reliable alternative to radioligand and electrophysiology techniques in the search and study of selective Kv1.x channel blockers of high scientific and medical importance.

Automatic analysis of diabetic peripheral neuropathy using multi-scale quantitative morphology of nerve fibres in corneal confocal microscopy imaging.

PubMed

Dabbah, M A; Graham, J; Petropoulos, I N; Tavakoli, M; Malik, R A

2011-10-01

Diabetic peripheral neuropathy (DPN) is one of the most common long term complications of diabetes. Corneal confocal microscopy (CCM) image analysis is a novel non-invasive technique which quantifies corneal nerve fibre damage and enables diagnosis of DPN. This paper presents an automatic analysis and classification system for detecting nerve fibres in CCM images based on a multi-scale adaptive dual-model detection algorithm. The algorithm exploits the curvilinear structure of the nerve fibres and adapts itself to the local image information. Detected nerve fibres are then quantified and used as feature vectors for classification using random forest (RF) and neural networks (NNT) classifiers. We show, in a comparative study with other well known curvilinear detectors, that the best performance is achieved by the multi-scale dual model in conjunction with the NNT classifier. An evaluation of clinical effectiveness shows that the performance of the automated system matches that of ground-truth defined by expert manual annotation. Copyright © 2011 Elsevier B.V. All rights reserved.

Time-Lapse, in Situ Imaging of Ice Crystal Growth Using Confocal Microscopy

PubMed Central

2016-01-01

Ice crystals nucleate and grow when a water solution is cooled below its freezing point. The growth velocities and morphologies of the ice crystals depend on many parameters, such as the temperature of ice growth, the melting temperature, and the interactions of solutes with the growing crystals. Three types of morphologies may appear: dendritic, cellular (or fingerlike), or the faceted equilibrium form. Understanding and controlling which type of morphology is formed is essential in several domains, from biology to geophysics and materials science. Obtaining, in situ, three dimensional observations without introducing artifacts due to the experimental technique is nevertheless challenging. Here we show how we can use laser scanning confocal microscopy to follow in real-time the growth of smoothed and faceted ice crystals in zirconium acetate solutions. Both qualitative and quantitative observations can be made. In particular, we can precisely measure the lateral growth velocity of the crystals, a measure otherwise difficult to obtain. Such observations should help us understand the influence of the parameters that control the growth of ice crystals in various systems. PMID:27917410

Confocal Raman microscopy of one dimensional ZnO nanostructures

NASA Astrophysics Data System (ADS)

Singamaneni, Srikanth; Gupta, Maneesh; Yang, Rusen; Wang, Zhong; Tsukruk, Vladimir

2009-03-01

ZnO nanostructures with various shapes (vertically aligned nanorods, nanobelts, nanohelixes, nanorings) have been synthesized using both vapor phase and solution growth methods. In the simplest example of a nanobelt, the fast growth direction can be either (21 1 0) or (011 0) or (0001). Here, we show that confocal Raman microscopy can be employed as a fast and nondestructive analytical technique to identify the crystal planes and reveal the relative orientation of the ZnO nanostructure. Various features of the Raman spectrum of ZnO nanostructures (presence of the A1(TO) mode, width of the E2 mode) were found to be sensitive to relative orientation of the incident source laser and the crystal plane. Furthermore, owing to the optical anisotropy of ZnO, Raman scattering from the substrate is modulated (either enhanced or suppressed with respect to the background) depending on the polarization of the incident light with respect to orientation of the nanobelt. The results presented here describe a novel method to nondestructively identify the growth, relative orientation, and the waveguiding properties of the ZnO nanostructures.

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

NASA Astrophysics Data System (ADS)

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

2009-01-01

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

Time-Lapse, in Situ Imaging of Ice Crystal Growth Using Confocal Microscopy.

PubMed

Marcellini, Moreno; Noirjean, Cecile; Dedovets, Dmytro; Maria, Juliette; Deville, Sylvain

2016-11-30

Ice crystals nucleate and grow when a water solution is cooled below its freezing point. The growth velocities and morphologies of the ice crystals depend on many parameters, such as the temperature of ice growth, the melting temperature, and the interactions of solutes with the growing crystals. Three types of morphologies may appear: dendritic, cellular (or fingerlike), or the faceted equilibrium form. Understanding and controlling which type of morphology is formed is essential in several domains, from biology to geophysics and materials science. Obtaining, in situ, three dimensional observations without introducing artifacts due to the experimental technique is nevertheless challenging. Here we show how we can use laser scanning confocal microscopy to follow in real-time the growth of smoothed and faceted ice crystals in zirconium acetate solutions. Both qualitative and quantitative observations can be made. In particular, we can precisely measure the lateral growth velocity of the crystals, a measure otherwise difficult to obtain. Such observations should help us understand the influence of the parameters that control the growth of ice crystals in various systems.

Confocal laser scanning microscopic photoconversion: a new method to stabilize fluorescently labeled cellular elements for electron microscopic analysis.

PubMed

Colello, Raymond J; Tozer, Jordan; Henderson, Scott C

2012-01-01

Photoconversion, the method by which a fluorescent dye is transformed into a stable, osmiophilic product that can be visualized by electron microscopy, is the most widely used method to enable the ultrastructural analysis of fluorescently labeled cellular structures. Nevertheless, the conventional method of photoconversion using widefield fluorescence microscopy requires long reaction times and results in low-resolution cell targeting. Accordingly, we have developed a photoconversion method that ameliorates these limitations by adapting confocal laser scanning microscopy to the procedure. We have found that this method greatly reduces photoconversion times, as compared to conventional wide field microscopy. Moreover, region-of-interest scanning capabilities of a confocal microscope facilitate the targeting of the photoconversion process to individual cellular or subcellular elements within a fluorescent field. This reduces the area of the cell exposed to light energy, thereby reducing the ultrastructural damage common to this process when widefield microscopes are employed. © 2012 by John Wiley & Sons, Inc.

Tackling the Challenges of Dynamic Experiments Using Liquid-Cell Transmission Electron Microscopy.

PubMed

Parent, Lucas R; Bakalis, Evangelos; Proetto, Maria; Li, Yiwen; Park, Chiwoo; Zerbetto, Francesco; Gianneschi, Nathan C

2018-01-16

Revolutions in science and engineering frequently result from the development, and wide adoption, of a new, powerful characterization or imaging technique. Beginning with the first glass lenses and telescopes in astronomy, to the development of visual-light microscopy, staining techniques, confocal microscopy, and fluorescence super-resolution microscopy in biology, and most recently aberration-corrected, cryogenic, and ultrafast (4D) electron microscopy, X-ray microscopy, and scanning probe microscopy in nanoscience. Through these developments, our perception and understanding of the physical nature of matter at length-scales beyond ordinary perception have been fundamentally transformed. Despite this progression in microscopy, techniques for observing nanoscale chemical processes and solvated/hydrated systems are limited, as the necessary spatial and temporal resolution presents significant technical challenges. However, the standard reliance on indirect or bulk phase characterization of nanoscale samples in liquids is undergoing a shift in recent times with the realization ( Williamson et al. Nat. Mater . 2003 , 2 , 532 - 536 ) of liquid-cell (scanning) transmission electron microscopy, LC(S)TEM, where picoliters of solution are hermetically sealed between electron-transparent "windows," which can be directly imaged or videoed at the nanoscale using conventional transmission electron microscopes. This Account seeks to open a discussion on the topic of standardizing strategies for conducting imaging experiments with a view to characterizing dynamics and motion of nanoscale materials. This is a challenge that could be described by critics and proponents alike, as analogous to doing chemistry in a lightning storm; where the nature of the solution, the nanomaterial, and the dynamic behaviors are all potentially subject to artifactual influence by the very act of our observation.

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.

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.

In Vivo Confocal Microscopy of the Ocular Surface: From Bench to Bedside

PubMed Central

Villani, Edoardo; Baudouin, Christophe; Efron, Nathan; Hamrah, Pedram; Kojima, Takashi; Patel, Sanjay V.; Pflugfelder, Stephen C.; Zhivov, Andrey; Dogru, Murat

2014-01-01

In vivo confocal microscopy (IVCM) is an emerging technology that provides minimally invasive, high resolution, steady-state assessment of the ocular surface at the cellular level. Several challenges still remain but, at present, IVCM may be considered a promising technique for clinical diagnosis and management. This mini-review summarizes some key findings in IVCM of the ocular surface, focusing on recent and promising attempts to move “from bench to bedside”. IVCM allows prompt diagnosis, disease course follow-up, and management of potentially blinding atypical forms of infectious processes, such as acanthamoeba and fungal keratitis. This technology has improved our knowledge of corneal alterations and some of the processes that affect the visual outcome after lamellar keratoplasty and excimer keratorefractive surgery. In dry eye disease, IVCM has provided new information on the whole-ocular surface morphofunctional unit. It has also improved understanding of pathophysiologic mechanisms and helped in the assessment of prognosis and treatment. IVCM is particularly useful in the study of corneal nerves, enabling description of the morphology, density, and disease- or surgically induced alterations of nerves, particularly the subbasal nerve plexus. In glaucoma, IVCM constitutes an important aid to evaluate filtering blebs, to better understand the conjunctival wound healing process, and to assess corneal changes induced by topical antiglaucoma medications and their preservatives. IVCM has significantly enhanced our understanding of the ocular response to contact lens wear. It has provided new perspectives at a cellular level on a wide range of contact lens complications, revealing findings that were not previously possible to image in the living human eye. The final section of this mini-review provides a focus on advances in confocal microscopy imaging. These include 2D wide-field mapping, 3D reconstruction of the cornea and automated image analysis. PMID:24215436

Preparation and characterization of ultraflat Pt facets by atom-height-resolved differential optical microscopy

NASA Astrophysics Data System (ADS)

Azhagurajan, M.; Wen, R.; Kim, Y. G.; Itoh, T.; Sashikata, K.; Itaya, K.

2015-01-01

We recently demonstrated that improvements to our technique, laser confocal microscopy with differential interference microscopy (LCM-DIM), has rendered it fully capable of resolving monatomic steps with heights of ca. 0.25 nm on Au(111) and Pd(111) surfaces, even as low as 0.14 nm on Si(100), in aqueous solution. In this paper, we describe in detail a method to prepare and characterize, via atomic-layer-resolved LCM-DIM, ultraflat Pt(111) and Pt(100) facets over a wide surface area. The preparation of ultraflat surfaces is important in the characterization at the atomic scale of electrochemical processes under reaction conditions. To showcase the elegance of LCM-DIM, the anodic dissolution of Pt in aqueous HCl is briefly recounted.

Micromirror structured illumination microscope for high-speed in vivo drosophila brain imaging.

PubMed

Masson, A; Pedrazzani, M; Benrezzak, S; Tchenio, P; Preat, T; Nutarelli, D

2014-01-27

Genetic tools and especially genetically encoded fluorescent reporters have given a special place to optical microscopy in drosophila neurobiology research. In order to monitor neural networks activity, high speed and sensitive techniques, with high spatial resolution are required. Structured illumination microscopies are wide-field approaches with optical sectioning ability. Despite the large progress made with the introduction of the HiLo principle, they did not meet the criteria of speed and/or spatial resolution for drosophila brain imaging. We report on a new implementation that took advantage of micromirror matrix technology to structure the illumination. Thus, we showed that the developed instrument exhibits a spatial resolution close to that of confocal microscopy but it can record physiological responses with a speed improved by more than an order a magnitude.

Decalin-assisted light emitting porous Si formation and its optical, surface and morphological properties

NASA Astrophysics Data System (ADS)

Karatutlu, Ali; Istengir, Sumeyra; Cosgun, Sedat; Seker, Isa; Unal, Bayram

2017-11-01

In this research paper, light emitting porous silicon (Lep-Si) samples were fabricated by a surfactant-mediated chemical stain etching solution in order to form homogenous luminescent nanostructures at room temperature. As an industrially important solvent, decalin (decahydronaphtalene) was used as a surfactant in the HF/HNO3 solutions in order to control the etching process. Morphological, surface and optical properties of the Lep-Si samples were examined using atomic force microscopy, X-ray photoelectron spectroscopy, photoluminescence (PL) spectroscopy, and laser scanning confocal microscopy (LSCM) techniques. These characterization techniques were correlated with the various etching times including depth dependent luminescence profiles for the first time. We report the optimum conditions for production of the most efficient Lep-Si using decalin (decahydronaphtalene) and possible structural origins of light emission using the depth dependent luminescence measurements.

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.

Carbon Dioxide Laser Ablation of Basal Cell Carcinoma with Visual Guidance by Reflectance Confocal Microscopy: A Proof of Principle Pilot Study

PubMed Central

Hibler, B.P.; Sierra, H.; Cordova, M.; Phillips, W.; Rajadhyaksha, M.; Nehal, K.S.; Rossi, A.M.

2016-01-01

Background Laser ablation is an alternative, non-surgical treatment modality for low-risk basal cell carcinoma (BCC); however, lack of confirmative tumour destruction or residual tumour presence has been a limiting factor to adoption. Reflectance confocal microscopy (RCM) provides non-invasive, cellular-level resolution imaging of the skin and is capable of identifying tumour. Objective To evaluate the use of RCM to guide carbon dioxide (CO2) laser ablation of BCC, confirm destruction, and correlate findings with histology. Methods RCM was used pre-ablation to evaluate for features of BCC. Ablation was performed with a CO2 laser, and the response rapidly assessed using handheld RCM to evaluate for residual tumour. Confirmative pathology was used to verify confocal imaging. Results RCM imaging identified tumour pre-ablation with features not identified on normal, surrounding skin. Post-ablation, RCM documented complete removal of tumour in six cases and residual tumour in two. Histologic examination identified the ablated area and confirmed clearance of tumour in the six aforementioned cases and corroborated confocal findings for residual tumour in the other two cases. Conclusions We report successful treatment of superficial and nodular BCC using CO2 laser ablation augmented by RCM imaging for pre-ablation guidance and verification of tumour removal post-ablation. Akin to complete circumferential and deep margin control techniques, using RCM helps to map peripheral and deep BCC margins to hone in on areas exhibiting persistent tumour after ablation. CO2 laser ablation visually guided by RCM can help circumvent previously cited limiting factors of laser ablation for tumour destruction by providing cellular-level resolution imaging of tumour and margin assessment in between each laser pass and post-ablation. PMID:26800657

Carbon dioxide laser ablation of basal cell carcinoma with visual guidance by reflectance confocal microscopy: a proof-of-principle pilot study.

PubMed

Hibler, B P; Sierra, H; Cordova, M; Phillips, W; Rajadhyaksha, M; Nehal, K S; Rossi, A M

2016-06-01

Laser ablation is an alternative, nonsurgical treatment modality for low-risk basal cell carcinoma (BCC). However, lack of confirmative tumour destruction or residual tumour presence has been a limiting factor to its adoption. Reflectance confocal microscopy (RCM) provides noninvasive, cellular-level resolution imaging of the skin and is capable of identifying tumour. To evaluate the use of RCM to guide carbon dioxide (CO2 ) laser ablation of BCC, confirm destruction and correlate findings with histology. RCM was used preablation to evaluate for features of BCC. Ablation was performed with a CO2 laser, and the response rapidly assessed using handheld RCM to evaluate for residual tumour. Confirmative pathology was used to verify confocal imaging. Preablation RCM imaging identified tumour with features not identified on normal, surrounding skin. Postablation, RCM documented complete removal of tumour in six cases and residual tumour in two. Histological examination identified the ablated area and confirmed clearance of tumour in the six aforementioned cases and corroborated confocal findings for residual tumour in the other two cases. We report successful treatment of superficial and nodular BCC using CO2 laser ablation augmented by RCM imaging for preablation guidance and verification of tumour removal postablation. Akin to complete circumferential and deep margin control techniques, using RCM helps to map peripheral and deep BCC margins to hone in on areas exhibiting persistent tumour after ablation. CO2 laser ablation visually guided by RCM can help circumvent previously cited limiting factors of laser ablation for tumour destruction by providing cellular-level resolution imaging of tumour and margin assessment in between each laser pass and postablation. © 2016 British Association of Dermatologists.

Cytocompatibility and uptake of halloysite clay nanotubes.

PubMed

Vergaro, Viviana; Abdullayev, Elshad; Lvov, Yuri M; Zeitoun, Andre; Cingolani, Roberto; Rinaldi, Ross; Leporatti, Stefano

2010-03-08

Halloysite is aluminosilicate clay with hollow tubular structure of 50 nm external diameter and 15 nm diameter lumen. Halloysite biocompatibility study is important for its potential applications in polymer composites, bone implants, controlled drug delivery, and for protective coating (e.g., anticorrosion or antimolding). Halloysite nanotubes were added to different cell cultures for toxicity tests. Its fluorescence functionalization by aminopropyltriethosilane (APTES) and with fluorescently labeled polyelectrolyte layers allowed following halloysite uptake by the cells with confocal laser scanning microscopy (CLSM). Quantitative Trypan blue and MTT measurements performed with two neoplastic cell lines model systems as a function of the nanotubes concentration and incubation time indicate that halloysite exhibits a high level of biocompatibility and very low cytotoxicity, rendering it a good candidate for household materials and medicine. A combination of transmission electron microscopy (TEM), scanning electron microscopy (SEM), and scanning force microscopy (SFM) imaging techniques have been employed to elucidate the structure of halloysite nanotubes.

Corneal collagen cross-linking: a confocal, electron, and light microscopy study of eye bank corneas.

PubMed

Dhaliwal, Jasmeet S; Kaufman, Stephen C

2009-01-01

The purpose of this study was to evaluate morphological changes induced by corneal collagen cross-linking in a human ex vivo cornea, using confocal, electron, and light microscopy. The central epithelium was partially removed from ex vivo human corneal buttons. Riboflavin 0.1% solution was applied before ultraviolet A light treatment and then for every 2 minutes for 30 minutes while the corneas were exposed to ultraviolet A light at a wavelength of 370 nm and intensity of 3 mW/cm(2). Each cornea was evaluated using confocal, electron, and light microscopy. Confocal microscopy demonstrated normal-appearing corneas on their initial pretreatment examination, with reduced stromal detail. After treatment, a superficial layer of highly reflective spherical structures (4-10 microm) was observed. Many of these hyperreflective structures appeared up to a depth of 300 microm. The remainder of the corneal stroma and endothelium appeared normal. Electron microscopy showed keratocyte apoptotic changes to a depth of 300 microm. No observable pathologic changes were seen on histology. Based on clinical studies, corneal cross-linking is a promising treatment that appears to be safe and to halt ectatic corneal disease progression. Initial European studies used animal models to extrapolate human protocols. In conjunction with clinical studies, we believe that human ex vivo corneal studies provide a means to evaluate the structural and morphological changes associated with this procedure, within human corneas, in a manner that cannot be accomplished in vivo.

The Use of Intravital Two-Photon and Thick Section Confocal Imaging to Analyze B Lymphocyte Trafficking in Lymph Nodes and Spleen.

PubMed

Park, Chung; Hwang, Il-Young; Kehrl, John H

2018-01-01

Intravital two-photon laser scanning microscopy (TP-LSM) has allowed the direct observation of immune cells in intact organs of living animals. In the B cell biology field TP-LSM has detailed the movement of B cells in high endothelial venules and during their transmigration into lymph organs; described the movement and positioning of B cells within lymphoid organs; outlined the mechanisms by which antigen is delivered to B cells; observed B cell interacting with T cells, other cell types, and even with pathogens; and delineated the egress of B cells from the lymph node (LN) parenchyma into the efferent lymphatics. As the quality of TP-LSM improves and as new fluorescent probes become available additional insights into B cell behavior and function await new investigations. Yet intravital TP-LSM has some disadvantages including a lower resolution than standard confocal microscopy, a narrow imaging window, and a shallow depth of imaging. We have found that supplementing intravital TP-LSM with conventional confocal microscopy using thick LN sections helps to overcome some of these shortcomings. Here, we describe procedures for visualizing the behavior and trafficking of fluorescently labeled, adoptively transferred antigen-activated B cells within the inguinal LN of live mice using two-photon microscopy. Also, we introduce procedures for fixed thick section imaging using standard confocal microscopy, which allows imaging of fluorescently labeled cells deep in the LN cortex and in the spleen with high resolution.

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

Rapid diagnosis of tinea incognito using handheld reflectance confocal microscopy: a paradigm shift in dermatology?

PubMed

Navarrete-Dechent, Cristián; Bajaj, Shirin; Marghoob, Ashfaq A; Marchetti, Michael A

2015-06-01

Dermatophytoses are common skin infections. Traditional diagnostic tests such as skin scrapings for light microscopy examination, fungal cultures and biopsies remain imperfect due to false-negative test results, cost, time required to perform the procedure, time delays in test results and/or a requirement for an invasive procedure. Herein, we present a case of an 80-year-old female whose tinea incognito was non-invasively diagnosed within seconds using handheld reflectance confocal microscopy (RCM). As non-invasive skin imaging continues to improve, we expect light-based office microscopy to be replaced with technologies such as RCM, which has multiple and continually expanding diagnostic applications. © 2015 Blackwell Verlag GmbH.

Application of confocal laser microscopy for monitoring mesh implants in herniology

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

Zakharov, V P; Belokonev, V I; Bratchenko, I A

2011-04-30

The state of the surface of mesh implants and their encapsulation region in herniology is investigated by laser confocal microscopy. A correlation between the probability of developing relapses and the size and density of implant microdefects is experimentally shown. The applicability limits of differential reverse scattering for monitoring the post-operation state of implant and adjacent tissues are established based on model numerical experiments. (optical technologies in biophysics and medicine)

Discovery of a New Cellular Motion and Its Relevance to Breast Cancer and Involution

DTIC Science & Technology

2014-02-01

motion (CAMo), live cell imaging , confocal microscopy Overall Project Summary: During this first year of funding we have concentrated our work to...cell types in 3D cultures and in vivo. Subtask 1.1a: Real time live cell imaging using confocal microscopy will be used to image cellular movement...exciting as they are important steps in understanding behavior of normal myoepithelial cells using live cell imaging in physiologically

ATP concentration as possible marker of liver damage at leukaemia treatment: confocal microscopy-based experimental study and numerical simulations

NASA Astrophysics Data System (ADS)

Malashchenko, V.; Zyubin, A.; Babak, S.; Lavrova, A.

2017-04-01

We consider the method of confocal microscopy as a convenient instrument for determination of chemical compounds in biological tissues and cells. In particular, we study the dynamics of adenosine triphosphate (ATP) concentration that could be used as a bio-marker of energy metabolism pathologies at the treatment of acute lymphoblastic leukaemia (ALL). On the basis of data obtained by the confocal microscopy, the values of ATP concentration have been calculated for each case. Possible correlations with other characteristics of pathology processes obtained from plasma of leukemia patients show that ATP value could be a prognostic factor of the treatment success. The role of ATP in the drug metabolism switching is also discussed within the context of kinetic modelling of metabolism processes leading to the production of 6-Thioguanosine monophosphate, which is a principal acting agent in chemotherapy.

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.

Evaluation of human sclera after femtosecond laser ablation using two photon and confocal microscopy

NASA Astrophysics Data System (ADS)

Sun, Hui; Kurtz, Ronald; Juhasz, Tibor

2012-08-01

Glaucoma is the second-leading cause of blindness worldwide and is often associated with elevated intraocular pressure (IOP). Partial thickness intrascleral channels can be created with a femtosecond laser operating at a wavelength of 1700 nm. Such channels have the potential to increase outflow facility and reduce elevated IOP. Analysis of the dimensions and location of these channels is important in understanding their effects. We describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in human cadaver eyes. High-resolution images, hundreds of microns deep in the sclera, were obtained to allow determination of the shape and dimension of such channels. This demonstrates that concept of integrating femtosecond laser surgery, and two-photon and confocal imaging has the future potential for image-guided high-precision surgery in transparent and translucent tissue.

Calcium oxalate crystal growth modification; investigations with confocal Raman microscopy

NASA Astrophysics Data System (ADS)

McMulkin, Calum J.; Massi, Massimiliano; Jones, Franca

2017-06-01

Confocal Raman Microscopy (CRM) in combination with a photophysical investigation has been employed to give insight into the interaction between calcium oxalate monohydrate (COM) and a series of tetrazole containing crystal growth modifier's (CGM's), in conjunction with characterisation of morphological changes using scanning electron and optical microscopy. The tetrazole CGM's were found to interact by surface adsorption with minimal morphological changes to the COM crystals however, significant interactions via chemisorption were observed; it was discovered that the chemisorption is sufficiently strong for aggregation of the tetrazole species to occur within the crystal during crystallisation.

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

Single-cell Raman and fluorescence microscopy reveal the association of lipid bodies with phagosomes in leukocytes

PubMed Central

van Manen, Henk-Jan; Kraan, Yvonne M.; Roos, Dirk; Otto, Cees

2005-01-01

Cellular imaging techniques based on vibrational spectroscopy have become powerful tools in cell biology because the molecular composition of subcellular compartments can be visualized without the need for labeling. Using high-resolution, nonresonant confocal Raman microscopy on individual cells, we demonstrate here that lipid bodies (LBs) rich in arachidonate as revealed by their Raman spectra associate with latex bead-containing phagosomes in neutrophilic granulocytes. This finding was corroborated in macrophages and in PLB-985 cells, which can be induced to differentiate into neutrophil-like cells, by selective staining of LBs and visualization by confocal fluorescence microscopy. We further show that the accumulation of LBs near phagosomes is mediated at least in part by the flavohemoprotein gp91phox (in which “phox” is phagocyte oxidase), because different LB distributions around phagocytosed latex beads were observed in WT and gp91phox-deficient PLB-985 cells. gp91phox, which accumulates in the phagosomal membrane, is the catalytic subunit of the leukocyte NADPH oxidase, a critical enzyme in the innate immune response. Finally, time-lapse fluorescence microscopy experiments on neutrophils revealed that the LB-phagosome association is transient, similar to the “kiss-and-run” behavior displayed by endosomes involved in phagosome maturation. Because arachidonic acid (AA) has been shown to be involved in NADPH oxidase activation and phagosome maturation in neutrophils and macrophages, respectively, the findings reported here suggest that LBs may provide a reservoir of AA for local activation of these essential leukocyte functions. PMID:16002471

Two-Photon Excitation, Fluorescence Microscopy, and Quantitative Measurement of Two-Photon Absorption Cross Sections

NASA Astrophysics Data System (ADS)

DeArmond, Fredrick Michael

As optical microscopy techniques continue to improve, most notably the development of super-resolution optical microscopy which garnered the Nobel Prize in Chemistry in 2014, renewed emphasis has been placed on the development and use of fluorescence microscopy techniques. Of particular note is a renewed interest in multiphoton excitation due to a number of inherent properties of the technique including simplified optical filtering, increased sample penetration, and inherently confocal operation. With this renewed interest in multiphoton fluorescence microscopy, comes an increased demand for robust non-linear fluorescent markers, and characterization of the associated tool set. These factors have led to an experimental setup to allow a systematized approach for identifying and characterizing properties of fluorescent probes in the hopes that the tool set will provide researchers with additional information to guide their efforts in developing novel fluorophores suitable for use in advanced optical microscopy techniques as well as identifying trends for their synthesis. Hardware was setup around a software control system previously developed. Three experimental tool sets were set up, characterized, and applied over the course of this work. These tools include scanning multiphoton fluorescence microscope with single molecule sensitivity, an interferometric autocorrelator for precise determination of the bandwidth and pulse width of the ultrafast Titanium Sapphire excitation source, and a simplified fluorescence microscope for the measurement of two-photon absorption cross sections. Resulting values for two-photon absorption cross sections and two-photon absorption action cross sections for two standardized fluorophores, four commercially available fluorophores, and ten novel fluorophores are presented as well as absorption and emission spectra.

Resin-dentin interface of Scotchbond Multi-Purpose dentin adhesive.

PubMed

Griffiths, B M; Watson, T F

1995-08-01

To evaluate the interface between dentin, Scotchbond Multi-Purpose (SMP), and resin composite (Z100) using confocal fluorescence microscopy. Novel techniques were employed to view and record at video rate the application of the SMP primer and adhesive onto the dentin surface. The subsurface morphology of the dentin/restorative interface was also studied. To facilitate this study, the components of the adhesive system were labeled with fluorescent dyes. In an ideal situation, the primer and adhesive penetrated the dentin tubules, a distinct hybrid zone was formed and a thin layer of adhesive was observed at the interfacial region. However, the primed dentin surface was sensitive to disruption by the adhesive application brush during its placement, so that parts of the primer layer could be incorporated into the adhesive. This disruption could only be seen using confocal microscopy and a fluorescence labeling technique. Delamination of the primer layer was not observed when the adhesive film was thinned by air blowing. The application of an air stream to the cavity surface increased the penetration of the primer and adhesive along the dentin tubules, but also increased the thickness of the adhesive within irregularities on the cavity surface and at the cavity line angle. The cause of the problems in the handling properties of SMP may be the difference in viscosity between the two components (primer and adhesive) at the adhesive interface.

Impact of Dermoscopy and Reflectance Confocal Microscopy on the Histopathologic Diagnosis of Lentigo Maligna/Lentigo Maligna Melanoma.

PubMed

Mataca, Ema; Migaldi, Mario; Cesinaro, Anna M

2018-06-15

Equivocal pigmented lesions of the head are usually biopsied to avoid inappropriate treatment. Clinical approach has evolved from simple visual examination to sophisticated techniques for selecting the biopsy sites. This study aimed to retrospectively evaluate the efficiency of dermoscopy (DE) and reflectance confocal microscopy (RCM) in sampling a histopathologically representative focus of lentigo maligna/lentigo maligna melanoma. Punch biopsies and surgical excisions of 72 patients, 37 men and 35 women (median age 70.6 years, range 39-90 years), affected by lentigo maligna/lentigo maligna melanoma of the head, sent from a single dermatology clinic, were reviewed for the presence of 5 histopathologic criteria: atypical junctional melanocytes, increased junctional melanocytes, follicular colonization, pagetoid spread and melanocytic junctional nests, plus other minor features. Forty-two patients were biopsied under DE and 30 under RCM guidance. Accuracy of the 2 techniques in sampling a representative tissue overlapped in most cases, although RCM selected sites to biopsy with more histopathologic criteria, in particular pagetoid spread and melanocytic nests. Interestingly, with RCM, inflammation and melanophages were observed more in biopsy than in excision. False positive cases were not registered. Compared with the sampling at naked eye, our results show that DE and RCM help selecting the most appropriate areas for biopsies, thus allowing not only more robust histopathologic diagnoses, but also a more accurate microstaging of tumor.

In vivo assessment of the structure of skin microcirculation by reflectance confocal-laser-scanning microscopy

NASA Astrophysics Data System (ADS)

Sugata, Keiichi; Osanai, Osamu; Kawada, Hiromitsu

2012-02-01

One of the major roles of the skin microcirculation is to supply oxygen and nutrition to the surrounding tissue. Regardless of the close relationship between the microcirculation and the surrounding tissue, there are few non-invasive methods that can evaluate both the microcirculation and its surrounding tissue at the same site. We visualized microcapillary plexus structures in human skin using in vivo reflectance confocal-laser-scanning microscopy (CLSM), Vivascope 3000® (Lucid Inc., USA) and Image J software (National Institutes of Health, USA) for video image processing. CLSM is a non-invasive technique that can visualize the internal structure of the skin at the cellular level. In addition to internal morphological information such as the extracellular matrix, our method reveals capillary structures up to the depth of the subpapillary plexus at the same site without the need for additional optical systems. Video images at specific depths of the inner forearm skin were recorded. By creating frame-to-frame difference images from the video images using off-line video image processing, we obtained images that emphasize the brightness depending on changes of intensity coming from the movement of blood cells. Merging images from different depths of the skin elucidates the 3-dimensional fine line-structure of the microcirculation. Overall our results show the feasibility of a non-invasive, high-resolution imaging technique to characterize the skin microcirculation and the surrounding tissue.

The Use of Spinning-Disk Confocal Microscopy for the Intravital Analysis of Platelet Dynamics in Response to Systemic and Local Inflammation

PubMed Central

Jenne, Craig N.; Wong, Connie H. Y.; Petri, Björn; Kubes, Paul

2011-01-01

Platelets are central players in inflammation and are an important component of the innate immune response. The ability to visualize platelets within the live host is essential to understanding their role in these processes. Past approaches have involved adoptive transfer of labelled platelets, non-specific dyes, or the use of fluorescent antibodies to tag platelets in vivo. Often, these techniques result in either the activation of the platelet, or blockade of specific platelet receptors. In this report, we describe two new methods for intravital visualization of platelet biology, intravenous administration of labelled anti-CD49b, which labels all platelets, and CD41-YFP transgenic mice, in which a percentage of platelets express YFP. Both approaches label endogenous platelets and allow for their visualization using spinning-disk confocal fluorescent microscopy. Following LPS-induced inflammation, we were able to measure a significant increase in both the number and size of platelet aggregates observed within the vasculature of a number of different tissues. Real-time observation of these platelet aggregates reveals them to be large, dynamic structures that are continually expanding and sloughing-off into circulation. Using these techniques, we describe for the first time, platelet recruitment to, and behaviour within numerous tissues of the mouse, both under control conditions and following LPS induced inflammation. PMID:21949865

The Intracellular Trafficking Pathway of Transferrin

PubMed Central

Mayle, Kristine M.; Le, Alexander M.; Kamei, Daniel T.

2011-01-01

Background Transferrin (Tf) is an iron-binding protein that facilitates iron-uptake in cells. Iron-loaded Tf first binds to the Tf receptor (TfR) and enters the cell through clathrin-mediated endocytosis. Inside the cell, Tf is trafficked to early endosomes, delivers iron, and then is subsequently directed to recycling endosomes to be taken back to the cell surface. Scope of Review We aim to review the various methods and techniques that researchers have employed for elucidating the Tf trafficking pathway and the cell-machinery components involved. These experimental methods can be categorized as microscopy, radioactivity, and surface plasmon resonance (SPR). Major Conclusions Qualitative experiments, such as total internal reflectance fluorescence (TIRF), electron, laser-scanning confocal, and spinning-disk confocal microscopy, have been utilized to determine the roles of key components in the Tf trafficking pathway. These techniques allow temporal resolution and are useful for imaging Tf endocytosis and recycling, which occur on the order of seconds to minutes. Additionally, radiolabeling and SPR methods, when combined with mathematical modeling, have enabled researchers to estimate quantitative kinetic parameters and equilibrium constants associated with Tf binding and trafficking. General Significance Both qualitative and quantitative data can be used to analyze the Tf trafficking pathway. The valuable information that is obtained about the Tf trafficking pathway can then be combined with mathematical models to identify design criteria to improve the ability of Tf to deliver anticancer drugs. PMID:21968002

Blinking correlation in nanocrystal quantum dots probed with novel laser scanning confocal microscopy methods

NASA Astrophysics Data System (ADS)

Hefti, Ryan Alf

Semiconductor quantum dots have a vast array of applications: as fluorescent labels in biological systems, as physical or chemical sensors, as components in photovoltaic technology, and in display devices. An attribute of nearly every quantum dot is its blinking, or fluorescence intermittency, which tends to be a disadvantage in most applications. Despite the fact that blinking has been a nearly universal phenomenon among all types of fluorescent constructs, it is more prevalent in quantum dots than in traditional fluorophores. Furthermore, no unanimously accepted model of quantum dot blinking yet exists. The work encompassed by this dissertation began with an in-depth study of molecular motor protein dynamics in a variety of environments using two specially developed techniques, both of which feature applicability to live cell systems. Parked-beam confocal microscopy was utilized to increase temporal resolution of molecular motor motion dynamics by an order of magnitude over other popular methods. The second technique, fast-scanning confocal microscopy (FSCM), was used for long range observation of motor proteins. While using FSCM on motor protein assays, we discovered an unusual phenomenon. Single quantum dots seemingly communicated with neighboring quantum dots, indicated by a distinct correlation in their blinking patterns. In order to explain this novel correlation phenomenon, the majority of blinking models developed thus far would suggest a dipole-dipole interaction or a Coulomb interaction between singly charged quantum dots. However, our results indicate that the interaction energy is higher than supported by current models, thereby prompting a renewed examination. We propose that the blinking correlation we observed is due to a Coulomb interaction on the order of 3-4 elementary charges per quantum dot and that multiple charging of individual quantum dots may be required to plunge them into a non-emissive state. As a result of charging, charge carriers are displaced into a wide distribution of trap sites in the surrounding matrix, resulting in the expected power-law probability distribution of off times ubiquitous in quantum dots. Our discovery also implies that quantum dot blinking can be controlled, advocating the creation of switchable nanoscale emitters.

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.

Applications of microscopy in Salmonella research.

PubMed

Malt, Layla M; Perrett, Charlotte A; Humphrey, Suzanne; Jepson, Mark A

2015-01-01

Salmonella enterica is a Gram-negative enteropathogen that can cause localized infections, typically resulting in gastroenteritis, or systemic infection, e.g., typhoid fever, in humans and many other animals. Understanding the mechanisms by which Salmonella induces disease has been the focus of intensive research. This has revealed that Salmonella invasion requires dynamic cross-talk between the microbe and host cells, in which bacterial adherence rapidly leads to a complex sequence of cellular responses initiated by proteins translocated into the host cell by a type 3 secretion system. Once these Salmonella-induced responses have resulted in bacterial invasion, proteins translocated by a second type 3 secretion system initiate further modulation of cellular activities to enable survival and replication of the invading pathogen. Elucidation of the complex and highly dynamic pathogen-host interactions ultimately requires analysis at the level of single cells and single infection events. To achieve this goal, researchers have applied a diverse range of microscopy techniques to analyze Salmonella infection in models ranging from whole animal to isolated cells and simple eukaryotic organisms. For example, electron microscopy and high-resolution light microscopy techniques such as confocal microscopy can reveal the precise location of Salmonella and its relationship to cellular components. Widefield light microscopy is a simpler approach with which to study the interaction of bacteria with host cells and often has advantages for live cell imaging, enabling detailed analysis of the dynamics of infection and cellular responses. Here we review the use of imaging techniques in Salmonella research and compare the capabilities of different classes of microscope to address specific types of research question. We also provide protocols and notes on some microscopy techniques used routinely in our own research.

A Pulse Coupled Neural Network Segmentation Algorithm for Reflectance Confocal Images of Epithelial Tissue

PubMed Central

Malik, Bilal H.; Jabbour, Joey M.; Maitland, Kristen C.

2015-01-01

Automatic segmentation of nuclei in reflectance confocal microscopy images is critical for visualization and rapid quantification of nuclear-to-cytoplasmic ratio, a useful indicator of epithelial precancer. Reflectance confocal microscopy can provide three-dimensional imaging of epithelial tissue in vivo with sub-cellular resolution. Changes in nuclear density or nuclear-to-cytoplasmic ratio as a function of depth obtained from confocal images can be used to determine the presence or stage of epithelial cancers. However, low nuclear to background contrast, low resolution at greater imaging depths, and significant variation in reflectance signal of nuclei complicate segmentation required for quantification of nuclear-to-cytoplasmic ratio. Here, we present an automated segmentation method to segment nuclei in reflectance confocal images using a pulse coupled neural network algorithm, specifically a spiking cortical model, and an artificial neural network classifier. The segmentation algorithm was applied to an image model of nuclei with varying nuclear to background contrast. Greater than 90% of simulated nuclei were detected for contrast of 2.0 or greater. Confocal images of porcine and human oral mucosa were used to evaluate application to epithelial tissue. Segmentation accuracy was assessed using manual segmentation of nuclei as the gold standard. PMID:25816131

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.

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

Miranda, Adelaide; De Beule, Pieter A. A., E-mail: pieter.de-beule@inl.int; Martins, Marco

Combined microscopy techniques offer the life science research community a powerful tool to investigate complex biological systems and their interactions. Here, we present a new combined microscopy platform based on fluorescence optical sectioning microscopy through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). The illumination scheme of the DSD microscope unit, contrary to standard single or multi-point confocal microscopes, provides a time-independent illumination of the AFM cantilever. This enables a distortion-free simultaneous operation of fluorescence optical sectioning microscopy and atomic force microscopy with standard probes. In this context, we discussmore » sample heating due to AFM cantilever illumination with fluorescence excitation light. Integration of a DSD fluorescence optical sectioning unit with an AFM platform requires mitigation of mechanical noise transfer of the spinning disk. We identify and present two solutions to almost annul this noise in the AFM measurement process. The new combined microscopy platform is applied to the characterization of a DOPC/DOPS (4:1) lipid structures labelled with a lipophilic cationic indocarbocyanine dye deposited on a mica substrate.« less

Fast evaluation of 69 basal cell carcinomas with ex vivo fluorescence confocal microscopy: criteria description, histopathological correlation, and interobserver agreement.

PubMed

Bennàssar, Antoni; Carrera, Cristina; Puig, Susana; Vilalta, Antoni; Malvehy, Josep

2013-07-01

Fluorescence confocal microscopy (FCM) represents a first step toward a rapid "bedside pathology" in the Mohs surgery setting and in other fields of general pathology. To describe and validate FCM criteria for the main basal cell carcinoma (BCC) subtypes and to demonstrate the overall agreement with classic pathologic analysis of hematoxylin-eosin-stained samples. DESIGN A total of 69 BCCs from 66 patients were prospectively imaged using ex vivo FCM. Confocal mosaics were evaluated in real time and compared with classic pathologic analysis. Department of Dermatology, Hospital Clínic of Barcelona, Barcelona, Spain, between November 2010 and July 2011. Patients with BCC attending the Mohs Surgery Unit. Presence or absence of BCC and histological subtype (superficial, nodular, and infiltrating) in the confocal mosaics. Eight criteria for BCC were described, evaluated, and validated. Although there were minor differences among BCC subtypes, the most BCC-defining criteria were peripheral palisading, clefting, nuclear pleomorphism, and presence of stroma. These criteria were validated with independent observers (κ values >0.7 [corrected] for most criteria). We herein propose, describe, and validate FCM criteria for BCC diagnosis. Fluorescence confocal microscopy is an attractive alternative to histopathologic analysis of frozen sections during Mohs surgery because large areas of freshly excised tissue can be assessed in real time without the need for tissue processing while minimizing labor and costs.

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

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.

The challenge of diagnosing seborrheic keratosis by reflectance confocal microscopy.

PubMed

Guo, A; Chen, J; Yang, C; Ding, Y; Zeng, Q; Tan, L

2018-05-24

Seborrheic keratosis (SK) is one of the most common skin tumors seen by dermatologists. It should be differentiated with many diseases, especially skin tumors. Reflectance confocal microscopy (RCM) has been applied for evaluation of SK. There are a few studies that describe the RCM of SK. The aim of the study was to find the challenge of diagnosing seborrheic keratosis by reflectance confocal microscopy. A total of 390 patients with a clinical suspicious diagnosis of seborrheic keratosis were enrolled in this study, and lesions from each patient were imaged with RCM. Thirty-seven of these patients performed a biopsy in order to be given a histological diagnosis. We retrospectively analyzed the outcomes of RCM diagnosis and histological diagnosis, and then found the RCM characteristics of biopsy-proven lesions. According to RCM images, 258 of 390 (66.2%) patients were diagnosed with SK, 97 of 390 (24.9%) patients could not be diagnosed by the dermatologist according to RCM. Of all 37 biopsied lesions, 23 were SK, 6 were actinic keratosis, 2 were basal cell carcinoma, and 2 were squamous cell carcinoma. It is challenge to diagnose seborrheic keratosis by reflectance confocal microscopy. It may due to the variable clinical and RCM appearances of SK, and limited depth of RCM. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Assessment of skin pigmentation by confocal microscopy: Influence of solar exposure and protection habits on cutaneous hyperchromias.

PubMed

Martini, Ana Paula M; Mercurio, Daiane G; Maia Campos, Patrícia M B G

2017-09-01

Cutaneous hyperchromias are disorders of skin pigmentation involving an increase of melanin production and its irregular accumulation in skin cells. It is known that the use of sunscreens helps to prevent changes in the skin pigmentation pattern, but the structural and morphological alterations that occur in the different types of hyperpigmentations need better elucidation. To assess the influence of solar exposure and protection habits on the pattern of skin pigmentation using reflectance confocal microscopy (RCM). Forty volunteers aged 18-39 years with skin hyperpigmentation participated in the study. Skin characterization was performed by imaging techniques and by assessing the habits of solar exposure and protection by applying questionnaires to the volunteers. RCM was used to record sequences of confocal sections at areas of interest and to examine cell shape and brightness in the basal cell layer of the lesion and in normal perilesional skin. Furthermore, high-resolution images were obtained for analysis of the spots. Sunlight influences the number and location of spots as the face of volunteers with higher solar exposure was covered with spots, whereas volunteers with less exposure had fewer spots located in the nose and cheeks region due to greater exposure of these areas to the sun. The data showed the importance of sun protection for preventing changes in the pattern of skin pigmentation, and RCM proved to be an important tool for skin characterization. © 2017 Wiley Periodicals, Inc.

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.

In vivo confocal microscopy for the oral cavity: Current state of the field and future potential.

PubMed

Maher, N G; Collgros, H; Uribe, P; Ch'ng, S; Rajadhyaksha, M; Guitera, P

2016-03-01

Confocal microscopy (CM) has been shown to correlate with oral mucosal histopathology in vivo. The purposes of this review are to summarize what we know so far about in vivo CM applications for oral mucosal pathologies, to highlight some current developments with CM devices relevant for oral applications, and to formulate where in vivo CM could hold further application for oral mucosal diagnosis and management. Ovid Medline® and/or Google® searches were performed using the terms 'microscopy, confocal', 'mouth neoplasms', 'mouth mucosa', 'leukoplakia, oral', 'oral lichen planus', 'gingiva', 'cheilitis', 'taste', 'inflammatory oral confocal', 'mucosal confocal' and 'confocal squamous cell oral'. In summary, inclusion criteria were in vivo use of any type of CM for the human oral mucosa and studies on normal or pathological oral mucosa. Experimental studies attempting to identify proteins of interest and microorganisms were excluded. In total 25 relevant articles were found, covering 8 main topics, including normal oral mucosal features (n=15), oral dysplasia or neoplasia (n=7), inflamed oral mucosa (n=3), taste impairment (n=3), oral autoimmune conditions (n=2), pigmented oral pathology/melanoma (n=1), delayed type hypersensitivity (n=1), and cheilitis glandularis (n=1). The evidence for using in vivo CM in these conditions is poor, as it is limited to mainly small descriptive studies. Current device developments for oral CM include improved probe design. The authors propose that future applications for in vivo oral CM may include burning mouth syndrome, intra-operative mapping for cancer surgery, and monitoring and targeted biopsies within field cancerization. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

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.

Microstructure of milk

USDA-ARS?s Scientific Manuscript database

The fat and protein in milk may be examined by scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy, and any bacteria present may be viewed by light microscopy. The fat exists as globules, the bulk of the protein is in the form of casein micelles, a...

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...

Hamstring Graft Technique for Stabilization of Canine Cranial Cruciate Ligament Deficient Stifles

PubMed Central

LOPEZ, MANDI J.; MARKEL, MARK D.; KALSCHEUR, VICKI; LU, YAN; MANLEY, PAUL A.

2007-01-01

Objective To investigate the harvest and application of hamstring grafts for canine cranial cruciate ligament (CrCL) reconstruction. Study Design Experimental study. Animals Four adult female hounds, weighing 26.3 ± 1.6 kg (mean ± SEM). Methods One stifle in each dog was randomly chosen for hamstring graft CrCL reconstruction after native CrCL transection. Arthroscopy was performed to evaluate graft integrity at 12 weeks. Gait analysis and stifle radiographs were performed preoperatively and up to 52 weeks after graft placement. Dogs were killed 12 (n = 2) or 52 weeks (n = 2) after CrCL reconstruction. Tissues were evaluated grossly and with light and confocal laser microscopy. Results Hamstring grafts were intact in all stifles at 12 weeks (n = 4) and 52 weeks (n = 2). Grossly, there was no osteoarthritis in stifles at 12 weeks and only chondrophytes along the trochlear ridges at 52 weeks. Minimal radiographic evidence of osteoarthritis developed in stifles with grafts during the study. Lameness in limbs with grafts resolved by 52 weeks. Graft tissue was highly vascular, ligamentized, and undergoing active remodeling at 12 weeks. Fifty-two weeks after graft placement, intraarticular graft tissue was well vascularized, mature, and encapsulated by synovium, and graft-bone interfaces were characterized by Sharpey’s fiber insertions. There was no evidence of graft necrosis using confocal laser microscopy at either time point. Conclusions The hamstring graft technique may be a viable method of canine CrCL reconstruction. Clinical Relevance Hamstring grafts may be an alternative technique for canine CrCL reconstruction. Further study is needed before clinical application. PMID:12866003

Scattering features for lung cancer detection in fibered confocal fluorescence microscopy images.

PubMed

Rakotomamonjy, Alain; Petitjean, Caroline; Salaün, Mathieu; Thiberville, Luc

2014-06-01

To assess the feasibility of lung cancer diagnosis using fibered confocal fluorescence microscopy (FCFM) imaging technique and scattering features for pattern recognition. FCFM imaging technique is a new medical imaging technique for which interest has yet to be established for diagnosis. This paper addresses the problem of lung cancer detection using FCFM images and, as a first contribution, assesses the feasibility of computer-aided diagnosis through these images. Towards this aim, we have built a pattern recognition scheme which involves a feature extraction stage and a classification stage. The second contribution relies on the features used for discrimination. Indeed, we have employed the so-called scattering transform for extracting discriminative features, which are robust to small deformations in the images. We have also compared and combined these features with classical yet powerful features like local binary patterns (LBP) and their variants denoted as local quinary patterns (LQP). We show that scattering features yielded to better recognition performances than classical features like LBP and their LQP variants for the FCFM image classification problems. Another finding is that LBP-based and scattering-based features provide complementary discriminative information and, in some situations, we empirically establish that performance can be improved when jointly using LBP, LQP and scattering features. In this work we analyze the joint capability of FCFM images and scattering features for lung cancer diagnosis. The proposed method achieves a good recognition rate for such a diagnosis problem. It also performs well when used in conjunction with other features for other classical medical imaging classification problems. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of imaging techniques to study the pathogenesis of biosafety level 2/3 infectious agents

PubMed Central

Rella, Courtney E.; Ruel, Nancy; Eugenin, Eliseo A.

2015-01-01

Despite significant advances in microbiology and molecular biology over the last decades, several infectious diseases remain global concerns, resulting in the death of millions of people worldwide each year. According to the Center for Disease Control (CDC) in 2012, there were 34 million people infected with HIV, 8.7 million new cases of tuberculosis, 500 million cases of hepatitis, and 50–100 million people infected with dengue. Several of these pathogens, despite high incidence, do not have reliable clinical detection methods. New or improved protocols have been generated to enhance detection and quantitation of several pathogens using high-end microscopy (light, confocal, and STORM microscopy) and imaging software. In the current manuscript, we discuss these approaches and the theories behind these methodologies. Thus, advances in imaging techniques will open new possibilities to discover therapeutic interventions to reduce or eliminate the devastating consequences of infectious diseases. PMID:24990818

Intracellular processing of poly(ethylene imine)/ribozyme complexes can be observed in living cells by using confocal laser scanning microscopy and inhibitor experiments.

PubMed

Merdan, Thomas; Kunath, Klaus; Fischer, Dagmar; Kopecek, Jindrich; Kissel, Thomas

2002-02-01

Critical steps in the subcellular processing of poly(ethylene imine)/nucleic acid complexes, especially endosomal/lysosomal escape, were visualized by using living cell confocal laser scanning microscopy (CSLM) to obtain an insight into their mechanism. Living cell confocal microscopy was used to examine the intracellular fate of poly(ethylene imine)/ribozyme and poly(L-lysine)/ribozyme complexes over time, in the presence of and without bafilomycin Al, a selective inhibitor of endosomal/lysosomal acidification. The compartment of complex accumulation was identified by confocal microscopy with a fluorescent acidotropic dye. To confirm microscopic data, luciferase reporter gene expression was determined under similar experimental conditions. Poly(ethylene imine)/ribozyme complexes accumulate in acidic vesicles, most probably lysosomes. Release of complexes occurs in a sudden event, very likely due to bursting of these organelles. After release, poly(ethylene imine) and ribozyme spread throughout the cell, during which slight differences in distribution between cytosol and nucleus are visible. No lysosomal escape was observed with poly(L-lysine)/ribozyme complexes or when poly(ethylene imine)/ ribozyme complexes were applied together with bafilomycin A1. Poly(ethylene imine)/plasmid complexes exhibited a high luciferase expression, which was reduced approximately 200-fold when lysosomal acidification was suppressed with bafilomycin A1. Our data provide, for the first time, direct experimental evidence for the escape of poly(ethylene imine)/nucleic acid complexes from the endosomal/lysosomal compartment. CLSM, in conjunction with living cell microscopy, is a promising tool for studying the subcellular fate of polyplexes in nucleic acid/gene delivery.

Confocal Raman characterization of different protein desorption behaviors from chromatographic particles.

PubMed

Xiao, Yuewu; Stone, Thomas; Moya, Wilson; Killian, Paul; Herget, Thomas

2014-01-21

Confocal Raman spectroscopy is a nondestructive analytical technique that combines the chemical information from vibrational spectroscopy with the spatial resolution of confocal microscopy. It was applied, for the first time, to measure protein desorption from chromatographic particles. Monoclonal antibody was loaded onto the Fractogel EMD SO3 (M) cation exchanger at either pH 5 or pH 4. Confocal Raman measurement suggests that only the protein loaded at pH 5 is able to release from chromatographic particles in the elution buffer. Detailed comparison of high-quality spectra indicates that, while proteins loaded at both pH values showed a predominant β-sheet conformation, protein loaded at pH 4 has a broader amide I band with more intensity in the >1680 cm(-1) region. This small but clear and reproducible amide I bandwidth increase is not observed for protein in the solution state at pH 4. No definitive assignment of the increased Raman intensity in the >1680 cm(-1) region could be made, but it might be related to structural changes involved in the association of protein molecules in the adsorbed state, which helps to explain the nearly 100% retention under elution conditions of the monoclonal antibody adsorbed at pH 4 in chromatographic particles.

An integrated instrumental setup for the combination of atomic force microscopy with optical spectroscopy.

PubMed

Owen, R J; Heyes, C D; Knebel, D; Röcker, C; Nienhaus, G U

2006-07-01

In recent years, the study of single biomolecules using fluorescence microscopy and atomic force microscopy (AFM) techniques has resulted in a plethora of new information regarding the physics underlying these complex biological systems. It is especially advantageous to be able to measure the optical, topographical, and mechanical properties of single molecules simultaneously. Here an AFM is used that is especially designed for integration with an inverted optical microscope and that has a near-infrared light source (850 nm) to eliminate interference between the optical experiment and the AFM operation. The Tip Assisted Optics (TAO) system consists of an additional 100 x 100-microm(2) X-Y scanner for the sample, which can be independently and simultaneously used with the AFM scanner. This allows the offset to be removed between the confocal optical image obtained with the sample scanner and the simultaneously acquired AFM topography image. The tip can be positioned exactly into the optical focus while the user can still navigate within the AFM image for imaging or manipulation of the sample. Thus the tip-enhancement effect can be maximized and it becomes possible to perform single molecule manipulation experiments within the focus of a confocal optical image. Here this is applied to simultaneous measurement of single quantum dot fluorescence and topography with high spatial resolution. (c) 2006 Wiley Periodicals, Inc.

Immobilization of cross linked Col-I-OPN bone matrix protein on aminolysed PCL surfaces enhances initial biocompatibility of human adipogenic mesenchymal stem cells (hADMSC)

NASA Astrophysics Data System (ADS)

Kim, Young-Hee; Jyoti, Md. Anirban; Song, Ho-Yeon

2014-06-01

In bone tissue engineering surface modification is considered as one of the important ways of fabricating successful biocompatible material. Addition of biologically active functionality on the surfaces has been tried for improving the overall biocompatibility of the system. In this study poly-ɛ-caprolactone film surfaces have been modified through aminolysis and immobilization process. Collagen type I (COL-I) and osteopontin (OPN), which play an important role in osteogenesis, was immobilized onto PCL films followed by aminolysis treatment using 1,6-hexanediamine. Characterization of animolysed and immobilized surfaces were done by a number techniques using scanning electron microscopy (SEM), FT-IR, XPS, ninhydrin staining, SDS-PAGE and confocal microscopy and compared between the modified and un-modified surfaces. Results of the successive experiments showed that aminolysis treatment was homogeneously achieved which helped to entrap or immobilize Col-I-OPN proteins on surfaces of PCL film. In vitro studies with human adipogenic mesenchymal stem cells (hADMSC) also confirmed the attachment and proliferation of cells was better in modified PCL surfaces than the unmodified surfaces. SEM, confocal microscopy and MTT assay showed a significant increase in cell spreading, attachment and proliferations on the biofunctionalized surfaces compared to the unmodified PCL surfaces at all-time points indicating the success of surface biofunctionalization.

DNA molecule stretching through thermo-electrophoresis and thermal convection in a heated converging-diverging microchannel.

PubMed

Hsieh, Shou-Shing; Chen, Jyun-Hong; Tsai, Cheng-Fung

2013-02-18

A novel DNA molecule stretching technique is developed and tested herein. Through a heated converging-diverging microchannel, thermal convection and thermophoresis induced by regional heating are shown to significantly elongate single DNA molecules; they are visualized via a confocal laser scanning microscopy. In addition, electrophoretic stretching is also implemented to examine the hybrid effect on the conformation and dynamics of single DNA molecules. The physical properties of the DNA molecules are secured via experimental measurements.

New diagnostics for melanoma detection: from artificial intelligence to RNA microarrays.

PubMed

Ahlgrimm-Siess, Verena; Laimer, Martin; Arzberger, Edith; Hofmann-Wellenhof, Rainer

2012-07-01

Early detection of melanoma remains crucial to ensuring a favorable prognosis. Dermoscopy and total body photography are well-established noninvasive aids that increase the diagnostic accuracy of dermatologists in their daily routine, beyond that of a naked-eye examination. New noninvasive diagnostic techniques, such as reflectance confocal microscopy, multispectral digital imaging and RNA microarrays, are currently being investigated to determine their utility for melanoma detection. This review presents emerging technologies for noninvasive melanoma diagnosis, and discusses their advantages and limitations.

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.

Confocal Raman and electronic microscopy studies on the topotactic conversion of calcium carbonate from Pomacea lineate shells into hydroxyapatite bioceramic materials in phosphate media.

PubMed

dePaula, S M; Huila, M F G; Araki, K; Toma, H E

2010-12-01

Conversion of Pomacea lineate shells into hydroxyapatite (HA) bioceramic materials was investigated by their in vitro treatment with phosphate solutions, at room temperature. Confocal Raman microscopy revealed that the conversion proceeds at distinct rates through the nacreous or periostracum sides of the shell. The conversion can be accelerated using powdered samples, yielding biocompatible materials of great interest in biomedicine. Copyright © 2010 Elsevier Ltd. All rights reserved.

Template confined synthesis of amorphous carbon nanotubes and its confocal Raman microscopy

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

Maity, Supratim; Roychowdhury, Tuhin; Chattopadhyay, Kalyan Kumar, E-mail: kalyan-chattopadhyay@yahoo.com

2014-04-24

Amorphous carbon nanotubes (aCNTs) were synthesized by AAO (anodic aluminum oxide) template at a temperature 500 °C in nitrogen atmosphere using the citric acid as a carbon source without the help of any catalyst particles. Morphological analysis of the as prepared samples was carried out by field emission scanning electron microscopy (FESEM). Confocal Raman imaging has been studied and an attempt has been made to find out the graphitic (sp{sup 2}) and disordered phase of the CNTs.

Confocal Raman spectroscopy and AFM for evaluation of sidewalls in type II superlattice FPAs

NASA Astrophysics Data System (ADS)

Rotter, T. J.; Busani, T.; Rathi, P.; Jaeckel, F.; Reyes, P. A.; Malloy, K. J.; Ukhanov, A. A.; Plis, E.; Krishna, S.; Jaime-Vasquez, M.; Baril, N. F.; Benson, J. D.; Tenne, D. A.

2015-06-01

We propose to utilize confocal Raman spectroscopy combined with high resolution atomic force microscopy (AFM) for nondestructive characterisation of the sidewalls of etched and passivated small pixel (24 μm×24 μm) focal plane arrays (FPA) fabricated using LW/LWIR InAs/GaSb type-II strained layer superlattice (T2SL) detector material. Special high aspect ratio Si and GaAs AFM probes, with tip length of 13 μm and tip aperture less than 7°, allow characterisation of the sidewall morphology. Confocal microscopy enables imaging of the sidewall profile through optical sectioning. Raman spectra measured on etched T2SL FPA single pixels enable us to quantify the non-uniformity of the mesa delineation process.

Micro-Raman spectroscopy as a tool for the characterization of silicon carbide in power semiconductor material processing

NASA Astrophysics Data System (ADS)

De Biasio, M.; Kraft, M.; Schultz, M.; Goller, B.; Sternig, D.; Esteve, R.; Roesner, M.

2017-05-01

Silicon carbide (SiC) is a wide band-gap semi-conductor material that is used increasingly for high voltage power devices, since it has a higher breakdown field strength and better thermal conductivity than silicon. However, in particular its hardness makes wafer processing difficult and many standard semi-conductor processes have to be specially adapted. We measure the effects of (i) mechanical processing (i.e. grinding of the backside) and (ii) chemical and thermal processing (i.e. doping and annealing), using confocal microscopy to measure the surface roughness of ground wafers and micro-Raman spectroscopy to measure the stresses induced in the wafers by grinding. 4H-SiC wafers with different dopings were studied before and after annealing, using depth-resolved micro-Raman spectroscopy to observe how doping and annealing affect: i.) the damage and stresses induced on the crystalline structure of the samples and ii.) the concentration of free electrical carriers. Our results show that mechanical, chemical and thermal processing techniques have effects on this semiconductor material that can be observed and characterized using confocal microscopy and high resolution micro Raman spectroscopy.

Spatial Distribution of Lead Iodide and Local Passivation on Organo-Lead Halide Perovskite.

PubMed

Chen, Sheng; Wen, Xiaoming; Yun, Jae S; Huang, Shujuan; Green, Martin; Jeon, Nam Joong; Yang, Woon Seok; Noh, Jun Hong; Seo, Jangwon; Seok, Sang Il; Ho-Baillie, Anita

2017-02-22

We identify nanoscale spatial distribution of PbI 2 on the (FAPbI 3 ) 0.85 (MAPbBr 3 ) 0.15 perovskite thin film and investigate the local passivation effect using confocal based optical microscopy of steady state and time-resolved photoluminescence (PL). Different from a typical scanning electron microscope (SEM) morphology study, confocal based PL spectroscopy and microscopy allow researchers to map the morphologies of both perovskite and PbI 2 grains simultaneously, by selectively detecting their characteristic fluorescent bands using band-pass filters. In this work, we compare the perovskite samples without and with excess PbI 2 incorporation and unambiguously reveal PbI 2 distribution for the PbI 2 -rich sample. In addition, using the nanoscale time-resolved PL technique we show that the PbI 2 -rich regions exhibit longer lifetime due to suppressed defect trapping, compared to the PbI 2 -poor regions. The measurement on the PbI 2 -rich sample indicates that the passivation effect of PbI 2 in perovskite film is effective, especially in localized regions. Hence, this finding is important for further improvement of the solar cells by considering the strategy of excess PbI 2 incorporation.

Measuring surface-area-to-volume ratios in soft porous materials using laser-polarized xenon interphase exchange nuclear magnetic resonance

NASA Technical Reports Server (NTRS)

Butler, J. P.; Mair, R. W.; Hoffmann, D.; Hrovat, M. I.; Rogers, R. A.; Topulos, G. P.; Walsworth, R. L.; Patz, S.

2002-01-01

We demonstrate a minimally invasive nuclear magnetic resonance (NMR) technique that enables determination of the surface-area-to-volume ratio (S/V) of soft porous materials from measurements of the diffusive exchange of laser-polarized 129Xe between gas in the pore space and 129Xe dissolved in the solid phase. We apply this NMR technique to porous polymer samples and find approximate agreement with destructive stereological measurements of S/V obtained with optical confocal microscopy. Potential applications of laser-polarized xenon interphase exchange NMR include measurements of in vivo lung function in humans and characterization of gas chromatography columns.

Functional photoacoustic microscopy of pH

NASA Astrophysics Data System (ADS)

Chatni, M. Rameez; Yao, Junjie; Danielli, Amos; Favazza, Christopher P.; Maslov, Konstantin I.; Wang, Lihong V.

2012-02-01

pH is a tightly regulated indicator of metabolic activity. In mammalian systems, imbalance of pH regulation may result from or result in serious illness. Even though the regulation system of pH is very robust, tissue pH can be altered in many diseases such as cancer, osteoporosis and diabetes mellitus. Traditional high-resolution optical imaging techniques, such as confocal microscopy, routinely image pH in cells and tissues using pH sensitive fluorescent dyes, which change their fluorescence properties with the surrounding pH. Since strong optical scattering in biological tissue blurs images at greater depths, high-resolution pH imaging is limited to penetration depths of 1mm. Here, we report photoacoustic microscopy (PAM) of commercially available pH-sensitive fluorescent dye in tissue phantoms. Using both opticalresolution photoacoustic microscopy (OR-PAM), and acoustic resolution photoacoustic microscopy (AR-PAM), we explored the possibility of recovering the pH values in tissue phantoms. In this paper, we demonstrate that PAM was capable of recovering pH values up to a depth of 2 mm, greater than possible with other forms of optical microscopy.

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.

In vitro excystation of Echinostoma paraensei (Digenea: Echinostomatidae) metacercariae assessed by light microscopy, morphometry and confocal laser scanning microscopy.

PubMed

Souza, Joyce; Garcia, Juberlan; Neves, Renata H; Machado-Silva, José Roberto; Maldonado, Arnaldo

2013-12-01

Trypsin and bile salts have been identified as important triggers for excystation of Echinostoma metacercariae. Although excystation in trematodes is a well-known phenomenon, some morphological developmental changes remain to be elucidated. In order to gain further insight into the in vitro development of metacercariae, we assayed different cultivating conditions: 0.5% trypsin and 0.5% bile salts; 1% trypsin and 1% bile salts; 1% trypsin and 0.5% bile salts; 0.5% bile salts; or 0.5% trypsin. By means of light microscopy and confocal microscopy, we characterized each encysted, activated, breached and excysted stage based on the morphological features. However, breached and excysted stages were not revealed in both bile salts and trypsin-free medium. Excretory concretions (25 ± 3.9) were visualized within excretory tubules, close to the ventral sucker and genital anlage. The oral sucker armed with spines and digestive system was similar to those of adult worms. The reproductive system is composed of a genital anlage and the cirrus sac primordium. In short, trypsin and bile salts associated were fundamental for the in vitro metacercariae excystation of Echinostoma paraensei. This article presents the first detailed information of all stages of metacercariae excystation obtained through light and confocal microscopy. Copyright © 2013. Published by Elsevier Inc.

Investigation of the nanodomain structure formation by piezoelectric force microscopy and Raman confocal microscopy in LiNbO3 and LiTaO3 crystals

NASA Astrophysics Data System (ADS)

Shur, V. Ya.; Zelenovskiy, P. S.; Nebogatikov, M. S.; Alikin, D. O.; Sarmanova, M. F.; Ievlev, A. V.; Mingaliev, E. A.; Kuznetsov, D. K.

2011-09-01

Piezoelectric force microscopy (PFM) and Raman confocal microscopy have been used for studying the nanodomain structures in congruent LiNbO3 and LiTaO3 crystals. The high-resolution nanodomain images at the surface were observed via PFM. Raman confocal microscopy has been used for the visualization of the nanodomain structures in the bulk via layer-by-layer scanning at various depths. It has been shown experimentally that the nanodomain images obtained at different depths correspond to domain images at the polar surface obtained at different moments: the deeper the nanodomain, the earlier the moment. Such a correlation was applied for the reconstruction of the evolution of the domain structures with charged domain walls. The studied domain structures were obtained in highly non-equilibrium switching conditions realized in LiNbO3 and LiTaO3 via pulse laser irradiation and the electric field poling of LiNbO3, with the surface layer modified by ion implantation. The revealed main stages of the domain structure evolution allow the authors to demonstrate that all geometrically different nanodomain structures observed in LiNbO3 and LiTaO3 appeared as a result of discrete switching.

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. ...

Multi-modal Registration for Correlative Microscopy using Image Analogies

PubMed Central

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

2014-01-01

Correlative microscopy is a methodology combining the functionality of light microscopy with the high resolution of electron microscopy and other microscopy technologies for the same biological specimen. In this paper, we propose an image registration method for correlative microscopy, which is challenging due to the distinct appearance of biological structures when imaged with different modalities. Our method is based on image analogies and allows to transform images of a given modality into the appearance-space of another modality. Hence, the registration between two different types of microscopy images can be transformed to a mono-modality image registration. We use a sparse representation model to obtain image analogies. The method makes use of corresponding image training patches of two different imaging modalities to learn a dictionary capturing appearance relations. We test our approach on backscattered electron (BSE) scanning electron microscopy (SEM)/confocal and transmission electron microscopy (TEM)/confocal images. We perform rigid, affine, and deformable registration via B-splines and show improvements over direct registration using both mutual information and sum of squared differences similarity measures to account for differences in image appearance. PMID:24387943

Tracking of cell nuclei for assessment of in vitro uptake kinetics in ultrasound-mediated drug delivery using fibered confocal fluorescence microscopy.

PubMed

Derieppe, Marc; de Senneville, Baudouin Denis; Kuijf, Hugo; Moonen, Chrit; Bos, Clemens

2014-10-01

Previously, we demonstrated the feasibility to monitor ultrasound-mediated uptake of a cell-impermeable model drug in real time with fibered confocal fluorescence microscopy. Here, we present a complete post-processing methodology, which corrects for cell displacements, to improve the accuracy of pharmacokinetic parameter estimation. Nucleus detection was performed based on the radial symmetry transform algorithm. Cell tracking used an iterative closest point approach. Pharmacokinetic parameters were calculated by fitting a two-compartment model to the time-intensity curves of individual cells. Cells were tracked successfully, improving time-intensity curve accuracy and pharmacokinetic parameter estimation. With tracking, 93 % of the 370 nuclei showed a fluorescence signal variation that was well-described by a two-compartment model. In addition, parameter distributions were narrower, thus increasing precision. Dedicated image analysis was implemented and enabled studying ultrasound-mediated model drug uptake kinetics in hundreds of cells per experiment, using fiber-based confocal fluorescence microscopy.

Use of direct fluorescence labeling and confocal microscopy to determine the biodistribution of two protein therapeutics, Cerezyme and Ceredase.

PubMed

Piepenhagen, Peter A; Vanpatten, Scott; Hughes, Heather; Waire, James; Murray, James; Andrews, Laura; Edmunds, Tim; O'Callaghan, Michael; Thurberg, Beth L

2010-07-01

Efficient targeting of therapeutic reagents to tissues and cell types of interest is critical to achieving therapeutic efficacy and avoiding unwanted side effects due to offtarget uptake. To increase assay efficiency and reduce the number of animals used per experiment during preclinical development, we used a combination of direct fluorescence labeling and confocal microscopy to simultaneously examine the biodistribution of two therapeutic proteins, Cerezyme and Ceredase, in the same animals. We show that the fluorescent tags do not interfere with protein uptake and localization. We are able to detect Cerezyme and Ceredase in intact cells and organs and demonstrate colocalization within target cells using confocal microscopy. In addition, the relative amount of protein internalized by different cell types can be quantified using cell type-specific markers and morphometric analysis. This approach provides an easy and straightforward means of assessing the tissue and cell type-specific biodistribution of multiple protein therapeutics in target organs using a minimal number of animals. (c) 2009 Wiley-Liss, Inc.

Usefulness of confocal microscopy in distinguishing between basal cell carcinoma and intradermal melanocytic nevus on the face.

PubMed

Gamo, R; Floristan, U; Pampín, A; Caro, D; Pinedo, F; López-Estebaranz, J L

2015-10-01

The clinical distinction between basal cell carcinoma (BCC) and intradermal melanocytic nevus lesions on the face can be difficult, particularly in young patients or patients with multiple nevi. Dermoscopy is a useful tool for analyzing characteristic dermoscopic features of BCC, such as cartwheel structures, maple leaf-like areas, blue-gray nests and dots, and ulceration. It also reveals arborizing telangiectatic vessels and prominent curved vessels, which are typical of BCC, and comma vessels, which are typical of intradermal melanocytic nevi. It is, however, not always easy to distinguish between these 2 conditions, even when dermoscopy is used. We describe 2 facial lesions that posed a clinical and dermoscopic challenge in two 38-year-old patients; confocal microscopy showed separation between tumor nests and stroma and polarized nuclei, which are confocal microscopy features of basal cell carcinoma. Copyright © 2014 Elsevier España, S.L.U. y AEDV. All rights reserved.

High throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy

PubMed Central

Dumitriu, Dani; Rodriguez, Alfredo; Morrison, John H.

2012-01-01

Morphological features such as size, shape and density of dendritic spines have been shown to reflect important synaptic functional attributes and potential for plasticity. Here we describe in detail a protocol for obtaining detailed morphometric analysis of spines using microinjection of fluorescent dyes, high resolution confocal microscopy, deconvolution and image analysis using NeuronStudio. Recent technical advancements include better preservation of tissue resulting in prolonged ability to microinject, and algorithmic improvements that compensate for the residual Z-smear inherent in all optical imaging. Confocal imaging parameters were probed systematically for the identification of both optimal resolution as well as highest efficiency. When combined, our methods yield size and density measurements comparable to serial section transmission electron microscopy in a fraction of the time. An experiment containing 3 experimental groups with 8 subjects in each can take as little as one month if optimized for speed, or approximately 4 to 5 months if the highest resolution and morphometric detail is sought. PMID:21886104

Combined FLIM and reflectance confocal microscopy for epithelial imaging

NASA Astrophysics Data System (ADS)

Jabbour, Joey M.; Cheng, Shuna; Shrestha, Sebina; Malik, Bilal; Jo, Javier A.; Applegate, Brian; Maitland, Kristen C.

2012-03-01

Current methods for detection of oral cancer lack the ability to delineate between normal and precancerous tissue with adequate sensitivity and specificity. The usual diagnostic mechanism involves visual inspection and palpation followed by tissue biopsy and histopathology, a process both invasive and time-intensive. A more sensitive and objective screening method can greatly facilitate the overall process of detection of early cancer. To this end, we present a multimodal imaging system with fluorescence lifetime imaging (FLIM) for wide field of view guidance and reflectance confocal microscopy for sub-cellular resolution imaging of epithelial tissue. Moving from a 12 x 12 mm2 field of view with 157 ìm lateral resolution using FLIM to 275 x 200 μm2 with lateral resolution of 2.2 μm using confocal microscopy, hamster cheek pouch model is imaged both in vivo and ex vivo. The results indicate that our dual modality imaging system can identify and distinguish between different tissue features, and, therefore, can potentially serve as a guide in early oral cancer detection..

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.

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

Fluorescence lifetime imaging microscopy using near-infrared contrast agents.

PubMed

Nothdurft, R; Sarder, P; Bloch, S; Culver, J; Achilefu, S

2012-08-01

Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging. © 2012 The Author Journal of Microscopy © 2012 Royal Microscopical Society.

Multiphoton imaging microscopy at deeper layers with adaptive optics control of spherical aberration.

PubMed

Bueno, Juan M; Skorsetz, Martin; Palacios, Raquel; Gualda, Emilio J; Artal, Pablo

2014-01-01

Despite the inherent confocality and optical sectioning capabilities of multiphoton microscopy, three-dimensional (3-D) imaging of thick samples is limited by the specimen-induced aberrations. The combination of immersion objectives and sensorless adaptive optics (AO) techniques has been suggested to overcome this difficulty. However, a complex plane-by-plane correction of aberrations is required, and its performance depends on a set of image-based merit functions. We propose here an alternative approach to increase penetration depth in 3-D multiphoton microscopy imaging. It is based on the manipulation of the spherical aberration (SA) of the incident beam with an AO device while performing fast tomographic multiphoton imaging. When inducing SA, the image quality at best focus is reduced; however, better quality images are obtained from deeper planes within the sample. This is a compromise that enables registration of improved 3-D multiphoton images using nonimmersion objectives. Examples on ocular tissues and nonbiological samples providing different types of nonlinear signal are presented. The implementation of this technique in a future clinical instrument might provide a better visualization of corneal structures in living eyes.

Enhanced Bioactivity of Internally Functionalized Cationic Dendrimers with PEG Cores

DTIC Science & Technology

2012-11-09

Miltenyi) by counting 10 000 events. Cell Culture and Confocal Imaging. HeLa (CCL-2) were purchased from ATCC and cultured following manufacturer’s...concentration of PI before confocal imaging. Internalization Assay and Colocalization Studies. To monitor dendrimer internalization, cells were incubated...calcein. After 2 h of incubation at 37 °C, cells were washed three times with PBS and then analyzed by confocal microscopy. Ethidium Bromide Intercalation

Intracellular Protein Delivery for Treating Breast Cancer

DTIC Science & Technology

2012-06-01

are efficiently internalized by mammalian cells lines as characterized by confocal microscopy, and rhodamine-labeled apoptin can be observed in the...To determine the cellular localization of delivered proteins, confocal images were taken with HeLa, MCF-7, or HEF cells incubated with 20 nM of S-S...and analyzed by Nikon NIS Element software. Fluorescence images were acquired on a Yokogawa spinning-disk confocal scanner system using a Nikon

UNC Pembroke Laser Scanning Confocal Microscopy Facility

DTIC Science & Technology

2016-04-29

cultures. INVASIVE FIRE ANTS Professor Lisa Kelly of UNC Pembroke has been trained on the new confocal system. Dr. Kelly’s research...interest in the trophic ecology of the invasive fire ant has begun to benefit from the wide field view and long working distances of a confocal imaging...of protein clearance pathways in living brain tissue cultures. INVASIVE FIRE ANTS Professor Lisa Kelly of UNC Pembroke has been trained on

Cassette Series Designed for Live-Cell Imaging of Proteins and High Resolution Techniques in Yeast

PubMed Central

Young, Carissa L.; Raden, David L.; Caplan, Jeffrey; Czymmek, Kirk; Robinson, Anne S.

2012-01-01

During the past decade, it has become clear that protein function and regulation are highly dependent upon intracellular localization. Although fluorescent protein variants are ubiquitously used to monitor protein dynamics, localization, and abundance; fluorescent light microscopy techniques often lack the resolution to explore protein heterogeneity and cellular ultrastructure. Several approaches have been developed to identify, characterize, and monitor the spatial localization of proteins and complexes at the sub-organelle level; yet, many of these techniques have not been applied to yeast. Thus, we have constructed a series of cassettes containing codon-optimized epitope tags, fluorescent protein variants that cover the full spectrum of visible light, a TetCys motif used for FlAsH-based localization, and the first evaluation in yeast of a photoswitchable variant – mEos2 – to monitor discrete subpopulations of proteins via confocal microscopy. This series of modules, complete with six different selection markers, provides the optimal flexibility during live-cell imaging and multicolor labeling in vivo. Furthermore, high-resolution imaging techniques include the yeast-enhanced TetCys motif that is compatible with diaminobenzidine photooxidation used for protein localization by electron microscopy and mEos2 that is ideal for super-resolution microscopy. We have examined the utility of our cassettes by analyzing all probes fused to the C-terminus of Sec61, a polytopic membrane protein of the endoplasmic reticulum of moderate protein concentration, in order to directly compare fluorescent probes, their utility and technical applications. Our series of cassettes expand the repertoire of molecular tools available to advance targeted spatiotemporal investigations using multiple live-cell, super-resolution or electron microscopy imaging techniques. PMID:22473760

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

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

Thermal maturity of Tasmanites microfossils from confocal laser scanning fluorescence microscopy

USGS Publications Warehouse

Hackley, Paul C.; Kus, Jolanta

2015-01-01

We report here, for the first time, spectral properties of Tasmanites microfossils determined by confocal laser scanning fluorescence microscopy (CLSM, using Ar 458 nm excitation). The Tasmanites occur in a well-characterized natural maturation sequence (Ro 0.48–0.74%) of Devonian shale (n = 3 samples) from the Appalachian Basin. Spectral property λmax shows excellent agreement (r2 = 0.99) with extant spectra from interlaboratory studies which used conventional fluorescence microscopy techniques. This result suggests spectral measurements from CLSM can be used to infer thermal maturity of fluorescent organic materials in geologic samples. Spectra of regions with high fluorescence intensity at fold apices and flanks in individual Tasmanites are blue-shifted relative to less-deformed areas in the same body that have lower fluorescence intensity. This is interpreted to result from decreased quenching moiety concentration at these locations, and indicates caution is needed in the selection of measurement regions in conventional fluorescence microscopy, where it is common practice to select high intensity regions for improved signal intensity and better signal to noise ratios. This study also documents application of CLSM to microstructural characterization of Tasmanites microfossils. Finally, based on an extant empirical relation between conventional λmax values and bitumen reflectance, λmax values from CLSM of Tasmanites microfossils can be used to calculate a bitumen reflectance equivalent value. The results presented herein can be used as a basis to broaden the future application of CLSM in the geological sciences into hydrocarbon prospecting and basin analysis.

[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.

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

PubMed

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

2018-02-01

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

Analysis of caspase-3 in ASTC-a-1 cells treated with mitomycin C using acceptor photobleaching techniques

NASA Astrophysics Data System (ADS)

Wang, Huiying; Chen, Tongsheng; Sun, Lei

2008-02-01

Caspase-3 is a key activated death protease, which catalyzes the specific cleavage of many cellular proteins and induces DNA cleavage eventually. In this report, cells were treated with mitomycin C (MMC) at different concentration and its activity was detected by cell counting kit (CCK-8). Based on results of CCK-8, cells were treated with 10μg/mL MMC and Hoechst 33258 has been used to observe cell apoptosis. Fluorescence resonance energy transfer (FRET) and confocal microscopy have been used to the effect of MMC on the caspase3 activation in living cells. Human lung adenocarcinoma cells (ASTC-a-1) was transfected with plasmid SCAT3 (pSCAT3)/CKAR FRET receptor. Acceptor photobleaching techniques of FRET plasmid has been used to destruct fluorophore of cells stably expressing SCAT3 reporter on a fluorescence confocal microscope. The activity of caspase3 can be analyzed by FRET dynamics of SCAT3 in living cells. Our results show that MM C can induce ASTC-a-1 cell apoptosis through activation of caspase3.

Influence of Temperature on Corrosion Behavior of 2A02 Al Alloy in Marine Atmospheric Environments

PubMed Central

Cao, Min; Liu, Li; Fan, Lei; Yu, Zhongfen; Li, Ying; Oguzie, Emeka E.; Wang, Fuhui

2018-01-01

The corrosion behavior of 2A02 Al alloy under 4 mg/cm2 NaCl deposition at different temperatures (from 30 to 80 °C) has been studied. This corrosion behavior was researched using mass-gain, scanning electron microscopy-SEM, laser scanning confocal microscopy-LSCM, X-ray photoelectron spectroscopy-XPS and other techniques. The results showed and revealed that the corrosion was maximal at 60 °C after 200 h of exposure. The increase of temperature not only affected the solubility of oxygen gas in the thin film, but also promoted the transport of ions (such as Cl−), and the formation of protective AlO(OH), which further affects the corrosion speed. PMID:29401690

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...

[In vivo reflectance confocal microscopy in dermatology: a proposal concerning French terminology].

PubMed

Kanitakis, J; Bahadoran, P; Braun, R; Debarbieux, S; Labeille, B; Perrot, J-L; Vabres, P

2013-11-01

Reflectance confocal microscopy (RCM) is a recently introduced non-invasive imaging technique allowing real-time examination of the skin in vivo. Whereas a substantial literature concerning RCM exists in English, so far there is no official terminology in French, despite the fact that an ever-growing number of French-speaking dermatologists now use this new imaging technique. The aim of the present study is to propose a French terminology for RCM in order to allow French-speaking dermatologists to communicate in a precise and homogeneous language on this topic. A group of French-speaking dermatologists with solid experience of RCM, members of the Non-invasive Cutaneous Imaging group of the French Society of Dermatology, endeavored to suggest terms in French concerning RCM. Each group member dealt with a specific paragraph. The members exchanged comments via email and the terminology was finalized during a meeting of the group members in Paris in June 2012. Descriptive terms referring to the RCM aspects of normal and diseased skin were proposed. Some of these already existed, being used in routine dermatopathology, while other specific terms were created or adapted from the English terminology. This terminology will allow French-speaking dermatologists using RCM to communicate their findings in a homogeneous language. It may be enriched in the future by the introduction of additional terms describing new aspects of both normal and, especially, diseased skin. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Perspective: Differential dynamic microscopy extracts multi-scale activity in complex fluids and biological systems

NASA Astrophysics Data System (ADS)

Cerbino, Roberto; Cicuta, Pietro

2017-09-01

Differential dynamic microscopy (DDM) is a technique that exploits optical microscopy to obtain local, multi-scale quantitative information about dynamic samples, in most cases without user intervention. It is proving extremely useful in understanding dynamics in liquid suspensions, soft materials, cells, and tissues. In DDM, image sequences are analyzed via a combination of image differences and spatial Fourier transforms to obtain information equivalent to that obtained by means of light scattering techniques. Compared to light scattering, DDM offers obvious advantages, principally (a) simplicity of the setup; (b) possibility of removing static contributions along the optical path; (c) power of simultaneous different microscopy contrast mechanisms; and (d) flexibility of choosing an analysis region, analogous to a scattering volume. For many questions, DDM has also advantages compared to segmentation/tracking approaches and to correlation techniques like particle image velocimetry. The very straightforward DDM approach, originally demonstrated with bright field microscopy of aqueous colloids, has lately been used to probe a variety of other complex fluids and biological systems with many different imaging methods, including dark-field, differential interference contrast, wide-field, light-sheet, and confocal microscopy. The number of adopting groups is rapidly increasing and so are the applications. Here, we briefly recall the working principles of DDM, we highlight its advantages and limitations, we outline recent experimental breakthroughs, and we provide a perspective on future challenges and directions. DDM can become a standard primary tool in every laboratory equipped with a microscope, at the very least as a first bias-free automated evaluation of the dynamics in a system.

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.

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.

Hydrocarbons in phlogopite from Kasenyi kamafugitic rocks (SW Uganda): cross-correlated AFM, confocal microscopy and Raman imaging

PubMed Central

Moro, Daniele; Valdrè, Giovanni; Mesto, Ernesto; Scordari, Fernando; Lacalamita, Maria; Ventura, Giancarlo Della; Bellatreccia, Fabio; Scirè, Salvatore; Schingaro, Emanuela

2017-01-01

This study presents a cross-correlated surface and near surface investigation of two phlogopite polytypes from Kasenyi kamafugitic rocks (SW Uganda) by means of advanced Atomic Force Microscopy (AFM), confocal microscopy and Raman micro-spectroscopy. AFM revealed comparable nanomorphology and electrostatic surface potential for the two mica polytypes. A widespread presence of nano-protrusions located on the mica flake surface was also observed, with an aspect ratio (maximum height/maximum width) from 0.01 to 0.09. Confocal microscopy showed these features to range from few nm to several μm in dimension, and shapes from perfectly circular to ellipsoidic and strongly elongated. Raman spectra collected across the bubbles showed an intense and convolute absorption in the range 3000–2800 cm−1, associated with weaker bands at 1655, 1438 and 1297 cm−1, indicating the presence of fluid inclusions consisting of aliphatic hydrocarbons, alkanes and cycloalkanes, with minor amounts of oxygenated compounds, such as carboxylic acids. High-resolution Raman images provided evidence that these hydrocarbons are confined within the bubbles. This work represents the first direct evidence that phlogopite, a common rock-forming mineral, may be a possible reservoir for hydrocarbons. PMID:28098185

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.

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

Transdermal Delivery of Functional Collagen Via Polyvinylpyrrolidone Microneedles

PubMed Central

Sun, Wenchao; Inayathullah, Mohammed; Manoukian, Martin A. C.; Malkovskiy, Andrey V.; Manickam, Sathish; Marinkovich, M. Peter; Lane, Alfred T.; Tayebi, Lobat; Seifalian, Alexander M.; Rajadas, Jayakumar

2017-01-01

Collagen makes up a large proportion of the human body, particularly the skin. As the body ages, collagen content decreases, resulting in wrinkled skin and decreased wound healing capabilities. This paper presents a method of delivering type I collagen into porcine and human skin utilizing a polyvinylpyrrolidone microneedle delivery system. The microneedle patches were made with concentrations of 1, 2, 4, and 8% type I collagen (w/w). Microneedle structures and the distribution of collagen were characterized using scanning electron microscopy and confocal microscopy. Patches were then applied on the porcine and human skin, and their effectiveness was examined using fluorescence microscopy. The results illustrate that this microneedle delivery system is effective in delivering collagen I into the epidermis and dermis of porcine and human skin. Since the technique presented in this paper is quick, safe, effective and easy, it can be considered as a new collagen delivery method for cosmetic and therapeutic applications. PMID:26066056

Diagnosing the Internal Architecture of Zeolite Ferrierite

PubMed Central

Schmidt, Joel E.; Hendriks, Frank C.; Lutz, Martin; Post, L. Christiaan; Fu, Donglong

2017-01-01

Abstract Large crystals of zeolite ferrierite (FER) are important model systems for spatially resolved catalysis and diffusion studies, though there is considerable variation in crystal habit depending on the chemical composition and employed synthesis conditions. A synergistic combination of techniques has been applied, including single crystal X‐ray diffraction, high‐temperature in situ confocal fluorescence microscopy, fluorescent probe molecules, wide‐field microscopy and atomic force microscopy to unravel the internal architecture of three distinct FER zeolites. Pyrolyzed template species can be used as markers for the 8‐membered ring direction as they are trapped in the terraced roof of the FER crystals. This happens as the materials grow in a layer‐by‐layer, defect‐free manner normal to the large crystal surface, and leads to a facile method to diagnose the pore system orientation, which avoids tedious single crystal X‐ray diffraction experiments. PMID:28809081

3D confocal reconstruction of gene expression in mouse.

PubMed

Hecksher-Sørensen, J; Sharpe, J

2001-01-01

Three-dimensional computer reconstructions of gene expression data will become a valuable tool in biomedical research in the near future. However, at present the process of converting in situ expression data into 3D models is a highly specialized and time-consuming procedure. Here we present a method which allows rapid reconstruction of whole-mount in situ data from mouse embryos. Mid-gestation embryos were stained with the alkaline phosphotase substrate Fast Red, which can be detected using confocal laser scanning microscopy (CLSM), and cut into 70 microm sections. Each section was then scanned and digitally reconstructed. Using this method it took two days to section, digitize and reconstruct the full expression pattern of Shh in an E9.5 embryo (a 3D model of this embryo can be seen at genex.hgu.mrc.ac.uk). Additionally we demonstrate that this technique allows gene expression to be studied at the single cell level in intact tissue.

Characteristics of subgingival calculus detection by multiphoton fluorescence microscopy

NASA Astrophysics Data System (ADS)

Tung, Oi-Hong; Lee, Shyh-Yuan; Lai, Yu-Lin; Chen, How-Foo

2011-06-01

Subgingival calculus has been recognized as a major cause of periodontitis, which is one of the main chronic infectious diseases of oral cavities and a principal cause of tooth loss in humans. Bacteria deposited in subgingival calculus or plaque cause gingival inflammation, function deterioration, and then periodontitis. However, subgingival calculus within the periodontal pocket is a complicated and potentially delicate structure to be detected with current dental armamentaria, namely dental x-rays and dental probes. Consequently, complete removal of subgingival calculus remains a challenge to periodontal therapies. In this study, the detection of subgingival calculus employing a multiphoton autofluorescence imaging method was characterized in comparison with a one-photon confocal fluorescence imaging technique. Feasibility of such a system was studied based on fluorescence response of gingiva, healthy teeth, and calculus with and without gingiva covered. The multiphoton fluorescence technology perceived the tissue-covered subgingival calculus that cannot be observed by the one-photon confocal fluorescence method.

Through the looking glass: Basics and principles of reflectance confocal microscopy.

PubMed

Que, Syril Keena T; Fraga-Braghiroli, Naiara; Grant-Kels, Jane M; Rabinovitz, Harold S; Oliviero, Margaret; Scope, Alon

2015-08-01

Reflectance confocal microscopy (RCM) offers high-resolution, noninvasive skin imaging and can help avoid obtaining unnecessary biopsy specimens. It can also increase efficiency in the surgical setting by helping to delineate tumor margins. Diagnostic criteria and several RCM algorithms have been published for the differentiation of benign and malignant neoplasms. We provide an overview of the basic principles of RCM, characteristic RCM features of normal skin and cutaneous neoplasms, and the limitations and future directions of RCM. Copyright © 2015 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

Long-depth imaging of specific gene expressions in whole-mount mouse embryos with single-photon excitation confocal fluorescence microscopy and FISH.

PubMed

Palmes-Saloma, C; Saloma, C

2000-07-01

Long-depth imaging of specific gene expression in the midgestation whole-mount mouse embryo (WME) is demonstrated with single-photon excitation (1PE) confocal fluorescence microscopy and fluorescence in situ hybridization. Expression domains of Pax-6 mRNA transcripts were labeled with an in situ hybridization probe that is a RNA sequence complementary to the cloned gene fragment and were rendered visible using two fluorochrome-conjugated antibodies that fluoresce at peak wavelengths of lambda(F) = 0.525 microm and lambda(F) = 0. 580 microm, respectively. Distributions of Pax-6 mRNA domains as deep as 1000 microm in the day 9.5 WME were imaged with a long-working-distance (13.6 mm) objective lens (magnification 5x). The scattering problem posed by the optically thick WME sample is alleviated by careful control of the detector pinhole size and the application of simple but fast postdetection image enhancement techniques, such as space and wavelength averaging to produce high-quality fluorescence images. A three-dimensional reconstruction that clearly shows the Pax-6 mRNA expression domains in the forebrain, diencephalon, optic cup, and spinal cord of the day 9.5 WME is obtained. The advantages of 1PE confocal fluorescence imaging over two-photon excitation fluorescence imaging are discussed for the case of long-depth imaging in highly scattering media. Imaging in midgestation WMEs at optical depths of more than 350 microm has not yet been realized with two-photon fluorescence excitation. Copyright 2000 Academic Press.

Fluorescence Confocal Microscopy for Ex Vivo Diagnosis of Conjunctival Tumors: A Pilot Study.

PubMed

Iovieno, Alfonso; Longo, Caterina; De Luca, Mariacarla; Piana, Simonetta; Fontana, Luigi; Ragazzi, Moira

2016-08-01

To evaluate the potential use of fluorescence confocal microscopy (FCM) for ex vivo diagnosis and excision margin assessment of conjunctival neoplasms. Validity study. setting: Single institution. Consecutive patients with clinically suspicious conjunctival lesions. Conjunctival lesions were excised in toto using a standard "no-touch technique" by a single surgeon (A.I.). Collected specimens were examined with a commercially available laser scanning fluorescence confocal microscope after immersion in a 0.6 mM solution of acridine orange dye for 10-20 seconds. Specimens were subsequently processed with standard histologic analysis. FCM diagnosis of the nature and extension of conjunctival lesions. Sixteen consecutive patients were included in the study (11 male, 5 female; mean age 58.1 ± 26.1 years, range 10-90 years). The median time needed to process and analyze a sample with FCM was 15 minutes. Eleven of 16 lesions were identified by FCM as squamous (2 benign papillomas, 2 grade 2 conjunctival intraepithelial neoplasias, 7 in situ squamous carcinomas) and 5 as nonsquamous (1 pingueculum, 1 dermolipoma, 2 melanocytic nevi, 1 melanoma). In all cases FCM was able to detect horizontal and vertical extension of the lesion. All FCM findings were confirmed by corresponding subsequent histologic examination. FCM provides a fast ex vivo preliminary diagnosis of suspicious conjunctival lesions with good histologic details and margin assessment, and may represent a novel tool for intraoperative and postsurgical management of conjunctival tumors. This is the first study to investigate ex vivo FCM application in ophthalmology. Copyright © 2016 Elsevier Inc. All rights reserved.

High resolution 3D imaging of synchrotron generated microbeams

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

Gagliardi, Frank M., E-mail: frank.gagliardi@wbrc.org.au; Cornelius, Iwan; Blencowe, Anton

2015-12-15

Purpose: Microbeam radiation therapy (MRT) techniques are under investigation at synchrotrons worldwide. Favourable outcomes from animal and cell culture studies have proven the efficacy of MRT. The aim of MRT researchers currently is to progress to human clinical trials in the near future. The purpose of this study was to demonstrate the high resolution and 3D imaging of synchrotron generated microbeams in PRESAGE® dosimeters using laser fluorescence confocal microscopy. Methods: Water equivalent PRESAGE® dosimeters were fabricated and irradiated with microbeams on the Imaging and Medical Beamline at the Australian Synchrotron. Microbeam arrays comprised of microbeams 25–50 μm wide with 200more » or 400 μm peak-to-peak spacing were delivered as single, cross-fire, multidirectional, and interspersed arrays. Imaging of the dosimeters was performed using a NIKON A1 laser fluorescence confocal microscope. Results: The spatial fractionation of the MRT beams was clearly visible in 2D and up to 9 mm in depth. Individual microbeams were easily resolved with the full width at half maximum of microbeams measured on images with resolutions of as low as 0.09 μm/pixel. Profiles obtained demonstrated the change of the peak-to-valley dose ratio for interspersed MRT microbeam arrays and subtle variations in the sample positioning by the sample stage goniometer were measured. Conclusions: Laser fluorescence confocal microscopy of MRT irradiated PRESAGE® dosimeters has been validated in this study as a high resolution imaging tool for the independent spatial and geometrical verification of MRT beam delivery.« less

Super-resolution fluorescence microscopy by stepwise optical saturation

PubMed Central

Zhang, Yide; Nallathamby, Prakash D.; Vigil, Genevieve D.; Khan, Aamir A.; Mason, Devon E.; Boerckel, Joel D.; Roeder, Ryan K.; Howard, Scott S.

2018-01-01

Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing. The method is based on the principle of stepwise optical saturation (SOS), where M steps of raw fluorescence images are linearly combined to generate an image with a M-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples. PMID:29675306

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.

Varifocal MOEMS fiber scanner for confocal endomicroscopy.

PubMed

Meinert, Tobias; Weber, Niklas; Zappe, Hans; Seifert, Andreas

2014-12-15

Based on an advanced silicon optical bench technology with integrated MOEMS (Micro-Opto-Electro-Mechanical-System) components, a piezo-driven fiber scanner for confocal microscopy has been developed. This highly-miniaturized technology allows integration into an endoscope with a total outer probe diameter of 2.5 mm. The system features a hydraulically-driven varifocal lens providing axial confocal scanning without any translational movement of components. The demonstrated resolutions are 1.7 μm laterally and 19 μm axially.

Three-dimensional Model of Tissue and Heavy Ions Effects

NASA Technical Reports Server (NTRS)

Ponomarev, Artem L.; Sundaresan, Alamelu; Huff, Janice L.; Cucinotta, Francis A.

2007-01-01

A three-dimensional tissue model was incorporated into a new Monte Carlo algorithm that simulates passage of heavy ions in a tissue box . The tissue box was given as a realistic model of tissue based on confocal microscopy images. The action of heavy ions on the cellular matrix for 2- or 3-dimensional cases was simulated. Cells were modeled as a cell culture monolayer in one example, where the data were taken directly from microscopy (2-d cell matrix), and as a multi-layer obtained from confocal microscopy (3-d case). Image segmentation was used to identify cells with precise areas/volumes in an irradiated cell culture monolayer, and slices of tissue with many cell layers. The cells were then inserted into the model box of the simulated physical space pixel by pixel. In the case of modeled tissues (3-d), the tissue box had periodic boundary conditions imposed, which extrapolates the technique to macroscopic volumes of tissue. For the real tissue (3-d), specific spatial patterns for cell apoptosis and necrosis are expected. The cell patterns were modeled based on action cross sections for apoptosis and necrosis estimated from current experimental data. A spatial correlation function indicating a higher spatial concentration of damaged cells from heavy ions relative to the low-LET radiation cell damage pattern is presented. The spatial correlation effects among necrotic cells can help studying microlesions in organs, and probable effects of directionality of heavy ion radiation on epithelium and endothelium.

Hydrohalite spatial distribution in frozen cell cultures measured using confocal Raman microscopy.

PubMed

Kreiner-Møller, Asger; Stracke, Frank; Zimmermann, Heiko

2014-08-01

Hydrohalite, a crystalline rock salt hydrate, (NaCl·2H2O), can form in cryopreservation samples under certain circumstances changing the local chemical environment of the preserved cells. Evidence of this crystalline phase was recently found by microspectroscopy measurements, and believed to form exclusively extracellular. We have studied the spatial distribution of hydrohalite in frozen mouse fibroblast cell samples by means of confocal Raman scanning microscopy (CRM). Hydrohalite has a unique Raman spectrum with several bands in the high frequency tail of the OH-stretching band which can be used for unambiguous identification. Hydrohalite can only form through eutectic crystallization in saline solutions without any cryoprotective agents and the spatial distribution thus gives a more detailed view on this crystallization process. This is important since eutectic crystallization has been empirically correlated to cell death, but the exact injury mechanism is unclear. By the means of colocalization of Raman bands we show that hydrohalite can indeed form intracellularly and is not a strictly extracellular phenomenon. We furthermore found that intracellular ice and intracellular hydrohalite very often coincide. Finally we show that the addition of 0.5 wt.% dimethyl sulfoxide (Me2SO) inhibits formation of hydrohalite. This study shows how Raman microscopy and successive analysis can be employed non-invasively within cryobiology to give additional chemical and structural information compared to conventional imaging techniques. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Live-Cell Imaging of the Adult Drosophila Ovary Using Confocal Microscopy.

PubMed

Shalaby, Nevine A; Buszczak, Michael

2017-01-01

The Drosophila ovary represents a key in vivo model used to study germline stem cell (GSC) maintenance and stem cell daughter differentiation because these cells and their somatic cell neighbors can be identified at single-cell resolution within their native environment. Here we describe a fluorescent-based technique for the acquisition of 4D datasets of the Drosophila ovariole for periods that can exceed 12 consecutive hours. Live-cell imaging facilitates the investigation of molecular and cellular dynamics that were not previously possible using still images.

Biogenicity and Syngeneity of Organic Matter in Ancient Sedimentary Rocks: Recent Advances in the Search for Evidence of Past Life

NASA Astrophysics Data System (ADS)

Oehler, Dorothy Z.; Cady, Sherry L.

2014-08-01

The past decade has seen an explosion of new technologies for assessment of biogenicity and syngeneity of carbonaceous material within sedimentary rocks. Advances have been made in techniques for analysis of in situ organic matter as well as for extracted bulk samples of soluble and insoluble (kerogen) organic fractions. The in situ techniques allow analysis of micrometer-to-sub-micrometer-scale organic residues within their host rocks and include Raman and fluorescence spectroscopy/imagery, confocal laser scanning microscopy, and forms of secondary ion/laser-based mass spectrometry, analytical transmission electron microscopy, and X-ray absorption microscopy/spectroscopy. Analyses can be made for chemical, molecular, and isotopic composition coupled with assessment of spatial relationships to surrounding minerals, veins, and fractures. The bulk analyses include improved methods for minimizing contamination and recognizing syngenetic constituents of soluble organic fractions as well as enhanced spectroscopic and pyrolytic techniques for unlocking syngenetic molecular signatures in kerogen. Together, these technologies provide vital tools for the study of some of the oldest and problematic carbonaceous residues and for advancing our understanding of the earliest stages of biological evolution on Earth and the search for evidence of life beyond Earth. We discuss each of these new technologies, emphasizing their advantages and disadvantages, applications, and likely future directions.

Early Corneal Cellular and Nerve Fiber Pathology in Young Patients With Type 1 Diabetes Mellitus Identified Using Corneal Confocal Microscopy.

PubMed

Szalai, Eszter; Deák, Eszter; Módis, László; Németh, Gábor; Berta, András; Nagy, Annamária; Felszeghy, Eniko; Káposzta, Rita; Malik, Rayaz A; Csutak, Adrienne

2016-03-01

The aim of this study was to quantify epithelial, stromal, and endothelial cell density, and subbasal nerve morphology in young patients with type 1 diabetes mellitus with and without diabetic retinopathy. A total of 28 young patients (mean age, 22.86 ± 9.05 years) with type 1 diabetes, with (n = 18) and without (n = 10) retinopathy, and 17 age-matched healthy control subjects (mean age, 26.53 ± 2.43 years) underwent corneal confocal microscopy (CCM). We found significantly lower epithelial (P < 0.0001) and endothelial (P = 0.001) cell densities and higher keratocyte cell density (P = 0.024) in patients with type 1 diabetes compared to controls. Significantly lower corneal nerve fiber density (P = 0.004), nerve branch density (P = 0.004), total nerve branch density (P = 0.04), and nerve fiber length (P = 0.001), and greater nerve fiber width (P = 0.04) were observed in patients with type 1 diabetes compared to control subjects. Significantly lower epithelial (P < 0.001) and endothelial (P = 0.02) cell densities, nerve branch density (P = 0.02), and nerve fiber length (P = 0.04), and significantly higher keratocyte cell density (P = 0.02) were found in patients with type 1 diabetes without retinopathy compared to control subjects. Corneal confocal microscopy identifies corneal cellular and small nerve fiber pathology in young patients with type 1 diabetes without retinopathy, which increases in severity in those with retinopathy. Corneal confocal microscopy appears to have considerable use as an imaging biomarker for early subclinical pathology in young patients with type 1 diabetes mellitus.

Dictionary-based image reconstruction for superresolution in integrated circuit imaging.

PubMed

Cilingiroglu, T Berkin; Uyar, Aydan; Tuysuzoglu, Ahmet; Karl, W Clem; Konrad, Janusz; Goldberg, Bennett B; Ünlü, M Selim

2015-06-01

Resolution improvement through signal processing techniques for integrated circuit imaging is becoming more crucial as the rapid decrease in integrated circuit dimensions continues. Although there is a significant effort to push the limits of optical resolution for backside fault analysis through the use of solid immersion lenses, higher order laser beams, and beam apodization, signal processing techniques are required for additional improvement. In this work, we propose a sparse image reconstruction framework which couples overcomplete dictionary-based representation with a physics-based forward model to improve resolution and localization accuracy in high numerical aperture confocal microscopy systems for backside optical integrated circuit analysis. The effectiveness of the framework is demonstrated on experimental data.

Pump-probe spectroscopy in organic semiconductors: monitoring fundamental processes of relevance in optoelectronics.

PubMed

Cabanillas-Gonzalez, Juan; Grancini, Giulia; Lanzani, Guglielmo

2011-12-08

In this review we highlight the contribution of pump-probe spectroscopy to understand elementary processes taking place in organic based optoelectronic devices. The techniques described in this article span from conventional pump-probe spectroscopy to electromodulated pump-probe and the state-of-the-art confocal pump-probe microscopy. The article is structured according to three fundamental processes (optical gain, charge photogeneration and charge transport) and the contribution of these techniques on them. The combination of these tools opens up new perspectives for assessing the role of short-lived excited states on processes lying underneath organic device operation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

NASA Astrophysics Data System (ADS)

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

2002-04-01

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

Nanoparticles as smart treatment-delivery systems in plants: assessment of different techniques of microscopy for their visualization in plant tissues.

PubMed

González-Melendi, P; Fernández-Pacheco, R; Coronado, M J; Corredor, E; Testillano, P S; Risueño, M C; Marquina, C; Ibarra, M R; Rubiales, D; Pérez-de-Luque, A

2008-01-01

The great potential of using nanodevices as delivery systems to specific targets in living organisms was first explored for medical uses. In plants, the same principles can be applied for a broad range of uses, in particular to tackle infections. Nanoparticles tagged to agrochemicals or other substances could reduce the damage to other plant tissues and the amount of chemicals released into the environment. To explore the benefits of applying nanotechnology to agriculture, the first stage is to work out the correct penetration and transport of the nanoparticles into plants. This research is aimed (a) to put forward a number of tools for the detection and analysis of core-shell magnetic nanoparticles introduced into plants and (b) to assess the use of such magnetic nanoparticles for their concentration in selected plant tissues by magnetic field gradients. Cucurbita pepo plants were cultivated in vitro and treated with carbon-coated Fe nanoparticles. Different microscopy techniques were used for the detection and analysis of these magnetic nanoparticles, ranging from conventional light microscopy to confocal and electron microscopy. Penetration and translocation of magnetic nanoparticles in whole living plants and into plant cells were determined. The magnetic character allowed nanoparticles to be positioned in the desired plant tissue by applying a magnetic field gradient there; also the graphitic shell made good visualization possible using different microscopy techniques. The results open a wide range of possibilities for using magnetic nanoparticles in general plant research and agronomy. The nanoparticles can be charged with different substances, introduced within the plants and, if necessary, concentrated into localized areas by using magnets. Also simple or more complex microscopical techniques can be used in localization studies.

Nanoparticles as Smart Treatment-delivery Systems in Plants: Assessment of Different Techniques of Microscopy for their Visualization in Plant Tissues

PubMed Central

González-Melendi, P.; Fernández-Pacheco, R.; Coronado, M. J.; Corredor, E.; Testillano, P. S.; Risueño, M. C.; Marquina, C.; Ibarra, M. R.; Rubiales, D.; Pérez-de-Luque, A.

2008-01-01

Background and Aims The great potential of using nanodevices as delivery systems to specific targets in living organisms was first explored for medical uses. In plants, the same principles can be applied for a broad range of uses, in particular to tackle infections. Nanoparticles tagged to agrochemicals or other substances could reduce the damage to other plant tissues and the amount of chemicals released into the environment. To explore the benefits of applying nanotechnology to agriculture, the first stage is to work out the correct penetration and transport of the nanoparticles into plants. This research is aimed (a) to put forward a number of tools for the detection and analysis of core-shell magnetic nanoparticles introduced into plants and (b) to assess the use of such magnetic nanoparticles for their concentration in selected plant tissues by magnetic field gradients. Methods Cucurbita pepo plants were cultivated in vitro and treated with carbon-coated Fe nanoparticles. Different microscopy techniques were used for the detection and analysis of these magnetic nanoparticles, ranging from conventional light microscopy to confocal and electron microscopy. Key Results Penetration and translocation of magnetic nanoparticles in whole living plants and into plant cells were determined. The magnetic character allowed nanoparticles to be positioned in the desired plant tissue by applying a magnetic field gradient there; also the graphitic shell made good visualization possible using different microscopy techniques. Conclusions The results open a wide range of possibilities for using magnetic nanoparticles in general plant research and agronomy. The nanoparticles can be charged with different substances, introduced within the plants and, if necessary, concentrated into localized areas by using magnets. Also simple or more complex microscopical techniques can be used in localization studies. PMID:17998213

Sensorless adaptive optics for isoSTED nanoscopy

NASA Astrophysics Data System (ADS)

Antonello, Jacopo; Hao, Xiang; Allgeyer, Edward S.; Bewersdorf, Joerg; Rittscher, Jens; Booth, Martin J.

2018-02-01

The presence of aberrations is a major concern when using fluorescence microscopy to image deep inside tissue. Aberrations due to refractive index mismatch and heterogeneity of the specimen under investigation cause severe reduction in the amount of fluorescence emission that is collected by the microscope. Furthermore, aberrations adversely affect the resolution, leading to loss of fine detail in the acquired images. These phenomena are particularly troublesome for super-resolution microscopy techniques such as isotropic stimulated-emission-depletion microscopy (isoSTED), which relies on accurate control of the shape and co-alignment of multiple excitation and depletion foci to operate as expected and to achieve the super-resolution effect. Aberrations can be suppressed by implementing sensorless adaptive optics techniques, whereby aberration correction is achieved by maximising a certain image quality metric. In confocal microscopy for example, one can employ the total image brightness as an image quality metric. Aberration correction is subsequently achieved by iteratively changing the settings of a wavefront corrector device until the metric is maximised. This simplistic approach has limited applicability to isoSTED microscopy where, due to the complex interplay between the excitation and depletion foci, maximising the total image brightness can lead to introducing aberrations in the depletion foci. In this work we first consider the effects that different aberration modes have on isoSTED microscopes. We then propose an iterative, wavelet-based aberration correction algorithm and evaluate its benefits.

In vivo laser confocal microscopy findings of radial keratoneuritis in patients with early stage Acanthamoeba keratitis.

PubMed

Kobayashi, Akira; Yokogawa, Hideaki; Yamazaki, Natsuko; Ishibashi, Yasuhisa; Oikawa, Yosaburo; Tokoro, Masaharu; Sugiyama, Kazuhisa

2013-07-01

To investigate in vivo corneal changes of keratoneuritis in early stage Acanthamoeba keratitis (AK) using in vivo laser confocal microscopy. Single-center, prospective, clinical study. Thirteen eyes (12 patients; 5 men and 7 women; mean age ± standard deviation, 22.3 ± 4.2 years) with keratoneuritis resulting from early stage AK were included in this study. In vivo laser confocal microscopy was performed, paying special attention to keratoneuritis. Selected confocal images of corneal layers were evaluated qualitatively for shape and degree of light reflection of abnormal cells and deposits. In all patients, Acanthamoeba cysts were observed clearly in the basal epithelial cell layer as highly reflective round particles with a diameter of 10 to 20 μm. Bowman's layer infiltration of Acanthamoeba cysts was observed in only 1 case, and no cases showed stromal or nerve infiltration of Acanthamoeba cysts. In the stroma, all cases showed highly reflective activated keratocytes forming a honeycomb pattern; these changes were significant around the keratoneuritis. Infiltration of inflammatory cells, possibly polymorphonuclear cells, was observed along with keratocyte bodies in all cases. Numerous highly reflective spindle-shaped materials were observed around the keratoneuritis. Most notably, highly reflective patchy lesions were observed around the keratoneuritis in 11 cases (84.6%). Inflammatory cells also were observed in the endothelial cell layer in 4 cases (30.8%). In vivo laser confocal microscopy identified consistent corneal abnormalities around keratoneuritis in early stage AK patients, of which highly reflective patchy lesions may be characteristic of keratoneuritis. Further morphologic studies of corneas with early stage AK in a larger number of patients may elucidate the clinical significance of radial keratoneuritis and may help us to understand the interaction between Acanthamoeba organisms and host corneal cells or nerves. Copyright © 2013 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

Investigation of the confocal wavefront sensor and its application to biological microscopy.

PubMed

Shaw, Michael; O'Holleran, Kevin; Paterson, Carl

2013-08-12

Wavefront sensing in the presence of background light sources is complicated by the need to restrict the effective depth of field of the wavefront sensor. This problem is particularly significant in direct wavefront sensing adaptive optic (AO) schemes for correcting imaging aberrations in biological microscopy. In this paper we investigate how a confocal pinhole can be used to reject out of focus light whilst still allowing effective wavefront sensing. Using a scaled set of phase screens with statistical properties derived from measurements of wavefront aberrations induced by C. elegans specimens, we investigate and quantify how the size of the pinhole and the aberration amplitude affect the transmitted wavefront. We suggest a lower bound for the pinhole size for a given aberration strength and quantify the optical sectioning provided by the system. For our measured aberration data we find that a pinhole of size approximately 3 Airy units represents a good compromise, allowing effective transmission of the wavefront and thin optical sections. Finally, we discuss some of the practical implications of confocal wavefront sensing for AO systems in microscopy.

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.

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

PubMed

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

2017-01-01

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

Metabolic changes of cultured DRG neurons induced by adenosine using confocal microscopy imaging

NASA Astrophysics Data System (ADS)

Zheng, Liqin; Huang, Yimei; Chen, Jiangxu; Wang, Yuhua; Yang, Hongqin; Zhang, Yanding; Xie, Shusen

2012-12-01

Adenosine exerts multiple effects on pain transmission in the peripheral nervous system. This study was performed to use confocal microscopy to evaluate whether adenosine could affect dorsal root ganglia (DRG) neurons in vitro and test which adenosine receptor mediates the effect of adenosine on DRG neurons. After adding adenosine with different concentration, we compared the metabolic changes by the real time imaging of calcium and mitochondria membrane potential using confocal microscopy. The results showed that the effect of 500 μM adenosine on the metabolic changes of DRG neurons was more significant than others. Furthermore, four different adenosine receptor antagonists were used to study which receptor mediated the influences of adenosine on the cultured DRG neurons. All adenosine receptor antagonists especially A1 receptor antagonist (DPCPX) had effect on the Ca2+ and mitochondria membrane potential dynamics of DRG neurons. The above studies demonstrated that the effect of adenosine which may be involved in the signal transmission on the sensory neurons was dose-dependent, and all the four adenosine receptors especially the A1R may mediate the transmission.