Science.gov

Sample records for 3-d confocal microscopy

  1. 3D imaging of neutron tracks using confocal microscopy

    NASA Astrophysics Data System (ADS)

    Gillmore, Gavin; Wertheim, David; Flowers, Alan

    2016-04-01

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

  2. Simultaneous multiplane imaging for 3D confocal microscopy using high-speed z-scanning multiplexing

    NASA Astrophysics Data System (ADS)

    Duocastella, Marti; Vicidomini, Giuseppe; Diaspro, Alberto

    2015-03-01

    One of the key frontiers in optical imaging is to maximize the spatial information retrieved from a sample while minimizing acquisition time. Confocal laser scanning microscopy is a powerful imaging modality that allows real-time and high-resolution acquisition of two-dimensional (2D) sections. However, in order to obtain information from threedimensional (3D) volumes it is currently limited by a stepwise process that consists of acquiring multiple 2D sections from different focal planes by slow z-focus translation. Here, we present a novel method that enables the capture of an entire 3D sample in a single step. Our approach is based on an acoustically-driven varifocal lens integrated in a commercial confocal system that enables axial focus scanning at speeds of 140 kHz or above. Such high-speed allows for one or multiple focus sweeps on a pixel by pixel basis. By using a fast acquisition card, we can assign the photons detected at each pixel to their corresponding focal plane allowing simultaneous multiplane imaging. We exemplify this novel 3D confocal microscopy technique by imaging different biological fluorescent samples and comparing them with those obtained using traditional z-scanners. Based on these results, we find that image quality in this novel approach is similar to that obtained with traditional confocal methods, while speed is only limited by signal-to-noise-ratio. As the sensitivity of photodetectors increases and more efficient fluorescent labeling is developed, this novel 3D method can result in significant reduction in acquisition time allowing the study of new fundamental processes in science.

  3. Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

    PubMed Central

    Bohórquez, Diego; Haque, Fariha; Medicetty, Satish; Liddle, Rodger A.

    2015-01-01

    Delineation of a cell’s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell’s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue. PMID:26273796

  4. Jamming of a soft granular system of hollow elastic shells in 3D using confocal microscopy

    NASA Astrophysics Data System (ADS)

    Jose, Jissy; van Blaaderen, Alfons; Imhof, Arnout

    2014-03-01

    We introduce a new system for jammed matter research consisting of monodisperse, fluorescent, hollow deformable shells, dispersed in an index matched solvent. The interesting fact about these elastic shells is that they undergo buckling: in each contact one of the shells receives an indentation from its neighbor under compressive stress. This kind of deformation is different from the soft granular systems experimentally studied so far like photo elastic disks, emulsions and foams, where the particles are flattened in the region of contact and conserve their volume. Using confocal microscopy and image analysis routines (ImageJ software) we identified the 3D position of the particles with sub pixel resolution. The force law to find the contact forces between pairs of particle is derived from the theory of elasticity of thin shells, where force is proportional to the square root of indentation depth. The distribution of normalized contact forces showed a similar trend like other jammed systems with a peak around the mean and a tail that decayed faster than exponential away from jamming threshold. Further, we also investigated the structure of the jammed packings and contact number distribution with distance to jamming.

  5. 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...

  6. 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...

  7. 3D Quantitative Confocal Laser Microscopy of Ilmenite Volume Distribution in Alpe Arami Olivine

    NASA Astrophysics Data System (ADS)

    Bozhilov, K. N.

    2001-12-01

    The deep origin of the Alpe Arami garnet lherzolite massif in the Swiss Alps proposed by Dobrzhinetskaya et al. (Science, 1996) has been a focus of heated debate. One of the lines of evidence supporting an exhumation from more than 200 km depth includes the abundance, distribution, and orientation of magnesian ilmenite rods in the oldest generation of olivine. This argument has been disputed in terms of the abundance of ilmenite and consequently the maximum TiO2 content in the discussed olivine. In order to address this issue, we have directly measured the volume fraction of ilmenite of the oldest generation of olivine by applying confocal laser scanning microscopy (CLSM). CLSM is a method which allows for three-dimensional imaging and quantitative volume determination by optical sectioning of the objects. The images for 3D reconstruction and measurements were acquired from petrographic thin sections in reflected laser light with 488 nm wavelength. Measurements of more than 80 olivine grains in six thin sections of our material yielded an average volume fraction of 0.31% ilmenite in the oldest generation of olivine from Alpe Arami. This translates into 0.23 wt.% TiO2 in olivine with error in determination of ±0.097 wt.%, a value significantly different from that of 0.02 to 0.03 wt.% TiO2 determined by Hacker et al. (Science, 1997) by a broad-beam microanalysis technique. During the complex geological history of the Alpe Arami massif, several events of metamorphism are recorded which all could have caused increased mobility of the mineral components. Evidence for loss of TiO2 from olivine is the tendency for high densities of ilmenite to be restricted to cores of old grains, the complete absence of ilmenite inclusions from the younger, recrystallized, generation of olivine, and reduction in ilmenite size and abundance in more serpentinized specimens. These observations suggest that only olivine grains with the highest concentrations of ilmenite are close to the

  8. Analyzing Remodeling of Cardiac Tissue: A Comprehensive Approach Based on Confocal Microscopy and 3D Reconstructions.

    PubMed

    Seidel, Thomas; Edelmann, J-C; Sachse, Frank B

    2016-05-01

    Microstructural characterization of cardiac tissue and its remodeling in disease is a crucial step in many basic research projects. We present a comprehensive approach for three-dimensional characterization of cardiac tissue at the submicrometer scale. We developed a compression-free mounting method as well as labeling and imaging protocols that facilitate acquisition of three-dimensional image stacks with scanning confocal microscopy. We evaluated the approach with normal and infarcted ventricular tissue. We used the acquired image stacks for segmentation, quantitative analysis and visualization of important tissue components. In contrast to conventional mounting, compression-free mounting preserved cell shapes, capillary lumens and extracellular laminas. Furthermore, the new approach and imaging protocols resulted in high signal-to-noise ratios at depths up to 60 µm. This allowed extensive analyzes revealing major differences in volume fractions and distribution of cardiomyocytes, blood vessels, fibroblasts, myofibroblasts and extracellular space in control vs. infarct border zone. Our results show that the developed approach yields comprehensive data on microstructure of cardiac tissue and its remodeling in disease. In contrast to other approaches, it allows quantitative assessment of all major tissue components. Furthermore, we suggest that the approach will provide important data for physiological models of cardiac tissue at the submicrometer scale. PMID:26399990

  9. Analyzing Remodeling of Cardiac Tissue: A Comprehensive Approach Based on Confocal Microscopy and 3D Reconstructions.

    PubMed

    Seidel, Thomas; Edelmann, J-C; Sachse, Frank B

    2016-05-01

    Microstructural characterization of cardiac tissue and its remodeling in disease is a crucial step in many basic research projects. We present a comprehensive approach for three-dimensional characterization of cardiac tissue at the submicrometer scale. We developed a compression-free mounting method as well as labeling and imaging protocols that facilitate acquisition of three-dimensional image stacks with scanning confocal microscopy. We evaluated the approach with normal and infarcted ventricular tissue. We used the acquired image stacks for segmentation, quantitative analysis and visualization of important tissue components. In contrast to conventional mounting, compression-free mounting preserved cell shapes, capillary lumens and extracellular laminas. Furthermore, the new approach and imaging protocols resulted in high signal-to-noise ratios at depths up to 60 µm. This allowed extensive analyzes revealing major differences in volume fractions and distribution of cardiomyocytes, blood vessels, fibroblasts, myofibroblasts and extracellular space in control vs. infarct border zone. Our results show that the developed approach yields comprehensive data on microstructure of cardiac tissue and its remodeling in disease. In contrast to other approaches, it allows quantitative assessment of all major tissue components. Furthermore, we suggest that the approach will provide important data for physiological models of cardiac tissue at the submicrometer scale.

  10. Influences of edges and steep slopes in 3D interference and confocal microscopy

    NASA Astrophysics Data System (ADS)

    Xie, Weichang; Hagemeier, Sebastian; Woidt, Carsten; Hillmer, Harmut; Lehmann, Peter

    2016-04-01

    Optical measurement techniques are widely applied in high-resolution contour, topography and roughness measurement. In this context vertical scanning white-light interferometers and confocal microscopes have become mature instruments over the last decades. The accuracy of measurement results is highly related not only to the type and physical properties of the measuring instruments, but also to the measurement object itself. This contribution focuses on measurement effects occurring at edges and height steps using white-light interferometers of different numerical apertures. If the edge is perfectly perpendicular, batwing effects appear at height steps. These batwings show maximum height if the height-to-wavelength-ratio (HWR) is about one forth or three forth, and they disappear if the HWR value is about an integer multiple of one half. The wavelength that is relevant in this context is the effective wavelength, i.e. the center wavelength of the illuminating light multiplied by a correction factor known as the numerical aperture correction. However, in practice the edges are usually not perfectly perpendicular. In this case, the measurement results depend also on the derivative of the surface height function and they may differ from theory and the prediction according to the HWR value. Measurements of such steps show systematical effects depending on the lateral resolution of the instrument. In this context, a Linnik interferometer with a magnification of 100x and NA = 0.9 is used to characterize the three dimensional topography of more or less rectangular calibration specimens and quasi-perpendicular structures produced by the nanoimprint technology. The Linnik interferometer is equipped with LED light sources emitting at different wavelengths, so that the HWR value can be changed. This is possible since the high NA objective lenses show a rather limited depth of focus such that the temporal coherence gating may be replaced by focal gating in this

  11. Influences of edges and steep slopes in 3D interference and confocal microscopy

    NASA Astrophysics Data System (ADS)

    Xie, Weichang; Hagemeier, Sebastian; Woidt, Carsten; Hillmer, Harmut; Lehmann, Peter

    2016-04-01

    Optical measurement techniques are widely applied in high-resolution contour, topography and roughness measurement. In this context vertical scanning white-light interferometers and confocal microscopes have become mature instruments over the last decades. The accuracy of measurement results is highly related not only to the type and physical properties of the measuring instruments, but also to the measurement object itself. This contribution focuses on measurement effects occurring at edges and height steps using white-light interferometers of different numerical apertures. If the edge is perfectly perpendicular, batwing effects appear at height steps. These batwings show maximum height if the height-to-wavelength-ratio (HWR) is about one forth or three forth, and they disappear if the HWR value is about an integer multiple of one half. The wavelength that is relevant in this context is the effective wavelength, i.e. the center wavelength of the illuminating light multiplied by a correction factor known as the numerical aperture correction. However, in practice the edges are usually not perfectly perpendicular. In this case, the measurement results depend also on the derivative of the surface height function and they may differ from theory and the prediction according to the HWR value. Measurements of such steps show systematical effects depending on the lateral resolution of the instrument. In this context, a Linnik interferometer with a magnification of 100x and NA = 0.9 is used to characterize the three dimensional topography of more or less rectangular calibration specimens and quasi-perpendicular structures produced by the nanoimprint technology. The Linnik interferometer is equipped with LED light sources emitting at different wavelengths, so that the HWR value can be changed. This is possible since the high NA objective lenses show a rather limited depth of focus such that the temporal coherence gating may be replaced by focal gating in this particular

  12. 3-D laser confocal microscopy study of the oxidation of NdFeB magnets in atmospheric conditions

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

    Neodymium iron boron (NdFeB) magnets are used in a number of important applications, such as generators in gearless wind turbines, motors in electric vehicles and electronic goods (e.g.- computer hard disk drives, HDD). Hydrogen can be used as a processing gas to separate and recycle scrap sintered Nd-Fe-B magnets from end-of-life products to form a powder suitable for recycling. However, the magnets are likely to have been exposed to atmospheric conditions prior to processing, and any oxidation could lead to activation problems for the hydrogen decrepitation reaction. Many previous studies on the oxidation of NdFeB magnets have been performed at elevated temperatures; however, few studies have been formed under atmospheric conditions. In this paper a combination of 3-D laser confocal microscopy and Raman spectroscopy have been used to assess the composition, morphology and rate of oxidation/corrosion on scrap sintered NdFeB magnets. Confocal microscopy has been employed to measure the growth of surface reaction products at room temperature, immediately after exposure to air. The results showed that there was a significant height increase at the triple junctions of the Nd-rich grain boundaries. Using Raman spectroscopy, the product was shown to consist of Nd2O3 and formed only on the Nd-rich triple junctions. The diffusion coefficient of the triple junction reaction product growth at 20 °C was determined to be approximately 4 × 10-13 cm2/sec. This value is several orders of magnitude larger than values derived from the diffusion controlled oxide growth observations at elevated temperatures in the literature. This indicates that the growth of the room temperature oxidation products are likely defect enhanced processes at the NdFeB triple junctions.

  13. Multiphotonic Confocal Microscopy 3D imaging: Application to mantle sulfides in sub-arc environment (Avacha Volcano, Kamchatka)

    NASA Astrophysics Data System (ADS)

    Antoine, Bénard; Luc-Serge, Doucet; Sabine, Palle; Dmitri A., Ionov

    2010-05-01

    Petrogenetic relations in igneous rocks are usually studied in natural samples using classical optical microscopy and subsequent geochemical data acquisition. Multiphotonic Laser Scanning Confocal Microscopy (MLSCM) can be a powerful tool to section geological materials optically with sub-micrometric resolution and then generate a three-dimensional (3D) reconstruction (ca. 106 μm3 stack). MLSCM is used here to investigate textural relations of Monosulfide Solid Solution (MSS) with silicate phases in fresh spinel harzburgite xenoliths from the andesitic Avacha volcano (Kamchatka, Russia). The xenoliths contain MSS disseminated in olivine and orthopyroxene (opx) neoblasts as well as MSS-rich quenched magmatic opx veins [1]. First, Reflection Mode (RM) was tested on vein sulfides in resin-impregnated thick (120 μm) polished rock sections. Then we used a combination of Differential Interference Contrast (DIC) with a transmitted light detector, two photons-excited fluorescence (2PEF) and Second Harmonic Generation (SHG). Sequential imaging feature of the Leica TCS-SP2 software was applied. The excitation laser used for 2PEF was a COHERENT MIRA 900 with a 76Hz repetition rate and 800nm wavelength. Image stacks were analysed using ImageJ software [2]. The aim of the tests was to try to discriminate sulfides in silicate matrix as a tool for a better assessment of equilibrium conditions between the two phases. Preliminary results show that Fe-Ni rich MSS from vein and host rock have a strong auto-fluorescence in the Near UV-VIS domain (392-715 nm) whereas silicate matrix is only revealed through DIC. SHG is obtained only from dense nanocentrosymmetrical structures such as embedded medium (organic matter like glue and resin). The three images were recorded sequentially enabling efficient discrimination between the different components of the rock slices. RM permits reconstruction of the complete 3D structure of the rock slice. High resolution (ca. 0.2 μm along X-Y axis vs

  14. Detection of latent fingerprints using high-resolution 3D confocal microscopy in non-planar acquisition scenarios

    NASA Astrophysics Data System (ADS)

    Kirst, Stefan; Vielhauer, Claus

    2015-03-01

    In digitized forensics the support of investigators in any manner is one of the main goals. Using conservative lifting methods, the detection of traces is done manually. For non-destructive contactless methods, the necessity for detecting traces is obvious for further biometric analysis. High resolutional 3D confocal laser scanning microscopy (CLSM) grants the possibility for a detection by segmentation approach with improved detection results. Optimal scan results with CLSM are achieved on surfaces orthogonal to the sensor, which is not always possible due to environmental circumstances or the surface's shape. This introduces additional noise, outliers and a lack of contrast, making a detection of traces even harder. Prior work showed the possibility of determining angle-independent classification models for the detection of latent fingerprints (LFP). Enhancing this approach, we introduce a larger feature space containing a variety of statistical-, roughness-, color-, edge-directivity-, histogram-, Gabor-, gradient- and Tamura features based on raw data and gray-level co-occurrence matrices (GLCM) using high resolutional data. Our test set consists of eight different surfaces for the detection of LFP in four different acquisition angles with a total of 1920 single scans. For each surface and angles in steps of 10, we capture samples from five donors to introduce variance by a variety of sweat compositions and application influences such as pressure or differences in ridge thickness. By analyzing the present test set with our approach, we intend to determine angle- and substrate-dependent classification models to determine optimal surface specific acquisition setups and also classification models for a general detection purpose for both, angles and substrates. The results on overall models with classification rates up to 75.15% (kappa 0.50) already show a positive tendency regarding the usability of the proposed methods for LFP detection on varying surfaces in non

  15. STXM goes 3D: digital reconstruction of focal stacks as novel approach towards confocal soft x-ray microscopy.

    PubMed

    Späth, Andreas; Scho Ll, Simon; Riess, Christian; Schmidtel, Daniel; Paradossi, Gaio; Raabe, Jo Rg; Hornegger, Joachim; Fink, Rainer H

    2014-09-01

    Fresnel zone plate based soft x-ray transmission microspectroscopy has developed into a routine technique for high-resolution elemental or chemical 2D imaging of thin film specimens. The availability of high resolution Fresnel lenses with short depth of focus offers the possibility of optical slicing (in the third dimension) by focus series with resolutions in the submicron regime. We introduce a 3D reconstruction algorithm that uses a variance-based metric to assign a focus measure as basis for volume rendering. The algorithm is applied to simulated geometries and opaque soft matter specimens thus enabling 3D visualization. These studies with z-resolution of few 100nm serve as important step towards the vision of a confocal transmission x-ray microscope.

  16. FluoRender: An Application of 2D Image Space Methods for 3D and 4D Confocal Microscopy Data Visualization in Neurobiology Research

    PubMed Central

    Wan, Yong; Otsuna, Hideo; Chien, Chi-Bin; Hansen, Charles

    2013-01-01

    2D image space methods are processing methods applied after the volumetric data are projected and rendered into the 2D image space, such as 2D filtering, tone mapping and compositing. In the application domain of volume visualization, most 2D image space methods can be carried out more efficiently than their 3D counterparts. Most importantly, 2D image space methods can be used to enhance volume visualization quality when applied together with volume rendering methods. In this paper, we present and discuss the applications of a series of 2D image space methods as enhancements to confocal microscopy visualizations, including 2D tone mapping, 2D compositing, and 2D color mapping. These methods are easily integrated with our existing confocal visualization tool, FluoRender, and the outcome is a full-featured visualization system that meets neurobiologists’ demands for qualitative analysis of confocal microscopy data. PMID:23584131

  17. FluoRender: An Application of 2D Image Space Methods for 3D and 4D Confocal Microscopy Data Visualization in Neurobiology Research.

    PubMed

    Wan, Yong; Otsuna, Hideo; Chien, Chi-Bin; Hansen, Charles

    2012-01-01

    2D image space methods are processing methods applied after the volumetric data are projected and rendered into the 2D image space, such as 2D filtering, tone mapping and compositing. In the application domain of volume visualization, most 2D image space methods can be carried out more efficiently than their 3D counterparts. Most importantly, 2D image space methods can be used to enhance volume visualization quality when applied together with volume rendering methods. In this paper, we present and discuss the applications of a series of 2D image space methods as enhancements to confocal microscopy visualizations, including 2D tone mapping, 2D compositing, and 2D color mapping. These methods are easily integrated with our existing confocal visualization tool, FluoRender, and the outcome is a full-featured visualization system that meets neurobiologists' demands for qualitative analysis of confocal microscopy data.

  18. 3D Imaging of Porous Media Using Laser Scanning Confocal Microscopy with Application to Microscale Transport Processes

    SciTech Connect

    Fredrich, J.T.

    1999-02-10

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

  19. [Artefacts of confocal microscopy].

    PubMed

    Vekshin, N L; Frolov, M S

    2014-01-01

    Typical artefacts caused by using confocal fluorescent microscopy while studying living cells are considered. The role of light scattering, mobility, staining, local concentrations, etc. is discussed.

  20. 3D digital image processing for biofilm quantification from confocal laser scanning microscopy: Multidimensional statistical analysis of biofilm modeling

    NASA Astrophysics Data System (ADS)

    Zielinski, Jerzy S.

    The dramatic increase in number and volume of digital images produced in medical diagnostics, and the escalating demand for rapid access to these relevant medical data, along with the need for interpretation and retrieval has become of paramount importance to a modern healthcare system. Therefore, there is an ever growing need for processed, interpreted and saved images of various types. Due to the high cost and unreliability of human-dependent image analysis, it is necessary to develop an automated method for feature extraction, using sophisticated mathematical algorithms and reasoning. This work is focused on digital image signal processing of biological and biomedical data in one- two- and three-dimensional space. Methods and algorithms presented in this work were used to acquire data from genomic sequences, breast cancer, and biofilm images. One-dimensional analysis was applied to DNA sequences which were presented as a non-stationary sequence and modeled by a time-dependent autoregressive moving average (TD-ARMA) model. Two-dimensional analyses used 2D-ARMA model and applied it to detect breast cancer from x-ray mammograms or ultrasound images. Three-dimensional detection and classification techniques were applied to biofilm images acquired using confocal laser scanning microscopy. Modern medical images are geometrically arranged arrays of data. The broadening scope of imaging as a way to organize our observations of the biophysical world has led to a dramatic increase in our ability to apply new processing techniques and to combine multiple channels of data into sophisticated and complex mathematical models of physiological function and dysfunction. With explosion of the amount of data produced in a field of biomedicine, it is crucial to be able to construct accurate mathematical models of the data at hand. Two main purposes of signal modeling are: data size conservation and parameter extraction. Specifically, in biomedical imaging we have four key problems

  1. Highlighting the impact of aging on type I collagen: label-free investigation using confocal reflectance microscopy and diffuse reflectance spectroscopy in 3D matrix model.

    PubMed

    Guilbert, Marie; Roig, Blandine; Terryn, Christine; Garnotel, Roselyne; Jeannesson, Pierre; Sockalingum, Ganesh D; Manfait, Michel; Perraut, François; Dinten, Jean-Marc; Koenig, Anne; Piot, Olivier

    2016-02-23

    During aging, alterations of extracellular matrix proteins contribute to various pathological phenotypes. Among these alterations, type I collagen cross-linking and associated glycation products accumulation over time detrimentally affects its physico-chemical properties, leading to alterations of tissue biomechanical stability. Here, different-age collagen 3D matrices using non-destructive and label-free biophotonic techniques were analysed to highlight the impact of collagen I aging on 3D constructs, at macroscopic and microscopic levels. Matrices were prepared with collagens extracted from tail tendons of rats (newborns, young and old adults) to be within the physiological aging process. The data of diffuse reflectance spectroscopy reveal that aging leads to an inhibition of fibril assembly and a resulting decrease of gel density. Investigations by confocal reflectance microscopy highlight poor-fibrillar structures in oldest collagen networks most likely related to the glycation products accumulation. Complementarily, an infrared analysis brings out marked spectral variations in the Amide I profile, specific of the peptidic bond conformation and for carbohydrates vibrations as function of collagen-age. Interestingly, we also highlight an unexpected behavior for newborn collagen, exhibiting poorly-organized networks and microscopic features close to the oldest collagen. These results demonstrate that changes in collagen optical properties are relevant for investigating the incidence of aging in 3D matrix models.

  2. Highlighting the impact of aging on type I collagen: label-free investigation using confocal reflectance microscopy and diffuse reflectance spectroscopy in 3D matrix model

    PubMed Central

    Terryn, Christine; Garnotel, Roselyne; Jeannesson, Pierre; Sockalingum, Ganesh D.; Manfait, Michel; Perraut, François; Dinten, Jean-Marc; Koenig, Anne; Piot, Olivier

    2016-01-01

    During aging, alterations of extracellular matrix proteins contribute to various pathological phenotypes. Among these alterations, type I collagen cross-linking and associated glycation products accumulation over time detrimentally affects its physico-chemical properties, leading to alterations of tissue biomechanical stability. Here, different-age collagen 3D matrices using non-destructive and label-free biophotonic techniques were analysed to highlight the impact of collagen I aging on 3D constructs, at macroscopic and microscopic levels. Matrices were prepared with collagens extracted from tail tendons of rats (newborns, young and old adults) to be within the physiological aging process. The data of diffuse reflectance spectroscopy reveal that aging leads to an inhibition of fibril assembly and a resulting decrease of gel density. Investigations by confocal reflectance microscopy highlight poor-fibrillar structures in oldest collagen networks most likely related to the glycation products accumulation. Complementarily, an infrared analysis brings out marked spectral variations in the Amide I profile, specific of the peptidic bond conformation and for carbohydrates vibrations as function of collagen-age. Interestingly, we also highlight an unexpected behavior for newborn collagen, exhibiting poorly-organized networks and microscopic features close to the oldest collagen. These results demonstrate that changes in collagen optical properties are relevant for investigating the incidence of aging in 3D matrix models. PMID:26885896

  3. Confocal microscopy in microgravity research

    NASA Astrophysics Data System (ADS)

    Goede, A. P. H.; Brakenhoff, G. J.; Woldringh, C. L.; Aalders, J. W. G.; Imhof, J. P.; van Kralingen, P.; Mels, W. A.; Schreinemakers, P.; Zegers, A.

    We have studied the application and the feasibility of confocal scanning laser microscopy (CSLM) in microgravity research. Its superior spatial resolution and 3D imaging capabilities and its use of light as a probe, render this instrument ideally suited for the study of living biological material on a (sub-)cellular level. In this paper a number of pertinent biological microgravity experiments is listed, concentrating on the direct observation of developing cells and cellular structures under microgravity condition. A conceptual instrument design is also presented, aimed at sounding rocket application followed by Biorack/Biolab application at a later stage.

  4. Deconvolution in 3-D optical microscopy.

    PubMed

    Shaw, P

    1994-09-01

    Fluorescent probes are becoming ever more widely used in the study of subcellular structure, and determination of their three-dimensional distributions has become very important. Confocal microscopy is now a common technique for overcoming the problem of out-of-focus flare in fluorescence imaging, but an alternative method uses digital image processing of conventional fluorescence images--a technique often termed 'deconvolution' or 'restoration'. This review attempts to explain image deconvolution in a non-technical manner. It is also applicable to 3-D confocal images, and can provide a further significant improvement in clarity and interpretability of such images. Some examples of the application of image deconvolution to both conventional and confocal fluorescence images are shown.

  5. Confocal microscopy and exfoliative cytology

    PubMed Central

    Reddy, Shyam Prasad; Ramani, Pratibha; Nainani, Purshotam

    2013-01-01

    Context: Early detection of potentially malignant lesions and invasive squamous-cell carcinoma in the oral cavity could be greatly improved through techniques that permit visualization of subtle cellular changes indicative of the neoplastic transformation process. One such technique is confocal microscopy. Combining rapidity with reliability, an innovative idea has been put forward using confocal microscope in exfoliative cytology. Aims: The main objective of this study was to assess confocal microscopy for cytological diagnosis and the results were compared with that of the standard PAP stain. Settings and Design: Confocal microscope, acridine orange (AO) stain, PAP (Papanicolaou) stain. The study was designed to assess confocal microscopy for cytological diagnosis. In the process, smears of patients with (clinically diagnosed and/or suspected) oral squamous cell carcinoma as well as those of controls (normal people) were stained with acridine orange and observed under confocal microscope. The results were compared with those of the standard PAP method. Materials and Methods: Samples of buccal mucosa smears from normal patients and squamous cell carcinoma patients were made, fixed in 100% alcohol, followed by AO staining. The corresponding set of smears was stained with PAP stain using rapid PAP stain kit. The results obtained were compared with those obtained with AO confocal microscopy. Results: The study had shown nuclear changes (malignant cells) in the smears of squamous cell carcinoma patients as increased intensity of fluorescence of the nucleus, when observed under confocal microscope. Acridine orange confocal microscopy showed good amount of sensitivity and specificity (93%) in identifying malignant cells in exfoliative cytological smears. Conclusion: Confocal microscopy was found to have good sensitivity in the identification of cancer (malignant) cells in exfoliative cytology, at par with the PAP method. The rapidity of processing and screening a

  6. Confocal microscopy in transmitted light

    NASA Astrophysics Data System (ADS)

    Dodt, Hans-Ulrich; Becker, Klaus

    2003-10-01

    We developed a confocal microscope for transmitted light to visualize fine details in phase objects like unstained biological specimens. The main difficulty of confocal microscopy in transmission is the alignment of illumination and detector pinholes. This alignment was achieved by using "electronic pinholes" on the detector side. As a first step, we were able to image cells in onion skin at greater depths and with higher resolution than by using conventional microscopy.

  7. Digital differential confocal microscopy based on spatial shift transformation.

    PubMed

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

    2014-11-01

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

  8. Digital differential confocal microscopy based on spatial shift transformation.

    PubMed

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

    2014-11-01

    Differential confocal microscopy is a particularly powerful surface profilometry technique in industrial metrology due to its high axial sensitivity and insensitivity to noise. However, the practical implementation of the technique requires the accurate positioning of point detectors in three-dimensions. We describe a simple alternative based on spatial transformation of a through-focus series of images obtained from a homemade beam scanning confocal microscope. This digital differential confocal microscopy approach is described and compared with the traditional Differential confocal microscopy approach. The ease of use of the digital differential confocal microscopy system is illustrated by performing measurements on a 3D standard specimen. PMID:25303106

  9. Novel application of confocal laser scanning microscopy and 3D volume rendering toward improving the resolution of the fossil record of charcoal.

    PubMed

    Belcher, Claire M; Punyasena, Surangi W; Sivaguru, Mayandi

    2013-01-01

    Variations in the abundance of fossil charcoals between rocks and sediments are assumed to reflect changes in fire activity in Earth's past. These variations in fire activity are often considered to be in response to environmental, ecological or climatic changes. The role that fire plays in feedbacks to such changes is becoming increasingly important to understand and highlights the need to create robust estimates of variations in fossil charcoal abundance. The majority of charcoal based fire reconstructions quantify the abundance of charcoal particles and do not consider the changes in the morphology of the individual particles that may have occurred due to fragmentation as part of their transport history. We have developed a novel application of confocal laser scanning microscopy coupled to image processing that enables the 3-dimensional reconstruction of individual charcoal particles. This method is able to measure the volume of both microfossil and mesofossil charcoal particles and allows the abundance of charcoal in a sample to be expressed as total volume of charcoal. The method further measures particle surface area and shape allowing both relationships between different size and shape metrics to be analysed and full consideration of variations in particle size and size sorting between different samples to be studied. We believe application of this new imaging approach could allow significant improvement in our ability to estimate variations in past fire activity using fossil charcoals.

  10. 3D multiplexed immunoplasmonics microscopy

    NASA Astrophysics Data System (ADS)

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-01

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed

  11. 3D multiplexed immunoplasmonics microscopy

    NASA Astrophysics Data System (ADS)

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-01

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed

  12. 3D multiplexed immunoplasmonics microscopy.

    PubMed

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-21

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K(+) channel subunit KV1.1) on human cancer CD44(+) EGFR(+) KV1.1(+) MDA-MB-231 cells and reference CD44(-) EGFR(-) KV1.1(+) 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third

  13. Non-destructive 3D Imaging of Extraterrestrial Materials by Synchrotron X-ray Micro- tomography (XR-CMT) and Laser Confocal Scanning Microscopy (LCSM): Beyond Pretty Pictures

    NASA Astrophysics Data System (ADS)

    Ebel, D. S.; Greenberg, M.

    2009-05-01

    We report scientific results made possible only by the use these two non-destructive 3D imaging techniques. XR-CMT provides 3D image reconstructions at spatial resolutions of 1 to 17 micron/voxel edge. We use XR- CMT to locate potential melt-inclusion-bearing phenocrysts in batches of 100-200 micron lunar fire-fountain spherules; to locate and visualize the morphology of 1-2mm size, irregular, unmelted Ca-, Al-rich inclusions (CAIs) and to quantify chondrule/matrix ratios and chondrule size distributions in 6x6x20mm chunks of carbonaceous chondrites; to quantify the modal abundance of opaque phases in similar sized Martian meteorite fragments, and in individual 1-2mm diameter chondrules from chondrites. LCSM provides 3D image stacks at resolutions < 100 nm/pixel. We are the only group creating deconvolved image stacks of 100 to over 1000 micron long comet particle tracks in aerogel keystones from the Stardust mission. We present measurements of track morphology in 3D, and locate high-value particles using complementary synchrotron x- ray fluorescence (XRF) examination. We show that bench-top LCSM extracts maximum information about tracks and particles rapidly and cheaply prior to destructive disassembly. Using XR-CMT we quantify, for the first time, the volumetric abundances of metal grains in 1-2 mm diameter CR chondrite chondrules. Metal abundances vary from 1 to 37 vol.% between 8 chondrules (and more by inspection), in a meteorite with solar (chondritic) Fe/Si ratio, indicating that chondrules formed and accreted locally from bulk solar composition material. They are 'complementary' to each other in Fe/Si ratios. Void spaces in chondritic CAIs and chondrules are shown to be a primary feature, not due to plucking during sectioning. CAI morphology in 3D reveals pre-accretionary impact features, and various types of mineralogical layering, seen in 3D, reveal the formation history of these building blocks of planets and asteroids. We also quantify the x

  14. 3D microscopy - new powerful tools in geomaterials characterization

    NASA Astrophysics Data System (ADS)

    Mauko Pranjić, Alenka; Mladenovič, Ana; Turk, Janez; Šajna, Aljoša; Čretnik, Janko

    2016-04-01

    Microtomography (microCT) is becoming more and more widely recognized in geological sciences as a powerful tool for the spatial characterization of rock and other geological materials. Together with 3D image analysis and other complementary techniques, it has the characteristics of an innovative and non-destructive 3D microscopical technique. On the other hand its main disadvantages are low availability (only a few geological laboratories are equipped with high resolution tomographs), the relatively high prices of testing connected with the use of an xray source, technical limitations connected to the resolution and imaging of certain materials, as well as timeconsuming and complex 3D image analysis, necessary for quantification of 3D tomographic data sets. In this work three examples are presented of optimal 3D microscopy analysis of geomaterials in construction such as porosity characterization of impregnated sandstone, aerated concrete and marble prone to bowing. Studies include processes of microCT imaging, 3D data analysis and fitting of data with complementary analysis, such as confocal microscopy, mercury porosimetry, gas sorption, optical/fluorescent microscopy and scanning electron microscopy. Present work has been done in the frame of national research project 3D and 4D microscopy development of new powerful tools in geosciences (ARRS J1-7148) funded by Slovenian Research Agency.

  15. Automated cellular pathology in noninvasive confocal microscopy

    NASA Astrophysics Data System (ADS)

    Ting, Monica; Krueger, James; Gareau, Daniel

    2014-03-01

    A computer algorithm was developed to automatically identify and count melanocytes and keratinocytes in 3D reflectance confocal microscopy (RCM) images of the skin. Computerized pathology increases our understanding and enables prevention of superficial spreading melanoma (SSM). Machine learning involved looking at the images to measure the size of cells through a 2-D Fourier transform and developing an appropriate mask with the erf() function to model the cells. Implementation involved processing the images to identify cells whose image segments provided the least difference when subtracted from the mask. With further simplification of the algorithm, the program may be directly implemented on the RCM images to indicate the presence of keratinocytes in seconds and to quantify the keratinocytes size in the en face plane as a function of depth. Using this system, the algorithm can identify any irregularities in maturation and differentiation of keratinocytes, thereby signaling the possible presence of cancer.

  16. 3DSEM: A 3D microscopy dataset.

    PubMed

    Tafti, Ahmad P; Kirkpatrick, Andrew B; Holz, Jessica D; Owen, Heather A; Yu, Zeyun

    2016-03-01

    The Scanning Electron Microscope (SEM) as a 2D imaging instrument has been widely used in many scientific disciplines including biological, mechanical, and materials sciences to determine the surface attributes of microscopic objects. However the SEM micrographs still remain 2D images. To effectively measure and visualize the surface properties, we need to truly restore the 3D shape model from 2D SEM images. Having 3D surfaces would provide anatomic shape of micro-samples which allows for quantitative measurements and informative visualization of the specimens being investigated. The 3DSEM is a dataset for 3D microscopy vision which is freely available at [1] for any academic, educational, and research purposes. The dataset includes both 2D images and 3D reconstructed surfaces of several real microscopic samples. PMID:26779561

  17. 3DSEM: A 3D microscopy dataset

    PubMed Central

    Tafti, Ahmad P.; Kirkpatrick, Andrew B.; Holz, Jessica D.; Owen, Heather A.; Yu, Zeyun

    2015-01-01

    The Scanning Electron Microscope (SEM) as a 2D imaging instrument has been widely used in many scientific disciplines including biological, mechanical, and materials sciences to determine the surface attributes of microscopic objects. However the SEM micrographs still remain 2D images. To effectively measure and visualize the surface properties, we need to truly restore the 3D shape model from 2D SEM images. Having 3D surfaces would provide anatomic shape of micro-samples which allows for quantitative measurements and informative visualization of the specimens being investigated. The 3DSEM is a dataset for 3D microscopy vision which is freely available at [1] for any academic, educational, and research purposes. The dataset includes both 2D images and 3D reconstructed surfaces of several real microscopic samples. PMID:26779561

  18. 3DSEM: A 3D microscopy dataset.

    PubMed

    Tafti, Ahmad P; Kirkpatrick, Andrew B; Holz, Jessica D; Owen, Heather A; Yu, Zeyun

    2016-03-01

    The Scanning Electron Microscope (SEM) as a 2D imaging instrument has been widely used in many scientific disciplines including biological, mechanical, and materials sciences to determine the surface attributes of microscopic objects. However the SEM micrographs still remain 2D images. To effectively measure and visualize the surface properties, we need to truly restore the 3D shape model from 2D SEM images. Having 3D surfaces would provide anatomic shape of micro-samples which allows for quantitative measurements and informative visualization of the specimens being investigated. The 3DSEM is a dataset for 3D microscopy vision which is freely available at [1] for any academic, educational, and research purposes. The dataset includes both 2D images and 3D reconstructed surfaces of several real microscopic samples.

  19. Confocal fluorescence microscopy and three-dimensional reconstruction.

    PubMed

    Wright, S J; Schatten, G

    1991-05-01

    Several recent technological advances have considerably improved the field of confocal fluorescence microscopy. Improvements in confocal microscope design, new fluorescent probes and indicators, more sensitive imaging devices, and computer advances which allow for data manipulation and storage provide a convenient method to acquire complex three-dimensional (3-D) architectural details which previously were difficult or impossible to obtain from biological specimens. Applications of the laser scanning and tandem scanning confocal microscopes offer the potential for gaining powerful insights into the complex relationship of cellular structure and function. Confocal microscopy generates optical sections free from out-of-focus blur. With the development of new visualization tools to render and display complex 3-D data, a set of optical sections taken at different focal planes can be three-dimensionally reconstructed to create an animated sequence which can reveal latent features of the specimen. The combination of confocal microscopy and 3-D reconstruction provides a powerful new imaging tool to advance knowledge about structural and functional cellular properties as they occur dynamically in three dimensions.

  20. 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...

  1. 3D microscopy for microfabrication quality control

    NASA Astrophysics Data System (ADS)

    Muller, Matthew S.; De Jean, Paul D.

    2015-03-01

    A novel stereo microscope adapter, the SweptVue, has been developed to rapidly perform quantitative 3D microscopy for cost-effective microfabrication quality control. The SweptVue adapter uses the left and right stereo channels of an Olympus SZX7 stereo microscope for sample illumination and detection, respectively. By adjusting the temporal synchronization between the illumination lines projected from a Texas Instruments DLP LightCrafter and the rolling shutter on a Point Grey Flea3 CMOS camera, micrometer-scale depth features can be easily and rapidly measured at up to 5 μm resolution on a variety of microfabricated samples. In this study, the build performance of an industrial-grade Stratasys Object 300 Connex 3D printer was examined. Ten identical parts were 3D printed with a lateral and depth resolution of 42 μm and 30 μm, respectively, using both a rigid and flexible Stratasys PolyJet material. Surface elevation precision and accuracy was examined over multiple regions of interest on plateau and hemispherical surfaces. In general, the dimensions of the examined features were reproducible across the parts built using both materials. However, significant systemic lateral and height build errors were discovered, such as: decreased heights when approaching the edges of plateaus, inaccurate height steps, and poor tolerances on channel width. For 3D printed parts to be used in functional applications requiring micro-scale tolerances, they need to conform to specification. Despite appearing identical, our 3D printed parts were found to have a variety of defects that the SweptVue adapter quickly revealed.

  2. Validation of image processing tools for 3-D fluorescence microscopy.

    PubMed

    Dieterlen, Alain; Xu, Chengqi; Gramain, Marie-Pierre; Haeberlé, Olivier; Colicchio, Bruno; Cudel, Christophe; Jacquey, Serge; Ginglinger, Emanuelle; Jung, Georges; Jeandidier, Eric

    2002-04-01

    3-D optical fluorescent microscopy becomes nowadays an efficient tool for volumic investigation of living biological samples. Using optical sectioning technique, a stack of 2-D images is obtained. However, due to the nature of the system optical transfer function and non-optimal experimental conditions, acquired raw data usually suffer from some distortions. In order to carry out biological analysis, raw data have to be restored by deconvolution. The system identification by the point-spread function is useful to obtain the knowledge of the actual system and experimental parameters, which is necessary to restore raw data. It is furthermore helpful to precise the experimental protocol. In order to facilitate the use of image processing techniques, a multi-platform-compatible software package called VIEW3D has been developed. It integrates a set of tools for the analysis of fluorescence images from 3-D wide-field or confocal microscopy. A number of regularisation parameters for data restoration are determined automatically. Common geometrical measurements and morphological descriptors of fluorescent sites are also implemented to facilitate the characterisation of biological samples. An example of this method concerning cytogenetics is presented.

  3. Confocal multiview light-sheet microscopy

    PubMed Central

    Medeiros, Gustavo de; Norlin, Nils; Gunther, Stefan; Albert, Marvin; Panavaite, Laura; Fiuza, Ulla-Maj; Peri, Francesca; Hiiragi, Takashi; Krzic, Uros; Hufnagel, Lars

    2015-01-01

    Selective-plane illumination microscopy has proven to be a powerful imaging technique due to its unsurpassed acquisition speed and gentle optical sectioning. However, even in the case of multiview imaging techniques that illuminate and image the sample from multiple directions, light scattering inside tissues often severely impairs image contrast. Here we combine multiview light-sheet imaging with electronic confocal slit detection implemented on modern camera sensors. In addition to improved imaging quality, the electronic confocal slit detection doubles the acquisition speed in multiview setups with two opposing illumination directions allowing simultaneous dual-sided illumination. Confocal multiview light-sheet microscopy eliminates the need for specimen-specific data fusion algorithms, streamlines image post-processing, easing data handling and storage. PMID:26602977

  4. Confocal microscopy imaging of solid tissue

    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 acquired images as a virtual 3-dimensional structure. In many ways CLSM offers an alternative to traditional sectioning ...

  5. 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...

  6. TelePresence Confocal Laser Scanning Microscopy.

    PubMed

    Youngblom, Janey H.; Youngblom, James J.; Wilkinson, Jerry

    2001-05-01

    The advent of the Internet has allowed the development of remote access capabilities to a growing variety and number of microscopy systems. To date, the confocal microscope has not been included among these systems. At the California State University (CSU) Confocal Microscopy Core Facility, we have established a remote access confocal laser scanning microscope facility that allows users with virtually any type of computer platform to connect to our system. Our Leica TCS NT confocal system is accessible to any authorized user via the Internet by using a free software program called VNC (Virtual Network Computing). Once connectivity is established, remote users are able to control virtually all the functions to conduct real-time image analysis and quantitative assessments of their specimen. They can also move the motorized stage to view different regions of their specimen by using a software program associated with the stage. At the end of the session, all files generated during the session can be downloaded to the user's computer from a link on the CSU confocal website. A number of safeguard features have been developed to ensure security and privacy of data acquired during a remote session. PMID:12597815

  7. Confocal filtering in cathodoluminescence microscopy of nanostructures

    SciTech Connect

    Narváez, Angela C. E-mail: j.p.hoogenboom@tudelft.nl; Weppelman, I. Gerward C.; Moerland, Robert J.; Hoogenboom, Jacob P. E-mail: j.p.hoogenboom@tudelft.nl; Kruit, Pieter

    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 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.

  8. Rapid observation of unfixed, unstained human skin biopsy specimens with confocal microscopy and visualization

    NASA Astrophysics Data System (ADS)

    Masters, Barry R.; Aziz, David J.; Gmitro, Arthur F.; Kerr, James H.; O'Grady, Terence C.; Goldman, Leon

    1997-10-01

    The use of reflected light confocal microscopy is proposed to rapidly observe unfixed, unstained biopsy specimens of human skin. Reflected light laser scanning confocal microscopy was used to compare a freshly excised, unfixed, unstained biopsy specimen, and in vivo human skin. Optical sections from the ex vivo biopsy specimen of human skin and in vivo human skin were converted to red-green anaglyphs for 3D visualization. Contrast was derived from intrinsic differences in the scattering properties of the organelles and cells within the tissue. Individual cellular layers were observed in both tissues from the surface to the papillary dermis. Confocal microscopy of an unfixed, unstained biopsy specimen showed cells and cell nuclei of the stratum spinosum. Confocal microscopy of in vivo human skin demonstrated optical sectioning through a hair shaft on the upper hand. The combination of reflected light confocal microscopy and 3D visualization with red-green anaglyphs provides a rapid technique for observing fresh biopsies of human skin.

  9. Simultaneous multiplane confocal microscopy using acoustic tunable lenses.

    PubMed

    Duocastella, Martí; Vicidomini, Giuseppe; Diaspro, Alberto

    2014-08-11

    Maximizing the amount of spatiotemporal information retrieved in confocal laser scanning microscopy is crucial to understand fundamental three-dimensional (3D) dynamic processes in life sciences. However, current 3D confocal microscopy is based on an inherently slow stepwise process that consists of acquiring multiple 2D sections at different focal planes by mechanical or optical z-focus translation. Here, we show that by using an acoustically-driven optofluidic lens integrated in a commercial confocal system we can capture an entire 3D image in a single step. Our method is based on continuous axial scanning at speeds as high as 140 kHz combined with fast readout. In this way, one or more focus sweeps are produced on a pixel by pixel basis and the detected photons can be assigned to their corresponding focal plane enabling simultaneous multiplane imaging. We exemplify this method by imaging calibration and biological fluorescence samples. These results open the door to exploring new fundamental processes in science with an unprecedented time resolution.

  10. High brightness LED in confocal microscopy

    NASA Astrophysics Data System (ADS)

    Vakili, Ali; Xiong, Daxi; Rajadhyaksha, Milind; DiMarzio, Charles A.

    2015-03-01

    We have introduced a novel illumination system for line scanning confocal microscopy. Confocal microscopy is a popular imaging tool in many applications specifically in medical imaging. Line scanning confocal microscopes have been proven to provide images with resolution comparable to point scanning microscopes. In the point scanning microscopes, the light is focused onto a diffraction limited spot. A pinhole is placed conjugate to the diffraction limited spot, in front of the detector to reject the light coming from out-of-focus planes. Therefore, confocal microscopy can provide optical sectioning. The size of the pinhole determines the amount of light that reaches the detector. A large pinhole results in a blurry image since more of the out-of-focus light contribute to the image. On the other hand, a smaller pinhole rejects more of the light, leading to a lower signal-to-noise ratio. Ideally it is desired to deliver a larger amount of optical power to the diffraction limited spot to increase the signal-to-noise ratio and have a smaller pinhole to reject more of the out-of-focus light. This is the property of the illumination system. In order to get a good signal-to noise ratio in the image, the light source has to provide sufficient radiance. We have introduced a new illumination system utilizing a high brightness LED in the line scanning confocal microscope. High brightness LEDs provide more optical power compared to ordinary LEDs from a smaller area; they have higher radiance. Preliminary results from our line scanning confocal microscope show that the high brightness LED is able to provide enough radiance to obtain an image with resolution comparable with the same microscope utilizing the laser diode. However, in high frame-rate application higher radiance or lower-noise detection system is required.

  11. Combining confocal microscopy with precise force-scope optical tweezers

    NASA Astrophysics Data System (ADS)

    Richardson, Andrew C.; Reihani, Nader; Oddershede, Lene B.

    2006-08-01

    We demonstrate an example of 'confocal-tweezers' wherein confocal images and precise optical force measurements, using photodiodes, are obtained simultaneously in the x-y plane without moving the objective lens. The optical trap is produced using a 1.064μm cw laser and is combined with Leica's TCS SP5 broadband confocal microscope to trap and image living cells. The unique method by which the confocal images are created facilitates the acquisition of images in areas far from the trapping location. In addition, because the scanning process involves moving galvanic mirrors independently of the objective, the trap is held stable in position and is not subject to any error in position for the x-y scan. We have successfully trapped and confocally imaged 80nm gold colloids, 150nm gold colloids and 1μm polystyrene beads whilst making quantitative measurements of the force applied by the trap on each bead. To the best of our knowledge this is the first time that anyone has combined precise force measuring optical tweezers with confocal microscopy. We also discuss some of the technical challenges involved in advancing the experimental set up to make quantitative force measurements in combination with 3D stacking. Having proven the potential of this system in 2D, we hope to develop it further to investigate the nano-mechanics of cell division through the attachment of gold beads to fluorescently labelled organelles in S. pombe yeast cells.

  12. Intravital confocal Raman microscopy with multiplexed SERS contrast agents

    NASA Astrophysics Data System (ADS)

    McVeigh, Patrick Z.; Wilson, Brian C.

    2012-03-01

    Intravital microscopy has been demonstrated to be a powerful technique for studying the delivery of contrast or therapeutic agents to tumours growing in a realistic 3D environment at high resolution. Surface enhanced Raman scattering (SERS)-active nanoparticle contrast agents provide the ability to improve in-vivo detection of tumour tissue through multiplex detection of their uniquely bright spectral lines. However, most work to date has been carried out in-vitro or in ex-vivo tissues. Here we present the results from confocal Raman microscopy in a dorsal skinfold window chamber in mice using SERS-active gold nanoparticle contrast agents directed towards an overexpressed tumour receptor tyrosine kinase.

  13. Spatially varying regularization of deconvolution in 3D microscopy.

    PubMed

    Seo, J; Hwang, S; Lee, J-M; Park, H

    2014-08-01

    Confocal microscopy has become an essential tool to explore biospecimens in 3D. Confocal microcopy images are still degraded by out-of-focus blur and Poisson noise. Many deconvolution methods including the Richardson-Lucy (RL) method, Tikhonov method and split-gradient (SG) method have been well received. The RL deconvolution method results in enhanced image quality, especially for Poisson noise. Tikhonov deconvolution method improves the RL method by imposing a prior model of spatial regularization, which encourages adjacent voxels to appear similar. The SG method also contains spatial regularization and is capable of incorporating many edge-preserving priors resulting in improved image quality. The strength of spatial regularization is fixed regardless of spatial location for the Tikhonov and SG method. The Tikhonov and the SG deconvolution methods are improved upon in this study by allowing the strength of spatial regularization to differ for different spatial locations in a given image. The novel method shows improved image quality. The method was tested on phantom data for which ground truth and the point spread function are known. A Kullback-Leibler (KL) divergence value of 0.097 is obtained with applying spatially variable regularization to the SG method, whereas KL value of 0.409 is obtained with the Tikhonov method. In tests on a real data, for which the ground truth is unknown, the reconstructed data show improved noise characteristics while maintaining the important image features such as edges.

  14. Confocal 3D DNA Cytometry: Assessment of Required Coefficient of Variation by Computer Simulation

    PubMed Central

    Ploeger, Lennert S.; Beliën, Jeroen A.M.; Poulin, Neal M.; Grizzle, William; van Diest, Paul J.

    2004-01-01

    Background: Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. So far, sample size has been limited by the time consuming nature of the technology. Since the power of DNA histograms to resolve different stemlines depends on both the sample size and the coefficient of variation (CV) of histogram peaks, interpretation of 3D CLSM DNA histograms might be hampered by both a small sample size and a large CV. The aim of this study was to analyze the required CV for 3D CLSM DNA histograms given a realistic sample size. Methods: By computer simulation, virtual histograms were composed for sample sizes of 20000, 10000, 5000, 1000, and 273 cells and CVs of 30, 25, 20, 15, 10 and 5%. By visual inspection, the histogram quality with respect to resolution of G0/1 and G2/M peaks of a diploid stemline was assessed. Results: As expected, the interpretability of DNA histograms deteriorated with decreasing sample sizes and higher CVs. For CVs of 15% and lower, a clearly bimodal peak pattern with well distinguishable G0/1 and G2/M peaks were still seen at a sample size of 273 cells, which is our current average sample size with 3D CLSM DNA cytometry. Conclusions: For unambiguous interpretation of DNA histograms obtained using 3D CLSM, a CV of at most 15% is tolerable at currently achievable sample sizes. To resolve smaller near diploid stemlines, a CV of 10% or better should be aimed at. With currently available 3D imaging technology, this CV is achievable. PMID:15371645

  15. Exploring the living cochlea using confocal microscopy.

    PubMed

    Ulfendahl, Mats; Boutet de Monvel, Jacques; Le Calvez, Sophie

    2002-01-01

    To obtain a more integrated view of the cellular behaviour of the cochlea it is essential to observe not only wider regions of the exposed turns but also to visualize structures below the reticular lamina. Using confocal microscopy and in vitro preparations of guinea pig and mouse inner ears, cellular structures within the intact organ of Corti can be visualized at high resolution. The approach thus offers a means to investigate detailed cellular events, e.g. structural reorganization following acoustic overstimulation. Confocal microscope images, although sharper than images acquired using regular light microscopy, are still subject to problems related to light scattering within the optical system and low signal-to-noise ratio. Significant image restoration can, however, be obtained by applying a combination of wavelet denoising techniques and deconvolution algorithms. Future work will focus both on more dynamical cellular events and on new in vivo models where the inner ear is visualized at a better functional state.

  16. Confocal Raman Microscopy in Pharmaceutical Development

    NASA Astrophysics Data System (ADS)

    Haefele, Thomas F.; Paulus, Kurt

    There is a wide range of applications of confocal Raman microscopy in pharmaceutical development. It is a powerful tool to probe the distribution of components within a formulation, to characterize homogeneity of pharmaceutical samples, to determine solid state of drug substances and excipients and to characterize contaminations and foreign particulates. The information obtained by confocal Raman microscopy is extremely useful, sometimes even crucial, for drug substance design, for the development of solid and liquid formulations, as a tool for process analytics and for patent infringements and counterfeit analysis. In this chapter, those aspects and applications will be presented, focusing on solid drug formulations. This chapter will also reveal the advantages and demonstrate the synergies of Raman mapping as compared to similar imaging methods such as SEM/EDX, NIR and MIR imaging.

  17. Comprehensive volumetric confocal microscopy with adaptive focusing

    PubMed Central

    Kang, DongKyun; Yoo, Hongki; Jillella, Priyanka; Bouma, Brett E.; Tearney, Guillermo J.

    2011-01-01

    Comprehensive microscopy of distal esophagus could greatly improve the screening and surveillance of esophageal diseases such as Barrett’s esophagus by providing histomorphologic information over the entire region at risk. Spectrally encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technology that can be configured to image the entire distal esophagus by helically scanning the beam using optics within a balloon-centering probe. It is challenging to image the human esophagus in vivo with balloon-based SECM, however, because patient motion and anatomic tissue surface irregularities decenter the optics, making it difficult to keep the focus at a predetermined location within the tissue as the beam is scanned. In this paper, we present a SECM probe equipped with an adaptive focusing mechanism that can compensate for tissue surface irregularity and dynamic focal variation. A tilted arrangement of the objective lens is employed in the SECM probe to provide feedback signals to an adaptive focusing mechanism. The tilted configuration also allows the probe to obtain reflectance confocal data from multiple depth levels, enabling the acquisition of three-dimensional volumetric data during a single scan of the probe. A tissue phantom with a surface area of 12.6 cm2 was imaged using the new SECM probe, and 8 large-area reflectance confocal microscopy images were acquired over the depth range of 56 μm in 20 minutes. Large-area SECM images of excised swine small intestine tissue were also acquired, enabling the visualization of villous architecture, epithelium, and lamina propria. The adaptive focusing mechanism was demonstrated to enable acquisition of in-focus images even when the probe was not centered and the tissue surface was irregular. PMID:21698005

  18. Segmentation of densely populated cell nuclei from confocal image stacks using 3D non-parametric shape priors.

    PubMed

    Ong, Lee-Ling S; Wang, Mengmeng; Dauwels, Justin; Asada, H Harry

    2014-01-01

    An approach to jointly estimate 3D shapes and poses of stained nuclei from confocal microscopy images, using statistical prior information, is presented. Extracting nuclei boundaries from our experimental images of cell migration is challenging due to clustered nuclei and variations in their shapes. This issue is formulated as a maximum a posteriori estimation problem. By incorporating statistical prior models of 3D nuclei shapes into level set functions, the active contour evolutions applied on the images is constrained. A 3D alignment algorithm is developed to build the training databases and to match contours obtained from the images to them. To address the issue of aligning the model over multiple clustered nuclei, a watershed-like technique is used to detect and separate clustered regions prior to active contour evolution. Our method is tested on confocal images of endothelial cells in microfluidic devices, compared with existing approaches.

  19. Estimation of single cell volume from 3D confocal images using automatic data processing

    NASA Astrophysics Data System (ADS)

    Chorvatova, A.; Cagalinec, M.; Mateasik, A.; Chorvat, D., Jr.

    2012-06-01

    Cardiac cells are highly structured with a non-uniform morphology. Although precise estimation of their volume is essential for correct evaluation of hypertrophic changes of the heart, simple and unified techniques that allow determination of the single cardiomyocyte volume with sufficient precision are still limited. Here, we describe a novel approach to assess the cell volume from confocal microscopy 3D images of living cardiac myocytes. We propose a fast procedure based on segementation using active deformable contours. This technique is independent on laser gain and/or pinhole settings and it is also applicable on images of cells stained with low fluorescence markers. Presented approach is a promising new tool to investigate changes in the cell volume during normal, as well as pathological growth, as we demonstrate in the case of cell enlargement during hypertension in rats.

  20. 3D differential phase contrast microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Michael; Tian, Lei; Waller, Laura

    2016-03-01

    We demonstrate three-dimensional (3D) optical phase and amplitude reconstruction based on coded source illumination using a programmable LED array. Multiple stacks of images along the optical axis are computed from recorded intensities captured by multiple images under off-axis illumination. Based on the first Born approximation, a linear differential phase contrast (DPC) model is built between 3D complex index of refraction and the intensity stacks. Therefore, 3D volume reconstruction can be achieved via a fast inversion method, without the intermediate 2D phase retrieval step. Our system employs spatially partially coherent illumination, so the transverse resolution achieves twice the NA of coherent systems, while axial resolution is also improved 2× as compared to holographic imaging.

  1. Combined Confocal and Magnetic Resonance Microscopy

    SciTech Connect

    Wind, Robert A.; Majors, Paul D.; Minard, Kevin R.; Ackerman, Eric J.; Daly, Don S.; Holtom, Gary R.; Thrall, Brian D.; Weber, Thomas J.

    2002-05-12

    Confocal and magnetic resonance microscopy are both used to study live cells in a minimally invasive way. Both techniques provide complementary information. Therefore, by examining cells simultaneously with both methodologies, more detailed information is obtained than is possible with each of the microscopes individually. In this paper two configurations of a combined confocal and magnetic resonance microscope described. In both cases the sample compartment is part of a temperature regulated perfusion system. The first configuration is capable of studying large single cells or three-dimensional cell agglomerates, whereas with the second configuration monolayers of mammalian cells can be investigated . Combined images are shown of Xenopus laevis frog oocytes, model JB6 tumor spheroids, and a single layer of Chinese hamster ovary cells. Finally, potential applications of the combined microscope are discussed.

  2. Digital confocal microscopy through a multimode fiber

    NASA Astrophysics Data System (ADS)

    Loterie, Damien; Farahi, Salma; Papadopoulos, Ioannis; Goy, Alexandre; Psaltis, Demetri; Moser, Christophe

    2015-09-01

    Acquiring high-contrast optical images deep inside biological tissues is still a challenging problem. Confocal microscopy is an important tool for biomedical imaging since it improves image quality by rejecting background signals. However, it suffers from low sensitivity in deep tissues due to light scattering. Recently, multimode fibers have provided a new paradigm for minimally invasive endoscopic imaging by controlling light propagation through them. Here we introduce a combined imaging technique where confocal images are acquired through a multimode fiber. We achieve this by digitally engineering the excitation wavefront and then applying a virtual digital pinhole on the collected signal. In this way, we are able to acquire images through the fiber with significantly increased contrast. With a fiber of numerical aperture 0.22, we achieve a lateral resolution of 1.5um, and an axial resolution of 12.7um. The point-scanning rate is currently limited by our spatial light modulator (20Hz).

  3. Probing individual molecules with confocal fluorescence microscopy.

    PubMed

    Nie, S; Chiu, D T; Zare, R N

    1994-11-11

    Confocal fluorescence microscopy coupled with a diffraction-limited laser beam and a high-efficiency detection system has been used to study the diffusive movement and emission process of individual fluorescent molecules in the liquid phase at room temperature. The high detection sensitivity achieved at fast data acquisition speeds (greater than 1 kilohertz) allows real-time observation of single-molecule fluorescence without statistical analysis. The results show fluorescence-cycle saturation at the single-molecule level and multiple recrossings of a single molecule into and out of the probe volume as well as the triplet state.

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

    SciTech Connect

    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.

  5. 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...

  6. Quantitative confocal microscopy: beyond a pretty picture.

    PubMed

    Jonkman, James; Brown, Claire M; Cole, Richard W

    2014-01-01

    Quantitative optical microscopy has become the norm, with the confocal laser-scanning microscope being the workhorse of many imaging laboratories. Generating quantitative data requires a greater emphasis on the accurate operation of the microscope itself, along with proper experimental design and adequate controls. The microscope, which is more accurately an imaging system, cannot be treated as a "black box" with the collected data viewed as infallible. There needs to be regularly scheduled performance testing that will ensure that quality data are being generated. This regular testing also allows for the tracking of metrics that can point to issues before they result in instrument malfunction and downtime. In turn, images must be collected in a manner that is quantitative with maximal signal to noise (which can be difficult depending on the application) without data clipping. Images must then be processed to correct for background intensities, fluorophore cross talk, and uneven field illumination. With advanced techniques such as spectral imaging, Förster resonance energy transfer, and fluorescence-lifetime imaging microscopy, experimental design needs to be carefully planned out and include all appropriate controls. Quantitative confocal imaging in all of these contexts and more will be explored within the chapter. PMID:24974025

  7. A linear programming approach to reconstructing subcellular structures from confocal images for automated generation of representative 3D cellular models

    PubMed Central

    Wood, Scott T.; Dean, Brian C.; Dean, Delphine

    2013-01-01

    This paper presents a novel computer vision algorithm to analyze 3D stacks of confocal images of fluorescently stained single cells. The goal of the algorithm is to create representative in silico model structures that can be imported into finite element analysis software for mechanical characterization. Segmentation of cell and nucleus boundaries is accomplished via standard thresholding methods. Using novel linear programming methods, a representative actin stress fiber network is generated by computing a linear superposition of fibers having minimum discrepancy compared with an experimental 3D confocal image. Qualitative validation is performed through analysis of seven 3D confocal image stacks of adherent vascular smooth muscle cells (VSMCs) grown in 2D culture. The presented method is able to automatically generate 3D geometries of the cell's boundary, nucleus, and representative F-actin network based on standard cell microscopy data. These geometries can be used for direct importation and implementation in structural finite element models for analysis of the mechanics of a single cell to potentially speed discoveries in the fields of regenerative medicine, mechanobiology, and drug discovery. PMID:23395283

  8. 3D electron microscopy of biological nanomachines: principles and applications.

    PubMed

    Sorzano, C O S; Jonic, S; Cottevieille, M; Larquet, E; Boisset, N; Marco, S

    2007-11-01

    Transmission electron microscopy is a powerful technique for studying the three-dimensional (3D) structure of a wide range of biological specimens. Knowledge of this structure is crucial for fully understanding complex relationships among macromolecular complexes and organelles in living cells. In this paper, we present the principles and main application domains of 3D transmission electron microscopy in structural biology. Moreover, we survey current developments needed in this field, and discuss the close relationship of 3D transmission electron microscopy with other experimental techniques aimed at obtaining structural and dynamical information from the scale of whole living cells to atomic structure of macromolecular complexes.

  9. Reflectance Confocal Microscopy for Inflammatory Skin Diseases.

    PubMed

    Agozzino, M; Gonzalez, S; Ardigò, M

    2016-10-01

    In vivo reflectance confocal microscopy (RCM) is a relatively novel non-invasive tool for microscopic evaluation of the skin used prevalently for diagnosis and management of skin tumour. Its axial resolution, its non-invasive and easy clinical application represents the goals for a large diffusion of this technique. During the last 15 years, RCM has been demonstrated to be able to increase the sensibility and sensitivity of dermoscopy in the diagnosis of skin tumours integrating in real time clinic, dermoscopic and microscopic information useful for the definition of malignancy. Despite to date, no large comparative studies on inflammatory skin diseases has been published in the literature, several papers already showed that RCM has a potential for the evaluation of the descriptive features of the most common inflammatory skin diseases as psoriasis, lupus erythematosus, contact dermatitis and others. The aim of the application of this technique in non-neoplastic skin diseases has been prevalently focused on the possibility of clinical diagnosis confirmation, as well as therapeutic management. Moreover, the use of RCM as driver for an optimised skin biopsy has been also followed in order to reduce the number of unsuccessful histopathological examination. In this review article we describe the confocal features of the major groups of inflammatory skin disorders focusing on psoriasiform dermatitis, interface dermatitis and spongiotic dermatitis. PMID:26996333

  10. Reflectance Confocal Microscopy for Inflammatory Skin Diseases.

    PubMed

    Agozzino, M; Gonzalez, S; Ardigò, M

    2016-10-01

    In vivo reflectance confocal microscopy (RCM) is a relatively novel non-invasive tool for microscopic evaluation of the skin used prevalently for diagnosis and management of skin tumour. Its axial resolution, its non-invasive and easy clinical application represents the goals for a large diffusion of this technique. During the last 15 years, RCM has been demonstrated to be able to increase the sensibility and sensitivity of dermoscopy in the diagnosis of skin tumours integrating in real time clinic, dermoscopic and microscopic information useful for the definition of malignancy. Despite to date, no large comparative studies on inflammatory skin diseases has been published in the literature, several papers already showed that RCM has a potential for the evaluation of the descriptive features of the most common inflammatory skin diseases as psoriasis, lupus erythematosus, contact dermatitis and others. The aim of the application of this technique in non-neoplastic skin diseases has been prevalently focused on the possibility of clinical diagnosis confirmation, as well as therapeutic management. Moreover, the use of RCM as driver for an optimised skin biopsy has been also followed in order to reduce the number of unsuccessful histopathological examination. In this review article we describe the confocal features of the major groups of inflammatory skin disorders focusing on psoriasiform dermatitis, interface dermatitis and spongiotic dermatitis.

  11. Nondestructive 3D confocal laser imaging with deconvolution of seven whole stardust tracks with complementary XRF and quantitative analysis

    SciTech Connect

    Greenberg, M.; Ebel, D.S.

    2009-03-19

    We present a nondestructive 3D system for analysis of whole Stardust tracks, using a combination of Laser Confocal Scanning Microscopy and synchrotron XRF. 3D deconvolution is used for optical corrections, and results of quantitative analyses of several tracks are presented. The Stardust mission to comet Wild 2 trapped many cometary and ISM particles in aerogel, leaving behind 'tracks' of melted silica aerogel on both sides of the collector. Collected particles and their tracks range in size from submicron to millimeter scale. Interstellar dust collected on the obverse of the aerogel collector is thought to have an average track length of {approx}15 {micro}m. It has been our goal to perform a total non-destructive 3D textural and XRF chemical analysis on both types of tracks. To that end, we use a combination of Laser Confocal Scanning Microscopy (LCSM) and X Ray Florescence (XRF) spectrometry. Utilized properly, the combination of 3D optical data and chemical data provides total nondestructive characterization of full tracks, prior to flattening or other destructive analysis methods. Our LCSM techniques allow imaging at 0.075 {micro}m/pixel, without the use of oil-based lenses. A full textural analysis on track No.82 is presented here as well as analysis of 6 additional tracks contained within 3 keystones (No.128, No.129 and No.140). We present a method of removing the axial distortion inherent in LCSM images, by means of a computational 3D Deconvolution algorithm, and present some preliminary experiments with computed point spread functions. The combination of 3D LCSM data and XRF data provides invaluable information, while preserving the integrity of the samples for further analysis. It is imperative that these samples, the first extraterrestrial solids returned since the Apollo era, be fully mapped nondestructively in 3D, to preserve the maximum amount of information prior to other, destructive analysis.

  12. Immunofluorescence and Confocal Microscopy of Neutrophils

    PubMed Central

    Allen, Lee-Ann H.

    2015-01-01

    Rapid recruitment of neutrophils to sites of infection and their ability to phagocytose and kill microbes is an important aspect of the innate immune response. Challenges associated with imaging of these cells include their short lifespan and small size and the fact that unstimulated cells are nonadherent. In addition, although cytoplasmic granules are plentiful, the abundance of many other organelles is diminished. Here we reprise methods for analysis of resting and activated cells using immunofluorescence and confocal microscopy, including kinetic analysis of phagosome maturation and degranulation, and detection of intraphagosomal superoxide accumulation. We describe approaches for rapid cell fixation and permeabilization that maximize antigen detection and discuss other variables that also affect data interpretation and image quality (such as cell spreading, degranulation, and phagocytosis). Finally, we show that these methods are also applicable to studies of neutrophil interactions with the extracellular matrix. PMID:24504957

  13. Confocal reference free traction force microscopy

    PubMed Central

    Bergert, Martin; Lendenmann, Tobias; Zündel, Manuel; Ehret, Alexander E.; Panozzo, Daniele; Richner, Patrizia; Kim, David K.; Kress, Stephan J. P.; Norris, David J.; Sorkine-Hornung, Olga; Mazza, Edoardo; Poulikakos, Dimos; Ferrari, Aldo

    2016-01-01

    The mechanical wiring between cells and their surroundings is fundamental to the regulation of complex biological processes during tissue development, repair or pathology. Traction force microscopy (TFM) enables determination of the actuating forces. Despite progress, important limitations with intrusion effects in low resolution 2D pillar-based methods or disruptive intermediate steps of cell removal and substrate relaxation in high-resolution continuum TFM methods need to be overcome. Here we introduce a novel method allowing a one-shot (live) acquisition of continuous in- and out-of-plane traction fields with high sensitivity. The method is based on electrohydrodynamic nanodrip-printing of quantum dots into confocal monocrystalline arrays, rendering individually identifiable point light sources on compliant substrates. We demonstrate the undisrupted reference-free acquisition and quantification of high-resolution continuous force fields, and the simultaneous capability of this method to correlatively overlap traction forces with spatial localization of proteins revealed using immunofluorescence methods. PMID:27681958

  14. Imaging white adipose tissue with confocal microscopy.

    PubMed

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

    2014-01-01

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

  15. Digital confocal microscopy through a multimode fiber

    NASA Astrophysics Data System (ADS)

    Loterie, Damien; Farahi, Salma; Papadopoulos, Ioannis; Goy, Alexandre; Psaltis, Demetri; Moser, Christophe

    2015-09-01

    Confocal laser-scanning microscopy is a well-known optical imaging method where a pinhole is used in the illumination and detection pathways of a normal microscope, in order to selectively excite and detect a particular focal volume. The advantage of this method is a significant increase in contrast, due to the rejection of background contributions to the signal. Here, we propose to apply this method in the context of multimode fiber endoscopy. Due to modal scrambling, it is not possible to use a physical pinhole to filter light signals that have travel through multimode fibers. Instead, we use a transmission matrix approach to characterize the propagation of light through the fiber, and we apply the filtering operation in the digital domain.

  16. Towards Single Cell Traction Microscopy within 3D Collagen Matrices

    PubMed Central

    Hall, Matthew S.; Long, Rong; Feng, Xinzeng; Huang, YuLing; Hui, Chung-Yuen; Wu, Mingming

    2013-01-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell-ECM and cell-cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cells migration within collagen gels. PMID:23806281

  17. Microscopy in 3D: a biologist’s toolbox

    PubMed Central

    Fischer, Robert S.; Wu, Yicong; Kanchanawong, Pakorn; Shroff, Hari; Waterman, Clare M.

    2012-01-01

    The power of fluorescence microscopy to study cellular structures and macromolecular complexes spans a wide range of size scales, from studies of cell behavior and function in physiological, three-dimensional (3D) environments, to understanding the molecular architecture of organelles. At each length scale, the challenge in 3D imaging is to extract the most spatial and temporal resolution possible while limiting photodamage/bleaching to living cells. A number of advancements in 3D fluorescence microscopy now offer higher resolution, improved speed, and reduced photobleaching relative to traditional point-scanning microscopy methods. Here, we discuss a few specific microscopy modalities that we believe will be particularly advantageous in imaging cells and subcellular structures in physiologically relevant 3D environments. PMID:22047760

  18. Oscillating optical tweezer-based 3-D confocal microrheometer for investigating the intracellular micromechanics and structures

    NASA Astrophysics Data System (ADS)

    Ou-Yang, H. D.; Rickter, E. A.; Pu, C.; Latinovic, O.; Kumar, A.; Mengistu, M.; Lowe-Krentz, L.; Chien, S.

    2005-08-01

    Mechanical properties of living biological cells are important for cells to maintain their shapes, support mechanical stresses and move through tissue matrix. The use of optical tweezers to measure micromechanical properties of cells has recently made significant progresses. This paper presents a new approach, the oscillating optical tweezer cytorheometer (OOTC), which takes advantage of the coherent detection of harmonically modulated particle motions by a lock-in amplifier to increase sensitivity, temporal resolution and simplicity. We demonstrate that OOTC can measure the dynamic mechanical modulus in the frequency range of 0.1-6,000 Hz at a rate as fast as 1 data point per second with submicron spatial resolution. More importantly, OOTC is capable of distinguishing the intrinsic non-random temporal variations from random fluctuations due to Brownian motion; this capability, not achievable by conventional approaches, is particular useful because living systems are highly dynamic and often exhibit non-thermal, rhythmic behavior in a broad time scale from a fraction of a second to hours or days. Although OOTC is effective in measuring the intracellular micromechanical properties, unless we can visualize the cytoskeleton in situ, the mechanical property data would only be as informative as that of "Blind men and the Elephant". To solve this problem, we take two steps, the first, to use of fluorescent imaging to identify the granular structures trapped by optical tweezers, and second, to integrate OOTC with 3-D confocal microscopy so we can take simultaneous, in situ measurements of the micromechanics and intracellular structure in living cells. In this paper, we discuss examples of applying the oscillating tweezer-based cytorheometer for investigating cultured bovine endothelial cells, the identification of caveolae as some of the granular structures in the cell as well as our approach to integrate optical tweezers with a spinning disk confocal microscope.

  19. Toward single cell traction microscopy within 3D collagen matrices

    SciTech Connect

    Hall, Matthew S.; Long, Rong; Feng, Xinzeng; Huang, YuLing; Hui, Chung-Yuen; Wu, Mingming

    2013-10-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. - Highlights: • Review of the current state of the art in 3D cell traction force microscopy. • Bulk and micro-characterization of remodelable fibrous collagen gels. • Strategies for performing 3D cell traction microscopy within collagen gels.

  20. 3-D reconstruction of neurons from multichannel confocal laser scanning image series.

    PubMed

    Wouterlood, Floris G

    2014-01-01

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

  1. Fringe projection 3D microscopy with the general imaging model.

    PubMed

    Yin, Yongkai; Wang, Meng; Gao, Bruce Z; Liu, Xiaoli; Peng, Xiang

    2015-03-01

    Three-dimensional (3D) imaging and metrology of microstructures is a critical task for the design, fabrication, and inspection of microelements. Newly developed fringe projection 3D microscopy is presented in this paper. The system is configured according to camera-projector layout and long working distance lenses. The Scheimpflug principle is employed to make full use of the limited depth of field. For such a specific system, the general imaging model is introduced to reach a full 3D reconstruction. A dedicated calibration procedure is developed to realize quantitative 3D imaging. Experiments with a prototype demonstrate the accessibility of the proposed configuration, model, and calibration approach.

  2. Deep stroma investigation by confocal microscopy

    NASA Astrophysics Data System (ADS)

    Rossi, Francesca; Tatini, Francesca; Pini, Roberto; Valente, Paola; Ardia, Roberta; Buzzonetti, Luca; Canovetti, Annalisa; Malandrini, Alex; Lenzetti, Ivo; Menabuoni, Luca

    2015-03-01

    Laser assisted keratoplasty is nowadays largely used to perform minimally invasive surgery and partial thickness keratoplasty [1-3]. The use of the femtosecond laser enables to perform a customized surgery, solving the specific problem of the single patient, designing new graft profiles and partial thickness keratoplasty (PTK). The common characteristics of the PTKs and that make them eligible respect to the standard penetrating keratoplasty, are: the preservation of eyeball integrity, a reduced risk of graft rejection, a controlled postoperative astigmatism. On the other hand, the optimal surgical results after these PTKs are related to a correct comprehension of the deep stroma layers morphology, which can help in the identification of the correct cleavage plane during surgeries. In the last years some studies were published, giving new insights about the posterior stroma morphology in adult subjects [4,5]. In this work we present a study performed on two groups of tissues: one group is from 20 adult subjects aged 59 +/- 18 y.o., and the other group is from 15 young subjects, aged 12+/-5 y.o.. The samples were from tissues not suitable for transplant in patients. Confocal microscopy and Environmental Scanning Electron Microscopy (ESEM) were used for the analysis of the deep stroma. The preliminary results of this analysis show the main differences in between young and adult tissues, enabling to improve the knowledge of the morphology and of the biomechanical properties of human cornea, in order to improve the surgical results in partial thickness keratoplasty.

  3. 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

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

    PubMed

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

    2013-01-01

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

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

    PubMed

    Paddock, Stephen W; Eliceiri, Kevin W

    2014-01-01

    Confocal microscopy is an established light microscopical technique for imaging fluorescently labeled specimens with significant three-dimensional structure. Applications of confocal microscopy in the biomedical sciences include the imaging of the spatial distribution of macromolecules in either fixed or living cells, the automated collection of 3D data, the imaging of multiple labeled specimens and the measurement of physiological events in living cells. The laser scanning confocal microscope continues to be chosen for most routine work although a number of instruments have been developed for more specific applications. Significant improvements have been made to all areas of the confocal approach, not only to the instruments themselves, but also to the protocols of specimen preparation, to the analysis, the display, the reproduction, sharing and management of confocal images using bioinformatics techniques. PMID:24052346

  6. Reflectance confocal microscopy for inflammatory skin diseases.

    PubMed

    Ardigò, M; Prow, T; Agozzino, M; Soyer, P; Berardesca, E

    2015-10-01

    Reflectance confocal microscopy evaluation of inflammatory skin diseases represents a relatively new indication that, during the last 5 years, has shown an increasing interest with consequent progressive increment of publications in literature. The success of RCM in this filed of dermatology is directly related to the high needing of non-invasive techniques able to reduce the number of skin biopsies and support the clinical diagnosis and patient's management. RCM demonstrated to visualize microscopic descriptors of inflammatory and pigmentary skin conditions with good reproducibility between observer and high grade of correspondence with optical histology. Moreover, RCM has shown to provide sufficient data to support clinical diagnosis and differential diagnosis of inflammatory and pigmentary skin diseases. Recently, several works published in literature have opened the prospective to use RCM also for therapeutic follow-up in order to monitor the improvement of the microscopic parameters and help to prevent treatment side effects. In this review article we present some examples of RCM application in inflammatory and pigmentary diseases. PMID:26333554

  7. 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...

  8. Latest developments and opportunities for 3D analysis of biological samples by confocal μ-XRF

    NASA Astrophysics Data System (ADS)

    Perez, Roberto D.; Sánchez, Héctor J.; Perez, Carlos A.; Rubio, Marcelo

    2010-02-01

    X-ray fluorescence analysis performed with a primary radiation focused in the micrometer range is known as micro-X-ray fluorescence (μ-XRF). It is characterized by a penetration depth higher than other micro-analytical methods, reaching hundreds of micrometers in biological samples. This characteristic of the X-ray beam can be employed in 3D analysis. An innovative method to perform 3D analysis by μ-XRF is the so-called confocal setup. The confocal setup consists of X-ray lenses in the excitation as well as in the detection channel. In this configuration, a micro-volume defined by the overlap of the foci of both X-ray lenses is analyzed. Scanning this micro-volume through the sample can be used to perform a study in three dimensions. At present, X-ray lenses used in confocal μ-XRF experiments are mainly glass capillaries and polycapillaries. Glass capillaries are used in the excitation channel with sources of high photon flux like synchrotron radiation. Half polycapillaries or conical polycapillary concentrators are used almost exclusively in the detection channel. Spatial resolution of the confocal μ-XRF depends on the dimensions of the foci of both X-ray lenses. The overlap of these foci forms an ellipsoid which is the probing volume of the confocal setup. The axis length of the probing volume reported in confocal μ-XRF experiments are of order of few tens of micrometer. In our confocal setup, we used a commercial glass monocapillary in the excitation channel and a monolithic half polycapillary in the detection channel. The polycapillary was home-made by means of drawing of multibundles of glass capillaries in a heating furnace. The experiment was carried out at the beamline D09B-XRF of the Synchrotron Light National Laboratory (Laboratório Nacional de Luz Síncrotron, LNLS) using white beam. A model for the theoretical description of X-ray fluorescence intensity registered by confocal μ-XRF was introduced by Malzer and Kanngieβer [2005. A model for the

  9. 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.

  10. Subcellular Microanatomy by 3D Deconvolution Brightfield Microscopy: Method and Analysis Using Human Chromatin in the Interphase Nucleus

    PubMed Central

    Tadrous, Paul Joseph

    2012-01-01

    Anatomy has advanced using 3-dimensional (3D) studies at macroscopic (e.g., dissection, injection moulding of vessels, radiology) and microscopic (e.g., serial section reconstruction with light and electron microscopy) levels. This paper presents the first results in human cells of a new method of subcellular 3D brightfield microscopy. Unlike traditional 3D deconvolution and confocal techniques, this method is suitable for general application to brightfield microscopy. Unlike brightfield serial sectioning it has subcellular resolution. Results are presented of the 3D structure of chromatin in the interphase nucleus of two human cell types, hepatocyte and plasma cell. I show how the freedom to examine these structures in 3D allows greater morphological discrimination between and within cell types and the 3D structural basis for the classical “clock-face” motif of the plasma cell nucleus is revealed. Potential for further applications discussed. PMID:22567315

  11. 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...

  12. Dual-detection confocal microscopy: high-speed surface profiling without depth scanning

    NASA Astrophysics Data System (ADS)

    Lee, Dong-Ryoung; Gweon, Dae-Gab; Yoo, Hongki

    2016-03-01

    We propose a new method for three-dimensional (3-D) imaging without depth scanning that we refer to as the dual-detection confocal microscopy (DDCM). Compared to conventional confocal microscopy, DDCM utilizes two pinholes of different sizes. DDCM generates two axial response curves which have different stiffness according to the pinhole diameters. The two axial response curves can draw the characteristics curve of the system which shows the relationship between the axial position of the sample and the intensity ratio. Utilizing the characteristic curve, the DDCM reconstructs a 3-D surface profile with a single 2-D scanning. The height of each pixel is calculated by the intensity ratio of the pixel and the intensity ratio curve. Since the height information can be obtained directly from the characteristic curve without depth scanning, a major advantage of DDCM over the conventional confocal microscopy is a speed. The 3-D surface profiling time is dramatically reduced. Furthermore, DDCM can measure 3-D images without the influence of the sample condition since the intensity ratio is independent of the quantum yield and reflectance. We present two types of DDCM, such as a fluorescence microscopy and a reflectance microscopy. In addition, we extend the measurement range axially by varying the pupil function. Here, we demonstrate the working principle of DDCM and the feasibility of the proposed methods.

  13. Managing multiple image stacks from confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Zerbe, Joerg; Goetze, Christian H.; Zuschratter, Werner

    1999-05-01

    A major goal in neuroanatomy is to obtain precise information about the functional organization of neuronal assemblies and their interconnections. Therefore, the analysis of histological sections frequently requires high resolution images in combination with an overview about the structure. To overcome this conflict we have previously introduced a software for the automatic acquisition of multiple image stacks (3D-MISA) in confocal laser scanning microscopy. Here, we describe a Windows NT based software for fast and easy navigation through the multiple images stacks (MIS-browser), the visualization of individual channels and layers and the selection of user defined subregions. In addition, the MIS browser provides useful tools for the visualization and evaluation of the datavolume, as for instance brightness and contrast corrections of individual layers and channels. Moreover, it includes a maximum intensity projection, panning and zoom in/out functions within selected channels or focal planes (x/y) and tracking along the z-axis. The import module accepts any tiff-format and reconstructs the original image arrangement after the user has defined the sequence of images in x/y and z and the number of channels. The implemented export module allows storage of user defined subregions (new single image stacks) for further 3D-reconstruction and evaluation.

  14. 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.

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

    PubMed Central

    BESSELING, TH; JOSE, J; BLAADEREN, A VAN

    2015-01-01

    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. PMID:25444358

  16. 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...

  17. 3D super-resolution microscopy of bacterial division machinery

    NASA Astrophysics Data System (ADS)

    Vedyaykin, A. D.; Sabantsev, A. V.; Vishnyakov, I. E.; Morozova, N. E.; Polinovskaya, V. S.; Khodorkovskii, M. A.

    2016-08-01

    Super-resolution microscopy is a promising tool for the field of microbiology, as bacteria sizes are comparable to the resolution limit of light microscopy. Bacterial division machinery and FtsZ protein in particular attract much attention of scientists who use different super-resolution microscopy techniques, but most of the available data on FtsZ structures was obtained using two-dimensional (2D) super-resolution microscopy. Using 3D single-molecule localization microscopy (SMLM, namely dSTORM) to visualize FtsZ, we demonstrate that this approach allows more accurate interpretation of super-resolution images and provides new opportunities for the study of complex structures like bacterial divisome.

  18. Confocal microscopy via multimode fibers: fluorescence bandwidth

    NASA Astrophysics Data System (ADS)

    Loterie, Damien; Psaltis, Demetri; Moser, Christophe

    2016-03-01

    We recently described a method for confocal reflection imaging through fibers, as a way to increase contrast when imaging unstained biological specimens. Using a transmission matrix, focused spots can be created at the distal end of a fiber. The backscattered field coming back from the sample can be filtered using optical correlation to obtain spatial selectivity in the detection. In this proceedings article, we briefly review the working principle of this method, and we discuss how the scheme could be adapted to confocal fluorescence imaging. In particular, we show simulations of the achievable detection bandwidth when using step-index multimode fibers as imaging devices.

  19. 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 ...

  20. Stripe-shaped apertures in confocal microscopy.

    PubMed

    Shen, Shuhao; Zhu, Bingzhao; Zheng, Yao; Gong, Wei; Si, Ke

    2016-09-20

    We have theoretically verified that, compared with the aperture shapes of previous research, combining two stripe-shaped apertures in a confocal microscope with a finite-sized pinhole improves the axial resolution to a certain extent. Because different stripe shapes cause different effects, we also investigated the relationships among resolution, shapes, pinhole size, and the signal-to-background ratio.

  1. 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

  2. Multiplex acquisition approach for high speed 3D measurements with a chromatic confocal microscope

    NASA Astrophysics Data System (ADS)

    Taphanel, Miro; Zink, Ralf; Längle, Thomas; Beyerer, Jürgen

    2015-05-01

    A technical realization of a multispectral camera is proposed, by multiplexing a light source with six different spectra. A monochrome line scan camera with six pixel rows is used as detector. The special feature of this acquisition approach is its high speed capability. The scan speed is as high as the frame rate of the line scan camera and not affected by the multiplexing. As application a chromatic confocal microscope was build up. From a data acquisition perspective up to 284 million 3D points per second can be measured. A real time signal processing is proposed, too.

  3. Super-resolution spinning-disk confocal microscopy using optical photon reassignment.

    PubMed

    Azuma, Takuya; Kei, Takayuki

    2015-06-01

    Spinning-disk confocal microscopy is a proven technology for investigating 3D structures of biological specimens. Here we report a super-resolution method based on spinning-disk confocal microscopy that optically improves lateral resolution by a factor of 1.37 with a single exposure. Moreover, deconvolution yields twofold improvement over the diffraction limit. With the help of newly modified Nipkow disk which comprises pinholes and micro-lenses on the front and back respectively, emitted photons from specimen can be optically reassigned to the most probable locations they originate from. Consequently, the improvement in resolution is achieved preserving inherent sectioning capabilities of confocal microscopy. This extremely simple implementation will enable reliable observations at super high resolution in biomedical routine research.

  4. Bridging microscopes: 3D correlative light and scanning electron microscopy of complex biological structures.

    PubMed

    Lucas, Miriam S; Günthert, Maja; Gasser, Philippe; Lucas, Falk; Wepf, Roger

    2012-01-01

    The rationale of correlative light and electron microscopy (CLEM) is to collect data on different information levels--ideally from an identical area on the same sample--with the aim of combining datasets at different levels of resolution to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern three-dimensional (3D) imaging techniques in light and electron microscopy opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale and over various volume dimensions. Here, we present two alternative approaches to correlate 3D light microscopy (LM) data with scanning electron microscopy (SEM) volume data. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described. The advantages and potential applications are exemplarily shown on various biological samples, such as cells, individual organisms, human tissue, as well as plant tissue. The two CLEM approaches presented here are per se not mutually exclusive, but have their distinct advantages. Confocal laser scanning microscopy (CLSM) and focused ion beam-SEM (FIB-SEM) is most suitable for targeted 3D correlation of small volumes, whereas serial-section LM and SEM imaging has its strength in large-area or -volume screening and correlation. The second method can be combined with immunocytochemical methods. Both methods, however, have the potential to extract statistically relevant data of structural details for systems biology.

  5. The three-dimensional elemental distribution based on the surface topography by confocal 3D-XRF analysis

    NASA Astrophysics Data System (ADS)

    Yi, Longtao; Qin, Min; Wang, Kai; Lin, Xue; Peng, Shiqi; Sun, Tianxi; Liu, Zhiguo

    2016-09-01

    Confocal three-dimensional micro-X-ray fluorescence (3D-XRF) is a good surface analysis technology widely used to analyse elements and elemental distributions. However, it has rarely been applied to analyse surface topography and 3D elemental mapping in surface morphology. In this study, a surface adaptive algorithm using the progressive approximation method was designed to obtain surface topography. A series of 3D elemental mapping analyses in surface morphology were performed in laboratories to analyse painted pottery fragments from the Majiayao Culture (3300-2900 BC). To the best of our knowledge, for the first time, sample surface topography and 3D elemental mapping were simultaneously obtained. Besides, component and depth analyses were also performed using synchrotron radiation confocal 3D-XRF and tabletop confocal 3D-XRF, respectively. The depth profiles showed that the sample has a layered structure. The 3D elemental mapping showed that the red pigment, black pigment, and pottery coat contain a large amount of Fe, Mn, and Ca, respectively. From the 3D elemental mapping analyses at different depths, a 3D rendering was obtained, clearly showing the 3D distributions of the red pigment, black pigment, and pottery coat. Compared with conventional 3D scanning, this method is time-efficient for analysing 3D elemental distributions and hence especially suitable for samples with non-flat surfaces.

  6. Confocal and Two-Photon Microscopy: Foundations, Applications and Advances

    NASA Astrophysics Data System (ADS)

    Diaspro, Alberto

    2001-11-01

    Confocal and Two-Photon Microscopy Foundations, Applications, and Advances Edited by Alberto Diaspro Confocal and two-photon fluorescence microscopy has provided researchers with unique possibilities of three-dimensional imaging of biological cells and tissues and of other structures such as semiconductor integrated circuits. Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances provides clear, comprehensive coverage of basic foundations, modern applications, and groundbreaking new research developments made in this important area of microscopy. Opening with a foreword by G. J. Brakenhoff, this reference gathers the work of an international group of renowned experts in chapters that are logically divided into balanced sections covering theory, techniques, applications, and advances, featuring: In-depth discussion of applications for biology, medicine, physics, engineering, and chemistry, including industrial applications Guidance on new and emerging imaging technology, developmental trends, and fluorescent molecules Uniform organization and review-style presentation of chapters, with an introduction, historical overview, methodology, practical tips, applications, future directions, chapter summary, and bibliographical references Companion FTP site with full-color photographs The significant experience of pioneers, leaders, and emerging scientists in the field of confocal and two-photon excitation microscopy Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances is invaluable to researchers in the biological sciences, tissue and cellular engineering, biophysics, bioengineering, physics of matter, and medicine, who use these techniques or are involved in developing new commercial instruments.

  7. Fluorescence performance standards for confocal microscopy

    NASA Astrophysics Data System (ADS)

    Rüttinger, Steffen; Kapusta, Peter; Völlkopf, Volker; Koberling, Felix; Erdmann, Rainer; Macdonald, Rainer

    2010-02-01

    State of the art confocal microscopes offer diffraction limited (or even better) spatial resolution, highest (single molecule) sensitivity and ps-fluorescence lifetime measurement accuracy. For developers, manufacturers, as well as users of confocal microscopes it is mandatory to assign values to these qualities. In particular for users, it is often not easy to ascertain that the instrument is properly aligned as a large number of factors influence resolution or sensitivity. Therefore, we aspire to design a set of performance standards to be deployed on a day-to-day fashion in order to check the instruments characteristics. The main quantities such performance standard must address are: • Spatial resolution • Sensitivity • Fluorescence lifetime To facilitate the deployment and thus promote wide range adoption in day-to-day performance testing the corresponding standards have to be ready made, easy to handle and to store. The measurement procedures necessary should be available on as many different setups as possible and the procedures involved in their deployment should be as easy as possible. To this end, we developed two performance standards to accomplish the mentioned goals: • Resolution reference • Combined molecular brightness and fluorescence lifetime reference The first one is based on sub-resolution sized Tetra-SpeckTM fluorescent beads or alternatively on single molecules on a glass surface to image and to determine quantitatively the confocal volume, while the latter is a liquid sample containing fluorescent dyes of different concentrations and spectral properties. Both samples are sealed in order to ease their use and prolong their storage life. Currently long-term tests are performed to ascertain durability and road capabilities.

  8. Detection limits of confocal surface plasmon microscopy

    PubMed Central

    Pechprasarn, Suejit; Somekh, Michael G.

    2014-01-01

    This paper applies rigorous diffraction theory to evaluate the minimum mass sensitivity of a confocal optical microscope designed to excite and detect surface plasmons operating on a planar metallic substrate. The diffraction model is compared with an intuitive ray picture which gives remarkably similar predictions. The combination of focusing the surface plasmons and accurate phase measurement mean that under favorable but achievable conditions detection of small numbers of molecules is possible, however, we argue that reliable detection of single molecules will benefit from the use of structured surfaces. System configurations needed to optimize performance are discussed. PMID:24940537

  9. Sheet-scanned dual-axis confocal (SS-DAC) microscopy using Richardson-Lucy deconvolution

    PubMed Central

    Wang, Danni; Meza, Daphne; Wang, Yu; Gao, Liang; Liu, Jonathan T.C.

    2015-01-01

    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. PMID:26466290

  10. 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.

  11. Applied 3D printing for microscopy in health science research

    NASA Astrophysics Data System (ADS)

    Brideau, Craig; Zareinia, Kourosh; Stys, Peter

    2015-03-01

    The rapid prototyping capability offered by 3D printing is considered advantageous for commercial applications. However, the ability to quickly produce precision custom devices is highly beneficial in the research laboratory setting as well. Biological laboratories require the manipulation and analysis of delicate living samples, thus the ability to create custom holders, support equipment, and adapters allow the extension of existing laboratory machines. Applications include camera adapters and stage sample holders for microscopes, surgical guides for tissue preparation, and small precision tools customized to unique specifications. Where high precision is needed, especially the reproduction of fine features, a printer with a high resolution is needed. However, the introduction of cheaper, lower resolution commercial printers have been shown to be more than adequate for less demanding projects. For direct manipulation of delicate samples, biocompatible raw materials are often required, complicating the printing process. This paper will examine some examples of 3D-printed objects for laboratory use, and provide an overview of the requirements for 3D printing for this application. Materials, printing resolution, production, and ease of use will all be reviewed with an eye to producing better printers and techniques for laboratory applications. Specific case studies will highlight applications for 3D-printed devices in live animal imaging for both microscopy and Magnetic Resonance Imaging.

  12. Confocal Microscopy of Jammed Matter: From Elasticity to Granular Thermodynamics

    NASA Astrophysics Data System (ADS)

    Jorjadze, Ivane

    Packings of particles are ubiquitous in nature and are of interest not only to the scientific community but also to the food, pharmaceutical, and oil industries. In this thesis we use confocal microscopy to investigate packing geometry and stress transmission in 3D jammed particulate systems. By introducing weak depletion attraction we probe the accessible phase-space and demonstrate that a microscopic approach to jammed matter gives validity to statistical mechanics framework, which is intriguing because our particles are not thermally activated. We show that the fluctuations of the local packing parameters can be successfully captured by the recently proposed 'granocentric' model, which generates packing statistics according to simple stochastic processes. This model enables us to calculate packing entropy and granular temperature, the so-called 'compactivity', therefore, providing a basis for a statistical mechanics of granular matter. At a jamming transition point at which there are formed just enough number of contacts to guarantee the mechanical stability, theoretical arguments suggest a singularity which gives rise to the surprising scaling behavior of the elastic moduli and the microstructure, as observed in numerical simulations. Since the contact network in 3D is typically hidden from view, experimental test of the scaling law between the coordination number and the applied pressure is lacking in the literature. Our data show corrections to the linear scaling of the pressure with density which takes into account the creation of contacts. Numerical studies of vibrational spectra, in turn, reveal sudden features such as excess of low frequency modes, dependence of mode localization and structure on the pressure. Chapter four describes the first calculation of vibrational density of states from the experimental 3D data and is in qualitative agreement with the analogous computer simulations. We study the configurational role of the pressure and demonstrate

  13. Reflectance confocal microscopy in infectious diseases.

    PubMed

    Cinotti, E; Labeille, B; Cambazard, F; Perrot, J L

    2015-10-01

    In vivo reflectance confocal microscope (RCM) is a high-resolution non-invasive imaging technique that was initially focused on the diagnosis of skin cancers. A rising number of other indications have been later described for the diagnosis and management of inflammatory and infectious dermatological disorders. RCM can identify cutaneous parasites that are not visible to naked eye such as Sarcoptes scabiei and Demodex folliculorum and it allows to better identify the different body parts of bigger parasites such as ticks. Fungal filaments can also be identified as elongated bright structures in the cutaneous upper layers. RCM cannot observe virus directly. However, the cytopathic effect associated with some virus can be recognized. In addition of being helpful for the diagnosis and follow-up after treatment, thanks to its non-invasiveness, RCM allows pathophysiological studies. PMID:26129682

  14. Visualizing Cochlear Mechanics Using Confocal Microscopy

    NASA Astrophysics Data System (ADS)

    Ulfendahl, M.; Boutet de Monvel, J.; Fridberger, A.

    2003-02-01

    The sound-evoked vibration pattern of the hearing organ is based on complex mechanical interactions between different cellular structures. To explore the structural changes occurring within the organ of Corti during basilar-membrane motion, stepwise alterations of the scala tympani pressure were applied in an in vitro preparation of the guinea-pig temporal bone. Confocal images were acquired at each pressure level. In this way, the motion of several structures could be simultaneously observed with high resolution in a nearly intact system. Images were analyzed using a novel wavelet-based optical-flow estimation algorithm. Under the present experimental conditions, the reticular lamina moved as a stiff plate with a center of rotation in the region of the inner hair cells. The outer hair cells appeared non-rigid and the basal, synaptic regions of these cells displayed significant radial motion indicative of cellular bending and internal shearing.

  15. 3D optical sectioning with a new hyperspectral confocal fluorescence imaging system.

    SciTech Connect

    Nieman, Linda T.; Sinclair, Michael B.; Davidson, George S.; Van Benthem, Mark Hilary; Haaland, David Michael; Timlin, Jerilyn Ann; Sasaki, Darryl Yoshio; Bachand, George David; Jones, Howland D. T.

    2007-02-01

    A novel hyperspectral fluorescence microscope for high-resolution 3D optical sectioning of cells and other structures has been designed, constructed, and used to investigate a number of different problems. We have significantly extended new multivariate curve resolution (MCR) data analysis methods to deconvolve the hyperspectral image data and to rapidly extract quantitative 3D concentration distribution maps of all emitting species. The imaging system has many advantages over current confocal imaging systems including simultaneous monitoring of numerous highly overlapped fluorophores, immunity to autofluorescence or impurity fluorescence, enhanced sensitivity, and dramatically improved accuracy, reliability, and dynamic range. Efficient data compression in the spectral dimension has allowed personal computers to perform quantitative analysis of hyperspectral images of large size without loss of image quality. We have also developed and tested software to perform analysis of time resolved hyperspectral images using trilinear multivariate analysis methods. The new imaging system is an enabling technology for numerous applications including (1) 3D composition mapping analysis of multicomponent processes occurring during host-pathogen interactions, (2) monitoring microfluidic processes, (3) imaging of molecular motors and (4) understanding photosynthetic processes in wild type and mutant Synechocystis cyanobacteria.

  16. Shaping Field for 3D Laser Scanning Microscopy

    PubMed Central

    Colon, Jorge; Lim, Hyungsik

    2015-01-01

    Imaging deep tissue can be extremely inefficient when the region of interest is non-planar and buried in a thick sample, yielding a severely limited effective field of view (FOV). Here we describe a novel technique, namely adaptive field microscopy, which improves the efficiency of 3D imaging by controlling the image plane. The plane of scanning laser focus is continuously reshaped in situ to match the conformation of the sample. The practicality is demonstrated for ophthalmic imaging, where a large area of the corneal epithelium of intact mouse eye is captured in a single frame with subcellular resolution. PMID:26176454

  17. MEMS-BASED 3D CONFOCAL SCANNING MICROENDOSCOPE USING MEMS SCANNERS FOR BOTH LATERAL AND AXIAL SCAN.

    PubMed

    Liu, Lin; Wang, Erkang; Zhang, Xiaoyang; Liang, Wenxuan; Li, Xingde; Xie, Huikai

    2014-08-15

    A fiber-optic 3D confocal scanning microendoscope employing MEMS scanners for both lateral and axial scan was designed and constructed. The MEMS 3D scan engine achieved a lateral scan range of over ± 26° with a 2D MEMS scanning micromirror and a depth scan of over 400 μm with a 1D MEMS tunable microlens. The lateral resolution and axial resolution of this system were experimentally measured as 1.0 μm and 7.0 μm, respectively. 2D and 3D confocal reflectance images of micro-patterns, micro-particles, onion skins and acute rat brain tissue were obtained by this MEMS-based 3D confocal scanning microendoscope.

  18. Diffusion of photoacid generators by laser scanning confocal microscopy

    NASA Astrophysics Data System (ADS)

    Zhang, Ping L.; Webber, Stephen E.; Mendenhall, J.; Byers, Jeffrey D.; Chao, Keith K.

    1998-06-01

    Diffusion of the photogenerated acid during the period of time between exposure and development can cause contrast loss and ultimately loss of the latent image. This is especially relevant for chemically amplified photoresists that require a post-exposure baking step, which in turn facilitates acid diffusion due to the high temperature normally employed. It is thus important to develop techniques with good spatial resolution to monitor the photogeneration of acid. More precisely, we need techniques that provide two distinct types of information: spatial resolution on various length scales within the surface layer and also sufficient depth resolution so that one can observe the transition from very surface layer to bulk structure in the polymer blend coated on silicon substrate. Herein laser scanning confocal microscopy is used to evaluate the resist for the first time. We report the use of the confocal microscopy to map the pag/dye distribution in PHS matrices, with both reflectance images and fluorescence images. A laser beam is focused onto a small 3D volume element, termed a voxel. It is typically 200 nm X 200 nm laterally and 800 nm axially. The illuminated voxel is viewed such that only signals emanating from this voxel are detected, i.e., signal from outside the probed voxel is not detected. By adjusting the vertical position of the laser focal point, the voxel can be moved to the designated lateral plane to produce an image. Contrast caused by topology difference between the exposed and unexposed area can be eliminated. Bis-p-butylphenyl iodonium triflat (7% of polyhydroxystyrene) is used as photoacid generators. 5% - 18% (by weight, PHS Mn equals 13 k) resist in PGMEA solution is spin cast onto the treated quartz disk with thickness of 1.4 micrometers , 5 micrometers space/10 micrometers pitch chrome mask is used to generate the pattern with mercury DUV illumination. Fluoresceinamine, the pH-sensitive dye, is also used to enhance the contrast of

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

    SciTech Connect

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

    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 and 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.

  20. 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.; Havrilla, George Joseph

    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

  1. Automated 3-D Tracking of Centrosomes in Sequences of Confocal Image Stacks

    SciTech Connect

    Kerekes, Ryan A; Gleason, Shaun Scott; Trivedi, Dr. Niraj; Solecki, Dr. David

    2009-01-01

    In order to facilitate the study of neuron migration, we propose a method for 3-D detection and tracking of centrosomes in time-lapse confocal image stacks of live neuron cells. We combine Laplacian-based blob detection, adaptive thresholding, and the extraction of scale and roundness features to find centrosome-like objects in each frame. We link these detections using the joint probabilistic data association filter (JPDAF) tracking algorithm with a Newtonian state-space model tailored to the motion characteristics of centrosomes in live neurons. We apply our algorithm to image sequences containing multiple cells, some of which had been treated with motion-inhibiting drugs. We provide qualitative results and quantitative comparisons to manual segmentation and tracking results showing that our motion estimates closely agree with those generated by neurobiology experts.

  2. Sample drift correction in 3D fluorescence photoactivation localization microscopy

    NASA Astrophysics Data System (ADS)

    Mlodzianoski, Michael J.; Schreiner, John M.; Callahan, Steven P.; Smolková, Katarina; Dlasková, Andrea; Šantorová, Jitka; Ježek, Petr; Bewersdorf, Joerg

    2011-08-01

    The recent development of diffraction-unlimited far-field fluorescence microscopy has overcome the classical resolution limit of ~250 nm of conventional light microscopy by about a factor of ten. The improved resolution, however, reveals not only biological structures at an unprecedented resolution, but is also susceptible to sample drift on a much finer scale than previously relevant. Without correction, sample drift leads to smeared images with decreased resolution, and in the worst case to misinterpretation of the imaged structures. This poses a problem especially for techniques such as Fluorescence Photoactivation Localization Microscopy (FPALM/PALM) or Stochastic Optical Reconstruction Microscopy (STORM), which often require minutes recording time. Here we discuss an approach that corrects for three-dimensional (3D) drift in images of fixed samples without the requirement for fiduciary markers or instrument modifications. Drift is determined by calculating the spatial cross-correlation function between subsets of localized particles imaged at different times. Correction down to ~5 nm precision is achieved despite the fact that different molecules are imaged in each frame. We demonstrate the performance of our drift correction algorithm with different simulated structures and analyze its dependence on particle density and localization precision. By imaging mitochondria with Biplane FPALM we show our algorithm's feasibility in a practical application.

  3. Determination of the positions and orientations of concentrated rod-like colloids from 3D microscopy data.

    PubMed

    Besseling, T H; Hermes, M; Kuijk, A; de Nijs, B; Deng, T-S; Dijkstra, M; Imhof, A; van Blaaderen, A

    2015-05-20

    Confocal microscopy in combination with real-space particle tracking has proven to be a powerful tool in scientific fields such as soft matter physics, materials science and cell biology. However, 3D tracking of anisotropic particles in concentrated phases remains not as optimized compared to algorithms for spherical particles. To address this problem, we developed a new particle-fitting algorithm that can extract the positions and orientations of fluorescent rod-like particles from three dimensional confocal microscopy data stacks. The algorithm is tailored to work even when the fluorescent signals of the particles overlap considerably and a threshold method and subsequent clusters analysis alone do not suffice. We demonstrate that our algorithm correctly identifies all five coordinates of uniaxial particles in both a concentrated disordered phase and a liquid-crystalline smectic-B phase. Apart from confocal microscopy images, we also demonstrate that the algorithm can be used to identify nanorods in 3D electron tomography reconstructions. Lastly, we determined the accuracy of the algorithm using both simulated and experimental confocal microscopy data-stacks of diffusing silica rods in a dilute suspension. This novel particle-fitting algorithm allows for the study of structure and dynamics in both dilute and dense liquid-crystalline phases (such as nematic, smectic and crystalline phases) as well as the study of the glass transition of rod-like particles in three dimensions on the single particle level. PMID:25922931

  4. 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.

  5. Resolution improvement by 3D particle averaging in localization microscopy

    PubMed Central

    Broeken, Jordi; Johnson, Hannah; Lidke, Diane S.; Liu, Sheng; Nieuwenhuizen, Robert P.J.; Stallinga, Sjoerd; Lidke, Keith A.; Rieger, Bernd

    2015-01-01

    Inspired by recent developments in localization microscopy that applied averaging of identical particles in 2D for increasing the resolution even further, we discuss considerations for alignment (registration) methods for particles in general and for 3D in particular. We detail that traditional techniques for particle registration from cryo electron microscopy based on cross-correlation are not suitable, as the underlying image formation process is fundamentally different. We argue that only localizations, i.e. a set of coordinates with associated uncertainties, are recorded and not a continuous intensity distribution. We present a method that owes to this fact and that is inspired by the field of statistical pattern recognition. In particular we suggest to use an adapted version of the Bhattacharyya distance as a merit function for registration. We evaluate the method in simulations and demonstrate it on three-dimensional super-resolution data of Alexa 647 labelled to the Nup133 protein in the nuclear pore complex of Hela cells. From the simulations we find suggestions that for successful registration the localization uncertainty must be smaller than the distance between labeling sites on a particle. These suggestions are supported by theoretical considerations concerning the attainable resolution in localization microscopy and its scaling behavior as a function of labeling density and localization precision. PMID:25866640

  6. Taylor series expansion based multidimensional image reconstruction for confocal and 4pi microscopy

    NASA Astrophysics Data System (ADS)

    Dilipkumar, Shilpa; Pratim Mondal, Partha

    2013-08-01

    We propose and experimentally demonstrate a three-dimensional (3D) image reconstruction methodology based on Taylor series approximation (TSA) in a Bayesian image reconstruction formulation. TSA incorporates the requirement of analyticity in the image domain, and acts as a finite impulse response filter. This technique is validated on images obtained from widefield, confocal laser scanning fluorescence microscopy and two-photon excited 4pi (2PE-4pi) fluorescence microscopy. Studies on simulated 3D objects, mitochondria-tagged yeast cells (labeled with Mitotracker Orange) and mitochondrial networks (tagged with Green fluorescent protein) show a signal-to-background improvement of 40% and resolution enhancement from 360 to 240 nm. This technique can easily be extended to other imaging modalities (single plane illumination microscopy (SPIM), individual molecule localization SPIM, stimulated emission depletion microscopy and its variants).

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

    PubMed

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

    2016-09-01

    Based on the optical arrangement of a bipolar differential confocal microscopy (BDCM), laser differential fitting confocal microscopy (DFCM) is proposed in this paper using the feature of BDCM that a zero-crossing point (ZCP) of the axial response curve precisely corresponds to the focus of the system objective. A linear segment of the DFCM axial response around the ZCP is used to fit a straight line. Focus can be determined by solving the equations of the fitting lines, and then, the sample surface could be measured and reconstructed with a high resolution. Compared with the curve-fitting peak detection, which is an algorithm for focus detection widely used in conventional confocal microscopy, the line-fitting zero solution method used in DFCM has several advantages, such as high precision and sensitivity. Most importantly, precise focus detection can be realized using less data, i.e., DFCM has a high measurement efficiency. Furthermore, DFCM can effectively eliminate common-mode noise in a confocal microscopy system and has good noise suppression and disturbance resistance capability. PMID:27607265

  8. Measuring rotational diffusion of colloidal spheres with confocal microscopy.

    PubMed

    Liu, Bing; Böker, Alexander

    2016-07-13

    We report an experimental method to measure the translational and rotational dynamics of colloidal spheres in three dimensions with confocal microscopy and show that the experimental values reasonably agree with the theoretical values. This method can be extended to study rotational dynamics in concentrated colloidal systems and complex bio-systems. PMID:27353601

  9. 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...

  10. 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...

  11. FOOD SURFACE TEXTURE MEASUREMENT USING REFLECTIVE CONFOCAL LASER SCANNING MICROSCOPY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Confocal laser scanning microscopy (CLSM) was used in the reflection mode to characterize the surface texture (roughness) of sliced food surfaces. Sandpapers of grit size between 150 and 600 were used as the height reference to standardize the CLSM hardware settings. Sandpaper particle sizes were v...

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

    PubMed

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

    2016-09-01

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

  13. 3D Light-Sheet Fluorescence Microscopy of Cranial Neurons and Vasculature during Zebrafish Embryogenesis.

    PubMed

    Park, Ok Kyu; Kwak, Jina; Jung, Yoo Jung; Kim, Young Ho; Hong, Hyun-Seok; Hwang, Byung Joon; Kwon, Seung-Hae; Kee, Yun

    2015-11-01

    Precise 3D spatial mapping of cells and their connections within living tissues is required to fully understand developmental processes and neural activities. Zebrafish embryos are relatively small and optically transparent, making them the vertebrate model of choice for live in vivo imaging. However, embryonic brains cannot be imaged in their entirety by confocal or two-photon microscopy due to limitations in optical range and scanning speed. Here, we use light-sheet fluorescence microscopy to overcome these limitations and image the entire head of live transgenic zebrafish embryos. We simultaneously imaged cranial neurons and blood vessels during embryogenesis, generating comprehensive 3D maps that provide insight into the coordinated morphogenesis of the nervous system and vasculature during early development. In addition, blood cells circulating through the entire head, vagal and cardiac vasculature were also visualized at high resolution in a 3D movie. These data provide the foundation for the construction of a complete 4D atlas of zebrafish embryogenesis and neural activity.

  14. 3D Light-Sheet Fluorescence Microscopy of Cranial Neurons and Vasculature during Zebrafish Embryogenesis

    PubMed Central

    Park, Ok Kyu; Kwak, Jina; Jung, Yoo Jung; Kim, Young Ho; Hong, Hyun-Seok; Hwang, Byung Joon; Kwon, Seung-Hae; Kee, Yun

    2015-01-01

    Precise 3D spatial mapping of cells and their connections within living tissues is required to fully understand developmental processes and neural activities. Zebrafish embryos are relatively small and optically transparent, making them the vertebrate model of choice for live in vivo imaging. However, embryonic brains cannot be imaged in their entirety by confocal or two-photon microscopy due to limitations in optical range and scanning speed. Here, we use light-sheet fluorescence microscopy to overcome these limitations and image the entire head of live transgenic zebrafish embryos. We simultaneously imaged cranial neurons and blood vessels during embryogenesis, generating comprehensive 3D maps that provide insight into the coordinated morphogenesis of the nervous system and vasculature during early development. In addition, blood cells circulating through the entire head, vagal and cardiac vasculature were also visualized at high resolution in a 3D movie. These data provide the foundation for the construction of a complete 4D atlas of zebrafish embryogenesis and neural activity. PMID:26429501

  15. 3D structure tensor analysis of light microscopy data for validating diffusion MRI.

    PubMed

    Khan, Ahmad Raza; Cornea, Anda; Leigland, Lindsey A; Kohama, Steven G; Jespersen, Sune Nørhøj; Kroenke, Christopher D

    2015-05-01

    Diffusion magnetic resonance imaging (d-MRI) is a powerful non-invasive and non-destructive technique for characterizing brain tissue on the microscopic scale. However, the lack of validation of d-MRI by independent experimental means poses an obstacle to accurate interpretation of data acquired using this method. Recently, structure tensor analysis has been applied to light microscopy images, and this technique holds promise to be a powerful validation strategy for d-MRI. Advantages of this approach include its similarity to d-MRI in terms of averaging the effects of a large number of cellular structures, and its simplicity, which enables it to be implemented in a high-throughput manner. However, a drawback of previous implementations of this technique arises from it being restricted to 2D. As a result, structure tensor analyses have been limited to tissue sectioned in a direction orthogonal to the direction of interest. Here we describe the analytical framework for extending structure tensor analysis to 3D, and utilize the results to analyze serial image "stacks" acquired with confocal microscopy of rhesus macaque hippocampal tissue. Implementation of 3D structure tensor procedures requires removal of sources of anisotropy introduced in tissue preparation and confocal imaging. This is accomplished with image processing steps to mitigate the effects of anisotropic tissue shrinkage, and the effects of anisotropy in the point spread function (PSF). In order to address the latter confound, we describe procedures for measuring the dependence of PSF anisotropy on distance from the microscope objective within tissue. Prior to microscopy, ex vivo d-MRI measurements performed on the hippocampal tissue revealed three regions of tissue with mutually orthogonal directions of least restricted diffusion that correspond to CA1, alveus and inferior longitudinal fasciculus. We demonstrate the ability of 3D structure tensor analysis to identify structure tensor orientations that

  16. 3D structure tensor analysis of light microscopy data for validating diffusion MRI

    PubMed Central

    Khan, Ahmad Raza; Cornea, Anda; Leigland, Lindsey A.; Kohama, Steven G.; Jespersen, Sune Nørhøj; Kroenke, Christopher D.

    2015-01-01

    Diffusion magnetic resonance imaging (d-MRI) is a powerful non-invasive and non-destructive technique for characterizing brain tissue on the microscopic scale. However, the lack of validation of d-MRI by independent experimental means poses an obstacle to accurate interpretation of data acquired using this method. Recently, structure tensor analysis has been applied to light microscopy images, and this technique holds promise to be a powerful validation strategy for d-MRI. Advantages of this approach include its similarity to d-MRI in terms of averaging the effects of a large number of cellular structures, and its simplicity, which enables it to be implemented in a high-throughput manner. However, a drawback of previous implementations of this technique arises from it being restricted to 2D. As a result, structure tensor analyses have been limited to tissue sectioned in a direction orthogonal to the direction of interest. Here we describe the analytical framework for extending structure tensor analysis to 3D, and utilize the results to analyze serial image “stacks” acquired with confocal microscopy of rhesus macaque hippocampal tissue. Implementation of 3D structure tensor procedures requires removal of sources of anisotropy introduced in tissue preparation and confocal imaging. This is accomplished with image processing steps to mitigate the effects of anisotropic tissue shrinkage, and the effects of anisotropy in the point spread function (PSF). In order to address the latter confound, we describe procedures for measuring the dependence of PSF anisotropy on distance from the microscope objective within tissue. Prior to microscopy, ex vivo d-MRI measurements performed on the hippocampal tissue revealed three regions of tissue with mutually orthogonal directions of least restricted diffusion that correspond to CA1, alveus and inferior longitudinal fasciculus. We demonstrate the ability of 3D structure tensor analysis to identify structure tensor orientations

  17. Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

    PubMed Central

    Changou, Chun A.; Wolfson, Deanna L.; Ahluwalia, Balpreet Singh; Bold, Richard J.; Kung, Hsing-Jien; Chuang, Frank Y.S.

    2013-01-01

    Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12. Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of

  18. Quantitative analysis of autophagy using advanced 3D fluorescence microscopy.

    PubMed

    Changou, Chun A; Wolfson, Deanna L; Ahluwalia, Balpreet Singh; Bold, Richard J; Kung, Hsing-Jien; Chuang, Frank Y S

    2013-01-01

    Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine(1). This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)(1,10). Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)(1,2,3). Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation(4,5). Although the essential components of this pathway are well-characterized(6,7,8,9), many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy(11,12). Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early

  19. Quantitative analysis of autophagy using advanced 3D fluorescence microscopy.

    PubMed

    Changou, Chun A; Wolfson, Deanna L; Ahluwalia, Balpreet Singh; Bold, Richard J; Kung, Hsing-Jien; Chuang, Frank Y S

    2013-05-03

    Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine(1). This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)(1,10). Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)(1,2,3). Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation(4,5). Although the essential components of this pathway are well-characterized(6,7,8,9), many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy(11,12). Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early

  20. 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

  1. Confocal volume in laser Raman microscopy depth profiling

    SciTech Connect

    Maruyama, Yutaka; Kanematsu, Wataru

    2011-11-15

    To clarify the degradation of confocality in laser Raman microscopy depth profiling (optical sectioning) and the influence of pinhole filtering on it, we investigate the confocal volume in detail based on Gaussian beam optics and scalar wave optics. Theoretical depth profiles of a homogeneous transparent sample for four different pinhole sizes, which are computed using the measured incident beam waist radius w{sub 0} and only a few optical system specific parameters such as a numerical aperture (NA) and a focal length, show a good agreement with the corresponding measured depth profiles. The computed confocal volume demonstrates that the pinhole size affects the actual probe depth as well as the axial resolution and the total intensity loss.

  2. Confocal fluorometer for diffusion tracking in 3D engineered tissue constructs

    NASA Astrophysics Data System (ADS)

    Daly, D.; Zilioli, A.; Tan, N.; Buttenschoen, K.; Chikkanna, B.; Reynolds, J.; Marsden, B.; Hughes, C.

    2016-03-01

    We present results of the development of a non-contacting instrument, called fScan, based on scanning confocal fluorometry for assessing the diffusion of materials through a tissue matrix. There are many areas in healthcare diagnostics and screening where it is now widely accepted that the need for new quantitative monitoring technologies is a major pinch point in patient diagnostics and in vitro testing. With the increasing need to interpret 3D responses this commonly involves the need to track the diffusion of compounds, pharma-active species and cells through a 3D matrix of tissue. Methods are available but to support the advances that are currently only promised, this monitoring needs to be real-time, non-invasive, and economical. At the moment commercial meters tend to be invasive and usually require a sample of the medium to be removed and processed prior to testing. This methodology clearly has a number of significant disadvantages. fScan combines a fiber based optical arrangement with a compact, free space optical front end that has been integrated so that the sample's diffusion can be measured without interference. This architecture is particularly important due to the "wet" nature of the samples. fScan is designed to measure constructs located within standard well plates and a 2-D motion stage locates the required sample with respect to the measurement system. Results are presented that show how the meter has been used to evaluate movements of samples through collagen constructs in situ without disturbing their kinetic characteristics. These kinetics were little understood prior to these measurements.

  3. Three-Dimensional Visualization of Interfacial Phenomena Using Confocal Microscopy

    NASA Astrophysics Data System (ADS)

    Shieh, Ian C.

    Surfactants play an integral role in numerous functions ranging from stabilizing the emulsion in a favorite salad dressing to organizing the cellular components that make life possible. We are interested in lung surfactant, which is a mixture of lipids and proteins essential for normal respiration because it modulates the surface tension of the air-liquid interface of the thin fluid lining in the lungs. Through this surface tension modulation, lung surfactant ensures effortless lung expansion and prevents lung collapse during exhalation, thereby effecting proper oxygenation of the bloodstream. The function of lung surfactant, as well as numerous interfacial lipid systems, is not solely dictated by the behavior of materials confined to the two-dimensional interface. Rather, the distributions of materials in the liquid subphase also greatly influence the performance of interfacial films of lung surfactant. Therefore, to better understand the behavior of lung surfactant and other interfacial lipid systems, we require a three-dimensional characterization technique. In this dissertation, we have developed a novel confocal microscopy methodology for investigating the interfacial phenomena of surfactants at the air-liquid interface of a Langmuir trough. Confocal microscopy provides the excellent combination of in situ, fast, three-dimensional visualization of multiple components of the lung surfactant system that other characterization techniques lack. We detail the solutions to the numerous challenges encountered when imaging a dynamic air-liquid interface with a high-resolution technique like confocal microscopy. We then use confocal microscopy to elucidate the distinct mechanisms by which a polyelectrolyte (chitosan) and nonadsorbing polymer (polyethylene glycol) restore the function of lung surfactant under inhibitory conditions mimicking the effects of lung trauma. Beyond this physiological model, we also investigate several one- and two-component interfacial films

  4. Cement paste surface roughness analysis using coherence scanning interferometry and confocal microscopy

    SciTech Connect

    Apedo, K.L.; Munzer, C.; He, H.; Montgomery, P.; Serres, N.; Fond, C.; Feugeas, F.

    2015-02-15

    Scanning electron microscopy and scanning probe microscopy have been used for several decades to better understand the microstructure of cementitious materials. Very limited work has been performed to date to study the roughness of cementitious materials by optical microscopy such as coherence scanning interferometry (CSI) and chromatic confocal sensing (CCS). The objective of this paper is to better understand how CSI can be used as a tool to analyze surface roughness and topography of cement pastes. Observations from a series of images acquired using this technique on both polished and unpolished samples are described. The results from CSI are compared with those from a STIL confocal microscopy technique (SCM). Comparison between both optical techniques demonstrates the ability of CSI to measure both polished and unpolished cement pastes. - Highlights: • Coherence scanning interferometry (CSI) was used to analyze cement paste surfaces. • The results from the CSI were compared with those from a confocal microscopy. • 3D roughness parameters were obtained using the window resizing method. • Polished and unpolished cement pastes were studied.

  5. Three-dimensional resolution and contrast-enhanced confocal microscopy with array detection.

    PubMed

    Ge, Baoliang; Wang, Yifan; Huang, Yujia; Kuang, Cuifang; Fang, Yue; Xiu, Peng; Rong, Zihao; Liu, Xu

    2016-05-01

    What we believe is a novel method for improving confocal microscopy's resolution and contrast in 3D space is proposed. Based on a conventional confocal microscopy setup, we use an array detector composed of 32 photomultiplier tubes (PMTs) to replace one point-detector, where the location offset of each PMT caused a different effective point spread function (PSF). By applying array detection and the fluorescence emission difference method of an image with a solid PSF and another with a donut-shaped PSF, we can enhance lateral resolution about 27% in real time with only one scan, and improve the axial resolving ability by about 22% simultaneously. Experimental results of both fluorescent beads and living cells are presented to verify the applicability and effectiveness of our method. PMID:27128062

  6. Holographic microscopy for 3D tracking of bacteria

    NASA Astrophysics Data System (ADS)

    Nadeau, Jay; Cho, Yong Bin; El-Kholy, Marwan; Bedrossian, Manuel; Rider, Stephanie; Lindensmith, Christian; Wallace, J. Kent

    2016-03-01

    Understanding when, how, and if bacteria swim is key to understanding critical ecological and biological processes, from carbon cycling to infection. Imaging motility by traditional light microscopy is limited by focus depth, requiring cells to be constrained in z. Holographic microscopy offers an instantaneous 3D snapshot of a large sample volume, and is therefore ideal in principle for quantifying unconstrained bacterial motility. However, resolving and tracking individual cells is difficult due to the low amplitude and phase contrast of the cells; the index of refraction of typical bacteria differs from that of water only at the second decimal place. In this work we present a combination of optical and sample-handling approaches to facilitating bacterial tracking by holographic phase imaging. The first is the design of the microscope, which is an off-axis design with the optics along a common path, which minimizes alignment issues while providing all of the advantages of off-axis holography. Second, we use anti-reflective coated etalon glass in the design of sample chambers, which reduce internal reflections. Improvement seen with the antireflective coating is seen primarily in phase imaging, and its quantification is presented here. Finally, dyes may be used to increase phase contrast according to the Kramers-Kronig relations. Results using three test strains are presented, illustrating the different types of bacterial motility characterized by an enteric organism (Escherichia coli), an environmental organism (Bacillus subtilis), and a marine organism (Vibrio alginolyticus). Data processing steps to increase the quality of the phase images and facilitate tracking are also discussed.

  7. Three-dimensional measurement of cAMP gradients using hyperspectral confocal microscopy

    NASA Astrophysics Data System (ADS)

    Rich, Thomas C.; Annamdevula, Naga; Britain, Andrea L.; Mayes, Samuel; Favreau, Peter F.; Leavesley, Silas J.

    2016-03-01

    Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions over a wide range of timescales. Several lines of evidence have suggested that the distribution of cAMP within cells is not uniform, and that cAMP compartmentalization is largely responsible for signaling specificity within the cAMP signaling pathway. However, to date, no studies have experimentally measured three dimensional (3D) cAMP distributions within cells. Here we use both 2D and 3D hyperspectral microscopy to visualize cAMP gradients in endothelial cells from the pulmonary microvasculature (PMVECs). cAMP levels were measured using a FRETbased cAMP sensor comprised of a cAMP binding domain from EPAC sandwiched between FRET donors and acceptors -- Turquoise and Venus fluorescent proteins. Data were acquired using either a Nikon A1R spectral confocal microscope or custom spectral microscopy system. Analysis of hyperspectral image stacks from a single confocal slice or from summed images of all slices (2D analysis) indicated little or no cAMP gradients were formed within PMVECs under basal conditions or following agonist treatment. However, analysis of hyperspectral image stacks from 3D cellular geometries (z stacks) demonstrate marked cAMP gradients from the apical to basolateral membrane of PMVECs. These results strongly suggest that 2D imaging studies of cAMP compartmentalization -- whether epifluorescence or confocal microscopy -- may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D studies are required to assess mechanisms of signaling specificity.

  8. Confocal microscopy of skin cancers: Translational advances toward clinical utility

    PubMed Central

    Rajadhyaksha, Milind

    2014-01-01

    Recent advances in translational research in and technology for confocal microscopy of skin cancers, toward clinical applications, are described. Advances in translational research are in diagnosis of melanoma in vivo, pre-operative mapping of lentigo maligna melanoma margins to guide surgery and intra-operative imaging of residual basal cell carcinomas to guide shave-biopsy. Advances in technology include mosaicing microscopy for detection of basal cell carcinomas in large areas of excised tissue, toward rapid pathology-at-the-bedside, and development of small, simple and low-cost line-scanning confocal microscopes for worldwide use in diverse primary healthcare settings. Current limitations and future opportunities and challenges for both clinicians and technologists are discussed. PMID:19964286

  9. 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. PMID:9393664

  10. Modeling of fibrin gels based on confocal microscopy and light-scattering data.

    PubMed

    Magatti, Davide; Molteni, Matteo; Cardinali, Barbara; Rocco, Mattia; Ferri, Fabio

    2013-03-01

    Fibrin gels are biological networks that play a fundamental role in blood coagulation and other patho/physiological processes, such as thrombosis and cancer. Electron and confocal microscopies show a collection of fibers that are relatively monodisperse in diameter, not uniformly distributed, and connected at nodal points with a branching order of ∼3-4. Although in the confocal images the hydrated fibers appear to be quite straight (mass fractal dimension D(m) = 1), for the overall system 1confocal images, we developed a method to generate three-dimensional (3D) in silico gels made of cylindrical sticks of diameter d, density ρ, and average length , joined at randomly distributed nodal points. The resulting 3D network strikingly resembles real fibrin gels and can be sketched as an assembly of densely packed fractal blobs, i.e., regions of size ξ, where the fiber concentration is higher than average. The blobs are placed at a distance ξ0 between their centers of mass so that they are overlapped by a factor η =ξ/ξ0 and have D(m) ∼1.2-1.6. The in silico gels' structure is quantitatively analyzed by its 3D spatial correlation function g(3D)(r) and corresponding power spectrum I(q) = FFT(3D[g3D(r)]), from which ρ, d, D(m), η, and ξ0 can be extracted. In particular, ξ0 provides an excellent estimate of the gel mesh size. The in silico gels' I(q) compares quite well with real gels' elastic light-scattering measurements. We then derived an analytical form factor for accurately fitting the scattering data, which allowed us to directly recover the gels' structural parameters.

  11. Study of liquid jet instability by confocal microscopy

    NASA Astrophysics Data System (ADS)

    Yang, Lisong; Adamson, Leanne J.; Bain, Colin D.

    2012-07-01

    The instability of a liquid microjet was used to measure the dynamic surface tension of liquids at the surface ages of ≤1 ms using confocal microscopy. The reflected light from a laser beam at normal incidence to the jet surface is linear in the displacement of the surface near the confocal position, leading to a radial resolution of 4 nm and a dynamic range of 4 μm in the surface position, thus permitting the measurement of amplitude of oscillation at the very early stage of jet instability. For larger oscillations outside the linear region of the confocal response, the swell and neck position of the jet can be located separately and the amplitude of oscillation determined with an accuracy of 0.2 μm. The growth rate of periodically perturbed water and ethanol/water mixture jets with a 100-μm diameter nozzle and mean velocity of 5.7 m s-1 has been measured. The dynamic surface tension was determined from the growth rate of the instability with a linear, axisymmetric, constant property model. Synchronisation of the confocal imaging system with the perturbation applied to the jet permitted a detailed study of the temporal evolution of the neck into a ligament and eventually into a satellite drop.

  12. 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. PMID:24002008

  13. 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. PMID:26331288

  14. Cell cycle phase classification in 3D in vivo microscopy of Drosophila embryogenesis

    PubMed Central

    2011-01-01

    Background Cell divisions play critical roles in disease and development. The analysis of cell division phenotypes in high content image-based screening and time-lapse microscopy relies on automated nuclear segmentation and classification of cell cycle phases. Automated identification of the cell cycle phase helps biologists quantify the effect of genetic perturbations and drug treatments. Most existing studies have dealt with 2D images of cultured cells. Few, if any, studies have addressed the problem of cell cycle classification in 3D image stacks of intact tissues. Results We developed a workflow for the automated cell cycle phase classification in 3D time-series image datasets of live Drosophila embryos expressing the chromatin marker histone-GFP. Upon image acquisition by laser scanning confocal microscopy and 3D nuclear segmentation, we extracted 3D intensity, shape and texture features from interphase nuclei and mitotic chromosomes. We trained different classifiers, including support vector machines (SVM) and neural networks, to distinguish between 5 cell cycles phases (Interphase and 4 mitotic phases) and achieved over 90% accuracy. As the different phases occur at different frequencies (58% of samples correspond to interphase), we devised a strategy to improve the identification of classes with low representation. To investigate which features are required for accurate classification, we performed feature reduction and selection. We were able to reduce the feature set from 42 to 9 without affecting classifier performance. We observed a dramatic decrease of classification performance when the training and testing samples were derived from two different developmental stages, the nuclear divisions of the syncytial blastoderm and the cell divisions during gastrulation. Combining samples from both developmental stages produced a more robust and accurate classifier. Conclusions Our study demonstrates that automated cell cycle phase classification, besides 2D

  15. Automated identification of epidermal keratinocytes in reflectance confocal microscopy

    NASA Astrophysics Data System (ADS)

    Gareau, Dan

    2011-03-01

    Keratinocytes in skin epidermis, which have bright cytoplasmic contrast and dark nuclear contrast in reflectance confocal microscopy (RCM), were modeled with a simple error function reflectance profile: erf( ). Forty-two example keratinocytes were identified as a training set which characterized the nuclear size a = 8.6+/-2.8 μm and reflectance gradient b = 3.6+/-2.1 μm at the nuclear/cytoplasmic boundary. These mean a and b parameters were used to create a rotationally symmetric erf( ) mask that approximated the mean keratinocyte image. A computer vision algorithm used an erf( ) mask to scan RCM images, identifying the coordinates of keratinocytes. Applying the mask to the confocal data identified the positions of keratinocytes in the epidermis. This simple model may be used to noninvasively evaluate keratinocyte populations as a quantitative morphometric diagnostic in skin cancer detection and evaluation of dermatological cosmetics.

  16. Automated spherical aberration correction in scanning confocal microscopy

    NASA Astrophysics Data System (ADS)

    Yoo, H. W.; van Royen, M. E.; van Cappellen, W. A.; Houtsmuller, A. B.; Verhaegen, M.; Schitter, G.

    2014-12-01

    Mismatch between the refractive indexes of immersion media and glass coverslips introduces spherical aberrations in microscopes especially for high numerical aperture objectives. This contribution demonstrates an automated adjustment of the coverslip correction collar in scanning confocal microscopy to compensate for spherical aberrations due to coverslip thickness mismatch. With a motorized coverslip correction collar, the adjustment procedure consists of xz image scans, image processing, correction quality evaluation, the mismatch estimation, and eventually the optimal adjustment of the correction collar. For fast correction with less photodamage, coarse-fine Gaussian fitting algorithms are proposed and evaluated with various specimen for their estimation accuracy. The benefits of the proposed automated correction are demonstrated for various coverslips with biological specimens, showing the optimized resolution of the confocal microscope.

  17. 3-D stimulated emission depletion microscopy with programmable aberration correction.

    PubMed

    Lenz, Martin O; Sinclair, Hugo G; Savell, Alexander; Clegg, James H; Brown, Alice C N; Davis, Daniel M; Dunsby, Chris; Neil, Mark A A; French, Paul M W

    2014-01-01

    We present a stimulated emission depletion (STED) microscope that provides 3-D super resolution by simultaneous depletion using beams with both a helical phase profile for enhanced lateral resolution and an annular phase profile to enhance axial resolution. The 3-D depletion point spread function is realised using a single spatial light modulator that can also be programmed to compensate for aberrations in the microscope and the sample. We apply it to demonstrate the first 3-D super-resolved imaging of an immunological synapse between a Natural Killer cell and its target cell.

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

    SciTech Connect

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

    2008-11-17

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

  19. Precise quantification of silica and ceria nanoparticle uptake revealed by 3D fluorescence microscopy

    PubMed Central

    Torrano, Adriano A

    2014-01-01

    Summary Particle_in_Cell-3D is a powerful method to quantify the cellular uptake of nanoparticles. It combines the advantages of confocal fluorescence microscopy with fast and precise semi-automatic image analysis. In this work we present how this method was applied to investigate the impact of 310 nm silica nanoparticles on human vascular endothelial cells (HUVEC) in comparison to a cancer cell line derived from the cervix carcinoma (HeLa). The absolute number of intracellular silica nanoparticles within the first 24 h was determined and shown to be cell type-dependent. As a second case study, Particle_in_Cell-3D was used to assess the uptake kinetics of 8 nm and 30 nm ceria nanoparticles interacting with human microvascular endothelial cells (HMEC-1). These small nanoparticles formed agglomerates in biological medium, and the particles that were in effective contact with cells had a mean diameter of 417 nm and 316 nm, respectively. A significant particle size-dependent effect was observed after 48 h of interaction, and the number of intracellular particles was more than four times larger for the 316 nm agglomerates. Interestingly, our results show that for both particle sizes there is a maximum dose of intracellular nanoparticles at about 24 h. One of the causes for such an interesting and unusual uptake behavior could be cell division. PMID:25383274

  20. Multimodal confocal hyperspectral imaging microscopy with wavelength sweeping source

    NASA Astrophysics Data System (ADS)

    Kim, Young-Duk; Do, Dukho; Yoo, Hongki; Gweon, DaeGab

    2015-02-01

    There exist microscopes that are able to obtain the chemical properties of a sample, because there are some cases in which it is difficult to find out causality of a phenomenon by using only the structural information of a sample. Obtaining the chemical properties of a sample is important in biomedical imaging, because most biological phenomena include changes in the chemical properties of the sample. Hyperspectral imaging (HSI) is one of the popular imaging methods for characterizing materials and biological samples by measuring the reflectance or emission spectrum of the sample. Because all materials have a unique reflectance spectrum, it is possible to analyze material properties and detect changes in the chemical properties of a sample by measuring the spectral changes with respect to the original spectrum. Because of its ability to measure the spectrum of a sample, HSI is widely used in materials identification applications such as aerial reconnaissance and is the subject of various studies in microscopy. Although there are many advantages to using the method, conventional HSI has some limitations because of its complex configuration and slow speed. In this research we propose a new type of multimodal confocal hyperspectral imaging microscopy with fast image acquisition and a simple configuration that is capable of both confocal and HSI microscopies.

  1. 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

  2. Atherosclerotic plaque detection by confocal Brillouin and Raman microscopies

    NASA Astrophysics Data System (ADS)

    Meng, Zhaokai; Basagaoglu, Berkay; Yakovlev, Vladislav V.

    2015-02-01

    Atherosclerosis, the development of intraluminal plaque, is a fundamental pathology of cardiovascular system and remains the leading cause of morbidity and mortality worldwide. Biomechanical in nature, plaque rupture occurs when the mechanical properties of the plaque, related to the morphology and viscoelastic properties, are compromised, resulting in intraluminal thrombosis and reduction of coronary blood flow. In this report, we describe the first simultaneous application of confocal Brillouin and Raman microscopies to ex-vivo aortic wall samples. Such a non-invasive, high specific approach allows revealing a direct relationship between the biochemical and mechanical properties of atherosclerotic tissue.

  3. Characterization of a hazardous eyeliner (kohl) by confocal Raman microscopy.

    PubMed

    Jallad, Karim N; Hedderich, Hartmut G

    2005-09-30

    A new method of analyzing kohl, a cosmetic eyeliner, using confocal Raman microscopy is reported. This technique offers an important alternative to conventional spectroscopic techniques that provide elemental/atomic composition. Raman spectra of three kohl samples have been measured between 150 and 3000 cm(-1) at room temperature. The main component of two kohl samples was found to be lead(II) sulfide (PbS). Kohl is used as a traditional cosmetic and remedy in the Middle East, Far East, and Northern Africa. Since kohl products contain very high concentrations of lead, they constitute a risk for public health, particularly for children.

  4. [Confocal microscopy for the diagnostics of fungal keratitis].

    PubMed

    Daas, L; Viestenz, A; Bischoff, M; Hasenfus, A; Seitz, B

    2016-09-01

    Fungal keratitis is a rare but very serious eye disease in industrial nations with a frequency of 1-5 % of all forms of keratitis from microbial causes. We present two patients with keratitis of primary unknown cause. Using confocal microscopy fungal filaments could be identified that partially showed a parallel configuration (like "railway tracks"). Thus, the correct diagnosis can often be made and suitable therapy can be non-invasively initiated even before the results of in vitro cultivation (fungal culture), polymerase chain reaction (PCR) and histological investigations are available.

  5. 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.

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

    PubMed Central

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

    2008-01-01

    Background Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture. Methods Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale in silico image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM). Results We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates. Conclusion The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes. PMID:18627634

  7. Measurement of steep edges and undercuts in confocal microscopy.

    PubMed

    Mueller, T; Jordan, M; Schneider, T; Poesch, A; Reithmeier, E

    2016-05-01

    Confocal microscopy is widely used to measure the surface topography of specimen with a precision in the micrometer range. The measurement uncertainty and quality of the acquired data of confocal microscopy depends on various effects, such as optical aberrations, vibrations of the measurement setup and variations in the surface reflectivity. In this article, the influence of steep edges and undercuts on measurement results is examined. Steep edges on the specimen's surface lead to a reduced detector signal which influences the measurement accuracy and undercuts cause surface regions, which cannot be captured in a measurement. The article describes a method to overcome the negative effects of steep edges and undercuts by capturing several measurements of the surface with different angles between the surface and the optical axis of the objective. An algorithm is introduced which stitches different angle measurements together without knowledge of the exact position and orientation of the rotation axis. Thus, the measurement uncertainty due to steep edges and undercuts can be avoided without expensive high-precision rotation stages and time consuming adjustment of the measurement setup.

  8. 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..

  9. Towards drug quantification in human skin with confocal Raman microscopy.

    PubMed

    Franzen, Lutz; Selzer, Dominik; Fluhr, Joachim W; Schaefer, Ulrich F; Windbergs, Maike

    2013-06-01

    Understanding the penetration behaviour of drugs into human skin is a prerequisite for the rational development and evaluation of effective dermal drug delivery. The general procedure for the acquisition of quantitative drug penetration profiles in human skin is performed by sequential segmentation and extraction. Unfortunately, this technique is destructive, laborious and lacks spatial resolution. Confocal Raman microscopy bares the potential of a chemically selective, label free and nondestructive analysis. However, the acquisition of quantitative drug depth profiles within skin by Raman microscopy is impeded by imponderable signal attenuation inside the tissue. In this study, we present a chemical semi-solid matrix system simulating the optical properties of human skin. This system serves as a skin surrogate for investigation of Raman signal attenuation under controlled conditions. Caffeine was homogeneously incorporated within the skin surrogate, and Raman intensity depth profiles were acquired. A mathematical algorithm describing the Raman signal attenuation within the surrogate was derived from these profiles. Human skin samples were incubated with caffeine, and Raman intensity depth profiles were similarly acquired. The surrogate algorithm was successfully applied to correct the drug profiles in human skin for signal attenuation. For the first time, a mathematical algorithm was established, which allows correction of Raman signal attenuation in human skin, thus facilitating reliable drug quantification in human skin by confocal Raman spectroscopy.

  10. Emission characteristics of light-emitting diodes by confocal microscopy

    NASA Astrophysics Data System (ADS)

    Cheung, W. S.; Choi, H. W.

    2016-03-01

    The emission profiles of light-emitting diodes have typically be measured by goniophotometry. However this technique suffers from several drawbacks, including the inability to generate three-dimensional intensity profiles as well as poor spatial resolution. These limitations are particularly pronounced when the technique is used to compared devices whose emission patterns have been modified through surface texturing at the micrometer and nanometer scales,. In view of such limitations, confocal microscopy has been adopted for the study of emission characteristics of LEDs. This enables three-dimensional emission maps to be collected, from which two-dimensional cross-sectional emission profiles can be generated. Of course, there are limitations associated with confocal microscopy, including the range of emission angles that can be measured due to the limited acceptance angle of the objective. As an illustration, the technique has been adopted to compare the emission profiles of LEDs with different divergence angles using an objective with a numerical aperture of 0.8. It is found that the results are consistent with those obtained by goniophotometry when the divergence angle is less that the acceptance angle of the objective.

  11. Segmentation of skin strata in reflectance confocal microscopy depth stacks

    NASA Astrophysics Data System (ADS)

    Hames, Samuel C.; Ardigò, Marco; Soyer, H. Peter; Bradley, Andrew P.; Prow, Tarl W.

    2015-03-01

    Reflectance confocal microscopy is an emerging tool for imaging human skin, but currently requires expert human assessment. To overcome the need for human experts it is necessary to develop automated tools for automatically assessing reflectance confocal microscopy imagery. This work presents a novel approach to this task, using a bag of visual words approach to represent and classify en-face optical sections from four distinct strata of the skin. A dictionary of representative features is learned from whitened and normalised patches using hierarchical spherical k-means. Each image is then represented by extracting a dense array of patches and encoding each with the most similar element in the dictionary. Linear discriminant analysis is used as a simple linear classifier. The proposed framework was tested on 308 depth stacks from 54 volunteers. Parameters are tuned using 10 fold cross validation on a training sub-set of the data, and final evaluation was performed on a held out test set. The proposed method generated physically plausible profiles of the distinct strata of human skin, and correctly classified 81.4% of sections in the test set.

  12. Precise colloids with tunable interactions for confocal microscopy

    PubMed Central

    Kodger, Thomas E.; Guerra, Rodrigo E.; Sprakel, Joris

    2015-01-01

    Model colloidal systems studied with confocal microscopy have led to numerous insights into the physics of condensed matter. Though confocal microscopy is an extremely powerful tool, it requires a careful choice and preparation of the colloid. Uncontrolled or unknown variations in the size, density, and composition of the individual particles and interactions between particles, often influenced by the synthetic route taken to form them, lead to difficulties in interpreting the behavior of the dispersion. Here we describe the straightforward synthesis of copolymer particles which can be refractive index- and density-matched simultaneously to a non-plasticizing mixture of high dielectric solvents. The interactions between particles are accurately tuned by surface grafting of polymer brushes using Atom Transfer Radical Polymerization (ATRP), from hard-sphere-like to long-ranged electrostatic repulsion or mixed charge attraction. We also modify the buoyant density of the particles by altering the copolymer ratio while maintaining their refractive index match to the suspending solution resulting in well controlled sedimentation. The tunability of the inter-particle interactions, the low volatility of the solvents, and the capacity to simultaneously match both the refractive index and density of the particles to the fluid opens up new possibilities for exploring the physics of colloidal systems. PMID:26420044

  13. Precise colloids with tunable interactions for confocal microscopy

    NASA Astrophysics Data System (ADS)

    Kodger, Thomas E.; Guerra, Rodrigo E.; Sprakel, Joris

    2015-09-01

    Model colloidal systems studied with confocal microscopy have led to numerous insights into the physics of condensed matter. Though confocal microscopy is an extremely powerful tool, it requires a careful choice and preparation of the colloid. Uncontrolled or unknown variations in the size, density, and composition of the individual particles and interactions between particles, often influenced by the synthetic route taken to form them, lead to difficulties in interpreting the behavior of the dispersion. Here we describe the straightforward synthesis of copolymer particles which can be refractive index- and density-matched simultaneously to a non-plasticizing mixture of high dielectric solvents. The interactions between particles are accurately tuned by surface grafting of polymer brushes using Atom Transfer Radical Polymerization (ATRP), from hard-sphere-like to long-ranged electrostatic repulsion or mixed charge attraction. We also modify the buoyant density of the particles by altering the copolymer ratio while maintaining their refractive index match to the suspending solution resulting in well controlled sedimentation. The tunability of the inter-particle interactions, the low volatility of the solvents, and the capacity to simultaneously match both the refractive index and density of the particles to the fluid opens up new possibilities for exploring the physics of colloidal systems.

  14. Three-dimensional reconstructions from optical sections of thick mouse inner ears using confocal microscopy.

    PubMed

    Kopecky, B J; Duncan, J S; Elliott, K L; Fritzsch, B

    2012-12-01

    Three-dimensional (3D) reconstructions of the vertebrate inner ear have provided novel insights into the development of this complex organ. 3D reconstructions enable superior analysis of phenotypic differences between wild type and mutant ears but can result in laborious work when reconstructed from physically sectioned material. Although nondestructive optical sectioning light sheet microscopy may ultimately prove the ideal solution, these technologies are not yet commercially available, or in many instances are not monetarily feasible. Here we introduce a simple technique to image a fluorescently labelled ear at different stages throughout development at high resolution enabling 3D reconstruction of any component of the inner ear using confocal microscopy. We provide a step-by-step manual from tissue preparation to imaging to 3D reconstruction and analysis including a rationale and troubleshooting guide at each step for researchers with different equipment, protocols, and access to resources to successfully incorporate the principles of this method and customize them to their laboratory settings.

  15. Confocal reflectance quantitative phase microscopy system for cell biology studies (Conference Presentation)

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

    Quantitative phase microscopy (QPM), used to measure the refractive index, provides the optical path delay measurement at each point of the specimen under study and becomes an active field in biological science. In this work we present development of confocal reflection phase microscopy system to provide depth resolved quantitative phase information for investigation of intracellular structures and other biological specimen. The system hardware development is mainly divided into two major parts. First, creates a pinhole array for parallel confocal imaging of specimen at multiple locations simultaneously. Here a digital micro mirror device (DMD) is used to generate pinhole array by turning on a subset micro-mirrors arranged on a grid. Second is the detection of phase information of confocal imaging foci by using a common path interferometer. With this novel approach, it is possible to measure the nuclei membrane fluctuations and distinguish them from the plasma membrane fluctuations. Further, depth resolved quantitative phase can be correlated to the intracellular contents and 3D map of refractive index measurements.

  16. In Vivo Confocal Microscopy and Anterior Segment Optic Coherence Tomography Findings in Ocular Ochronosis

    PubMed Central

    Demirkilinc Biler, Elif; Guven Yilmaz, Suzan; Palamar, Melis; Hamrah, Pedram

    2015-01-01

    Purpose. To report clinical and in vivo confocal microscopy (IVCM) findings of two patients with ocular ochronosis secondary due to alkaptonuria. Materials and Methods. Complete ophthalmologic examinations, including IVCM (HRT II/Rostock Cornea Module, Heidelberg, Germany), anterior segment optical coherence tomography (AS-OCT) (Topcon 3D spectral-domain OCT 2000, Topcon Medical Systems, Paramus, NJ, USA), corneal topography (Pentacam, OCULUS Optikgeräte GmbH, Wetzlar, Germany), and anterior segment photography, were performed. Results. Biomicroscopic examination showed bilateral darkly pigmented lesions of the nasal and temporal conjunctiva and episclera in both patients. In vivo confocal microscopy of the lesions revealed prominent degenerative changes, including vacuoles and fragmentation of collagen fibers in the affected conjunctival lamina propria and episclera. Hyperreflective pigment granules in different shapes were demonstrated in the substantia propria beneath the basement membrane. AS-OCT of Case 1 demonstrated hyporeflective areas. Fundus examination was within normal limits in both patients, except tilted optic discs with peripapillary atrophy in one of the patients. Corneal topography, thickness, and macular OCT were normal bilaterally in both cases. Conclusion. The degenerative and anatomic changes due to ochronotic pigment deposition in alkaptonuria can be demonstrated in detail with IVCM and AS-OCT. Confocal microscopic analysis in ocular ochronosis may serve as a useful adjunct in diagnosis and monitoring of the disease progression. PMID:26788390

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

    PubMed

    Meller, Karl; Theiss, Carsten

    2006-03-01

    We describe a technical method of cell permeabilisation and embedding to study the organisation and distribution of intracellular proteins with aid of atomic force microscopy and confocal laser scanning microscopy in identical areas. While confocal laser scanning microscopy is useful for the identification of certain proteins subsequent labelling with markers or antibodies, atomic force microscopy allows the observation of macromolecular structures in fixed and living cells. To demonstrate the field of application of this preparatory technique, cells were permeabilised, fixed, and the actin cytoskeleton was stained with phalloidin-rhodamine. Confocal laser scanning microscopy was used to show the organisation of these microfilaments, e.g. geodesic dome structures. Thereafter, cells were embedded in Durcupan water-soluble resin, followed by UV-polymerisation of resin at 4 degrees C. This procedure allowed intracellular visualisation of the cell nucleus or cytoskeletal elements by atomic force microscopy, for instance to analyse the globular organisation of actin filaments. Therefore, this method offers a great potential to combine both microscopy techniques in order to understand and interpret intracellular protein relations, for example, the biochemical and morphological interaction of the cytoskeleton. PMID:16360280

  18. Advanced 3D Optical Microscopy in ENS Research.

    PubMed

    Vanden Berghe, Pieter

    2016-01-01

    Microscopic techniques are among the few approaches that have survived the test of time. Being invented half way the seventeenth century by Antonie van Leeuwenhoek and Robert Hooke, this technology is still essential in modern biomedical labs. Many microscopy techniques have been used in ENS research to guide researchers in their dissections and later to enable electrode recordings. Apart from this, microscopy has been instrumental in the identification of subpopulations of cells in the ENS, using a variety of staining methods. A significant step forward in the use of microscopy was the introduction of fluorescence approaches. Due to the fact that intense excitation light is now filtered away from the longer wavelength emission light, the contrast can be improved drastically, which helped to identify subpopulations of enteric neurons in a variety of species. Later functionalized fluorescent probes were used to measure and film activity in muscle and neuronal cells. Another important impetus to the use of microscopy was the discovery and isolation of the green fluorescent protein (GFP), as it gave rise to the development of many different color variants and functionalized constructs. Recent advances in microscopy are the result of a continuous search to enhance contrast between the item of interest and its background but also to improve resolving power to tell two small objects apart. In this chapter three different microscopy approaches will be discussed that can aid to improve our understanding of ENS function within the gut wall. PMID:27379646

  19. Advanced 3D Optical Microscopy in ENS Research.

    PubMed

    Vanden Berghe, Pieter

    2016-01-01

    Microscopic techniques are among the few approaches that have survived the test of time. Being invented half way the seventeenth century by Antonie van Leeuwenhoek and Robert Hooke, this technology is still essential in modern biomedical labs. Many microscopy techniques have been used in ENS research to guide researchers in their dissections and later to enable electrode recordings. Apart from this, microscopy has been instrumental in the identification of subpopulations of cells in the ENS, using a variety of staining methods. A significant step forward in the use of microscopy was the introduction of fluorescence approaches. Due to the fact that intense excitation light is now filtered away from the longer wavelength emission light, the contrast can be improved drastically, which helped to identify subpopulations of enteric neurons in a variety of species. Later functionalized fluorescent probes were used to measure and film activity in muscle and neuronal cells. Another important impetus to the use of microscopy was the discovery and isolation of the green fluorescent protein (GFP), as it gave rise to the development of many different color variants and functionalized constructs. Recent advances in microscopy are the result of a continuous search to enhance contrast between the item of interest and its background but also to improve resolving power to tell two small objects apart. In this chapter three different microscopy approaches will be discussed that can aid to improve our understanding of ENS function within the gut wall.

  20. Adaptive optics in digital micromirror based confocal microscopy

    NASA Astrophysics Data System (ADS)

    Pozzi, P.; Wilding, D.; Soloviev, O.; Vdovin, G.; Verhaegen, M.

    2016-03-01

    This proceeding reports early results in the development of a new technique for adaptive optics in confocal microscopy. The term adaptive optics refers to the branch of optics in which an active element in the optical system is used to correct inhomogeneities in the media through which light propagates. In its most classical form, mostly used in astronomical imaging, adaptive optics is achieved through a closed loop in which the actuators of a deformable mirror are driven by a wavefront sensor. This approach is severely limited in fluorescence microscopy, as the use of a wavefront sensor requires the presence of a bright, point like source in the field of view, a condition rarely satisfied in microscopy samples. Previously reported approaches to adaptive optics in fluorescence microscopy are therefore limited to the inclusion of fluorescent microspheres in the sample, to use as bright stars for wavefront sensors, or time consuming sensorless optimization procedures, requiring several seconds of optimization before the acquisition of a single image. We propose an alternative approach to the problem, implementing sensorless adaptive optics in a Programmable array microscope. A programmable array microscope is a microscope based on a digital micromirror device, in which the single elements of the micromirror act both as point sources and pinholes.

  1. Precision 3-D microscopy with intensity modulated fibre optic scanners

    NASA Astrophysics Data System (ADS)

    Olmos, P.

    2016-01-01

    Optical 3-D imagers constitute a family of precision and useful instruments, easily available on the market in a wide variety of configurations and performances. However, besides their cost they usually provide an image of the object (i.e. a more or less faithful representation of the reality) instead of a truly object's reconstruction. Depending on the detailed working principles of the equipment, this reconstruction may become a challenging task. Here a very simple yet reliable device is described; it is able to form images of opaque objects by illuminating them with an optical fibre and collecting the reflected light with another fibre. Its 3-D capability comes from the spatial filtering imposed by the fibres together with their movement (scanning) along the three directions: transversal (surface) and vertical. This unsophisticated approach allows one to model accurately the entire optical process and to perform the desired reconstruction, finding that information about the surface which is of interest: its profile and its reflectance, ultimately related to the type of material.

  2. Confocal Brillouin microscopy for three-dimensional mechanical imaging

    NASA Astrophysics Data System (ADS)

    Scarcelli, Giuliano; Yun, Seok Hyun

    2008-01-01

    Acoustically induced inelastic light scattering, first reported in 1922 by Brillouin, allows non-contact, direct readout of the viscoelastic properties of a material and has widely been investigated for material characterization, structural monitoring and environmental sensing. Extending the Brillouin technique from point sampling spectroscopy to imaging modality would open up new possibilities for mechanical imaging, but has been challenging because rapid spectrum acquisition is required. Here, we demonstrate a confocal Brillouin microscope based on a fully parallel spectrometer-a virtually imaged phased array-that improves the detection efficiency by nearly 100-fold over previous approaches. Using the system, we show the first cross-sectional Brillouin imaging based on elastic properties as the contrast mechanism and monitor fast dynamic changes in elastic modulus during polymer crosslinking. Furthermore, we report the first in situ biomechanical measurement of the crystalline lens in a mouse eye. These results suggest multiple applications of Brillouin microscopy in biomedical and biomaterial science.

  3. Monitoring Ubiquitin-Coated Bacteria via Confocal Microscopy.

    PubMed

    Lork, Marie; Delvaeye, Mieke; Gonçalves, Amanda; Van Hamme, Evelien; Beyaert, Rudi

    2016-01-01

    Salmonella is a gram-negative facultative intracellular pathogen that is capable of infecting a variety of hosts. Inside host cells, most Salmonella bacteria reside and replicate within Salmonella-containing vacuoles. They use virulence proteins to manipulate the host cell machinery for their own benefit and hijack the host cytoskeleton to travel toward the perinuclear area. However, a fraction of bacteria escapes into the cytosol where they get decorated with a dense layer of polyubiquitin, which labels the bacteria for clearance by autophagy. More specifically, autophagy receptor proteins recognize the ubiquitinated bacteria and deliver them to autophagosomes, which subsequently fuse to lysosomes. Here, we describe methods used to infect HeLa cells with Salmonella bacteria and to detect their ubiquitination via immunofluorescence and laser scanning confocal microscopy. PMID:27613040

  4. Confocal laser scanning microscopy with spatiotemporal structured illumination.

    PubMed

    Gao, Peng; Nienhaus, G Ulrich

    2016-03-15

    Confocal laser scanning microscopy (CLSM), which is widely utilized in the biological and biomedical sciences, is limited in spatial resolution due to diffraction to about half the light wavelength. Here we have combined structured illumination with CLSM to enhance its spatial resolution. To this end, we have used a spatial light modulator (SLM) to generate fringe patterns of different orientations and phase shifts in the excitation spot without any mechanical movement. We have achieved 1.8 and 1.7 times enhanced lateral and axial resolutions, respectively, by synthesizing the object spectrum along different illumination directions. This technique is thus a promising tool for high-resolution morphological or fluorescence imaging, especially in deep tissue. PMID:26977667

  5. Reflectance confocal microscopy for cutaneous infections and infestations.

    PubMed

    Cinotti, E; Perrot, J L; Labeille, B; Cambazard, F

    2016-05-01

    Reflectance confocal microscopy (RCM) is a high-resolution emerging imaging technique that allows non-invasive diagnosis of several cutaneous disorders. A systematic review of the literature on the use of RCM for the study of infections and infestations has been performed to evaluate the current use of this technique and its possible future applications in this field. RCM is particularly suitable for the identification of Sarcoptes scabies, Demodex folliculorum, Ixodes, Dermatophytes and Candida species in the clinical practice and for the follow-up after treatment. The cytopathic effect of herpes simplex virus, varicella zoster virus and molluscipoxvirus is also detectable by this imaging technique even in a pre-vesicular stage. In addition, thanks to its non-invasiveness, RCM allows pathophysiological studies. PMID:26387660

  6. Endocrine and metabolic disease: Confocal microscopy as a diagnostic aid

    PubMed Central

    Bhutani, Jaikrit; Chakinala, Raja Chandra; Bhutani, Sukriti; Sachdeva, Shruti

    2015-01-01

    Diabetes is a systemic disease associated with many complications. These can be prevented and managed effectively if detected promptly. Confocal microscopy (CFM) is a diagnostic tool which has the potential to help in early detection of disease and timely management. CFM has the potential to serve as an excellent noninvasive modality for in vivo imaging and morphological analysis, which can aid us in assessing and monitoring various infectious and pathological diseases at the cellular level. Besides ophthalmological indications, CFM has shown good sensitivity and specificity for identifying those at risk of neuropathy and foot ulceration, monitoring evolution and therapeutic response in a wide range of neuropathies apart from diabetic neuropathy. Through this communication, we aim to sensitize the endocrinologists towards cerebral cavernous malformation as a biomarker to evaluate potential outcomes and therapies in human diabetic neuropathy. PMID:25593848

  7. 3D fluorescence anisotropy imaging using selective plane illumination microscopy

    PubMed Central

    Hedde, Per Niklas; Ranjit, Suman; Gratton, Enrico

    2015-01-01

    Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein. PMID:26368202

  8. Confocal laser scanning microscopy in study of bone calcification

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

  9. Registration and 3D visualization of large microscopy images

    NASA Astrophysics Data System (ADS)

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

    2006-03-01

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

  10. Cell-selective knockout and 3D confocal image analysis reveals separate roles for astrocyte-and endothelial-derived CCL2 in neuroinflammation

    PubMed Central

    2014-01-01

    Background Expression of chemokine CCL2 in the normal central nervous system (CNS) is nearly undetectable, but is significantly upregulated and drives neuroinflammation during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis which is considered a contributing factor in the human disease. As astrocytes and brain microvascular endothelial cells (BMEC) forming the blood–brain barrier (BBB) are sources of CCL2 in EAE and other neuroinflammatory conditions, it is unclear if one or both CCL2 pools are critical to disease and by what mechanism(s). Methods Mice with selective CCL2 gene knockout (KO) in astrocytes (Astro KO) or endothelial cells (Endo KO) were used to evaluate the respective contributions of these sources to neuroinflammation, i.e., clinical disease progression, BBB damage, and parenchymal leukocyte invasion in a myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced EAE model. High-resolution 3-dimensional (3D) immunofluorescence confocal microscopy and colloidal gold immuno-electron microscopy were employed to confirm sites of CCL2 expression, and 3D immunofluorescence confocal microscopy utilized to assess inflammatory responses along the CNS microvasculature. Results Cell-selective loss of CCL2 immunoreactivity was demonstrated in the respective KO mice. Compared to wild-type (WT) mice, Astro KO mice showed reduced EAE severity but similar onset, while Endo KO mice displayed near normal severity but significantly delayed onset. Neither of the KO mice showed deficits in T cell proliferation, or IL-17 and IFN-γ production, following MOG35-55 exposure in vitro, or altered MOG-major histocompatibility complex class II tetramer binding. 3D confocal imaging further revealed distinct actions of the two CCL2 pools in the CNS. Astro KOs lacked the CNS leukocyte penetration and disrupted immunostaining of CLN-5 at the BBB seen during early EAE in WT mice, while Endo KOs uniquely displayed leukocytes stalled in the

  11. Photothermal confocal multicolor microscopy of nanoparticles and nanodrugs in live cells.

    PubMed

    Nedosekin, Dmitry A; Foster, Stephen; Nima, Zeid A; Biris, Alexandru S; Galanzha, Ekaterina I; Zharov, Vladimir P

    2015-08-01

    Growing biomedical applications of non-fluorescent nanoparticles (NPs) for molecular imaging, disease diagnosis, drug delivery, and theranostics require new tools for real-time detection of nanomaterials, drug nano-carriers, and NP-drug conjugates (nanodrugs) in complex biological environments without additional labeling. Photothermal (PT) microscopy (PTM) has enormous potential for absorption-based identification and quantification of non-fluorescent molecules and NPs at a single molecule and 1.4 nm gold NP level. Recently, we have developed confocal PTM providing three-dimensional (3D) mapping and spectral identification of multiple chromophores and fluorophores in live cells. Here, we summarize recent advances in the application of confocal multicolor PTM for 3D visualization of single and clustered NPs, alone and in individual cells. In particular, we demonstrate identification of functionalized magnetic and gold-silver NPs, as well as graphene and carbon nanotubes in cancer cells and among blood cells. The potential to use PTM for super-resolution imaging (down to 50 nm), real-time NP tracking, guidance of PT nanotherapy, and multiplex cancer markers targeting, as well as analysis of non-linear PT phenomena and amplification of nanodrug efficacy through NP clustering and nano-bubble formation are also discussed.

  12. Photothermal confocal multicolor microscopy of nanoparticles and nanodrugs in live cells.

    PubMed

    Nedosekin, Dmitry A; Foster, Stephen; Nima, Zeid A; Biris, Alexandru S; Galanzha, Ekaterina I; Zharov, Vladimir P

    2015-08-01

    Growing biomedical applications of non-fluorescent nanoparticles (NPs) for molecular imaging, disease diagnosis, drug delivery, and theranostics require new tools for real-time detection of nanomaterials, drug nano-carriers, and NP-drug conjugates (nanodrugs) in complex biological environments without additional labeling. Photothermal (PT) microscopy (PTM) has enormous potential for absorption-based identification and quantification of non-fluorescent molecules and NPs at a single molecule and 1.4 nm gold NP level. Recently, we have developed confocal PTM providing three-dimensional (3D) mapping and spectral identification of multiple chromophores and fluorophores in live cells. Here, we summarize recent advances in the application of confocal multicolor PTM for 3D visualization of single and clustered NPs, alone and in individual cells. In particular, we demonstrate identification of functionalized magnetic and gold-silver NPs, as well as graphene and carbon nanotubes in cancer cells and among blood cells. The potential to use PTM for super-resolution imaging (down to 50 nm), real-time NP tracking, guidance of PT nanotherapy, and multiplex cancer markers targeting, as well as analysis of non-linear PT phenomena and amplification of nanodrug efficacy through NP clustering and nano-bubble formation are also discussed. PMID:26133539

  13. A generalized Potts model for confocal microscopy images

    NASA Astrophysics Data System (ADS)

    Máté, Gabriell; Heermann, Dieter W.

    2015-01-01

    Much as being among the least invasive mainstream imaging technologies in life sciences, the resolution of confocal microscopy is limited. Imaged structures, e.g., chromatin-fiber loops, have diameters around or beyond the diffraction limit, and microscopy images show seemingly random spatial density distributions only. While such images are important because the organization of the chromosomes influences different cell mechanisms, many interesting questions can also be related to the observed patterns. These concern their spatial aspects, the role of randomness, the possibility of modeling these images with a random generative process, the interaction between the densities of adjacent loci, the length-scales of these influences, etc. We answer these questions by implementing a generalization of the Potts model. We show how to estimate the model parameters, test the performance of the estimation process and numerically prove that the obtained values converge to the ground truth. Finally, we generate images with a trained model and show that they compare well to real cell images.

  14. Virtual 3D microscopy using multiplane whole slide images in diagnostic pathology.

    PubMed

    Kalinski, Thomas; Zwönitzer, Ralf; Sel, Saadettin; Evert, Matthias; Guenther, Thomas; Hofmann, Harald; Bernarding, Johannes; Roessner, Albert

    2008-08-01

    To reproduce focusing in virtual microscopy, it is necessary to construct 3-dimensional (3D) virtual slides composed of whole slide images with different focuses. As focusing is frequently used for the assessment of Helicobacter pylori colonization in diagnostic pathology, we prepared virtual 3D slides with up to 9 focus planes from 144 gastric biopsy specimens with or without H pylori gastritis. The biopsy specimens were diagnosed in a blinded manner by 3 pathologists according to the updated Sydney classification using conventional microscopy, virtual microscopy with a single focus plane, and virtual 3D microscopy with 5 and 9 focus planes enabling virtual focusing. Regarding the classification of H pylori, we found a positive correlation between the number of focus planes used in virtual microscopy and the number of correct diagnoses as determined by conventional microscopy. Concerning H pylori positivity, the specificity and sensitivity of virtual 3D microscopy using virtual slides with 9 focus planes achieved a minimum of 0.95 each, which was approximately the same as in conventional microscopy. We consider virtual 3D microscopy appropriate for primary diagnosis of H pylori gastritis and equivalent to conventional microscopy.

  15. Astigmatic multifocus microscopy enables deep 3D super-resolved imaging

    PubMed Central

    Oudjedi, Laura; Fiche, Jean-Bernard; Abrahamsson, Sara; Mazenq, Laurent; Lecestre, Aurélie; Calmon, Pierre-François; Cerf, Aline; Nöllmann, Marcelo

    2016-01-01

    We have developed a 3D super-resolution microscopy method that enables deep imaging in cells. This technique relies on the effective combination of multifocus microscopy and astigmatic 3D single-molecule localization microscopy. We describe the optical system and the fabrication process of its key element, the multifocus grating. Then, two strategies for localizing emitters with our imaging method are presented and compared with a previously described deep 3D localization algorithm. Finally, we demonstrate the performance of the method by imaging the nuclear envelope of eukaryotic cells reaching a depth of field of ~4µm. PMID:27375935

  16. Astigmatic multifocus microscopy enables deep 3D super-resolved imaging.

    PubMed

    Oudjedi, Laura; Fiche, Jean-Bernard; Abrahamsson, Sara; Mazenq, Laurent; Lecestre, Aurélie; Calmon, Pierre-François; Cerf, Aline; Nöllmann, Marcelo

    2016-06-01

    We have developed a 3D super-resolution microscopy method that enables deep imaging in cells. This technique relies on the effective combination of multifocus microscopy and astigmatic 3D single-molecule localization microscopy. We describe the optical system and the fabrication process of its key element, the multifocus grating. Then, two strategies for localizing emitters with our imaging method are presented and compared with a previously described deep 3D localization algorithm. Finally, we demonstrate the performance of the method by imaging the nuclear envelope of eukaryotic cells reaching a depth of field of ~4µm.

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

    PubMed Central

    Gualda, Emilio J.; Simão, Daniel; Pinto, Catarina; Alves, Paula M.; Brito, Catarina

    2014-01-01

    The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment. PMID:25161607

  18. Study of the collagen structure in the superficial zone and physiological state of articular cartilage using a 3D confocal imaging technique

    PubMed Central

    Wu, Jian P; Kirk, Thomas B; Zheng, Ming H

    2008-01-01

    Introduction The collagen structure in the superficial zone of articular cartilage is critical to the tissue's durability. Early osteoarthritis is often characterized with fissures on the articular surface. This is closely related to the disruption of the collagen network. However, the traditional histology can not offer visualization of the collagen structure in articular cartilage because it uses conventional optical microscopy that does not have insufficient imaging resolution to resolve collagen from proteoglycans in hyaline articular cartilage. This study examines the 3D collagen network of articular cartilage scored from 0 to 2 in the scoring system of International Cartilage Repair Society, and aims to develop a 3D histology for assessing early osteoarthritis. Methods Articular cartilage was visually classified into five physiological groups: normal cartilage, aged cartilage, cartilage with artificial and natural surface disruption, and fibrillated. The 3D collagen matrix of the cartilage was acquired using a 3D imaging technique developed previously. Traditional histology was followed to grade the physiological status of the cartilage in the scoring system of International Cartilage Repair Society. Results Normal articular cartilage contains interwoven collagen bundles near the articular surface, approximately within the lamina splendens. However, its collagen fibres in the superficial zone orient predominantly in a direction spatially oblique to the articular surface. With age and disruption of the articular surface, the interwoven collagen bundles are gradually disappeared, and obliquely oriented collagen fibres change to align predominantly in a direction spatially perpendicular to the articular surface. Disruption of the articular surface is well related to the disappearance of the interwoven collagen bundles. Conclusion A 3D histology has been developed to supplement the traditional histology and study the subtle changes in the collagen network in the

  19. A segmentation method for 3D visualization of neurons imaged with a confocal laser scanning microscope

    NASA Astrophysics Data System (ADS)

    Anderson, Jeffrey R.; Barrett, Steven F.; Wilcox, Michael J.

    2005-04-01

    Our understanding of the world around us is based primarily on three-dimensional information because of the environment in which we live and interact. Medical or biological image information is often collected in the form of two-dimensional, serial section images. As such, it is difficult for the observer to mentally reconstruct the three dimensional features of each object. Although many image rendering software packages allow for 3D views of the serial sections, they lack the ability to segment, or isolate different objects in the data set. Segmentation is the key to creating 3D renderings of distinct objects from serial slice images, like separate pieces to a puzzle. This paper describes a segmentation method for objects recorded with serial section images. The user defines threshold levels and object labels on a single image of the data set that are subsequently used to automatically segment each object in the remaining images of the same data set, while maintaining boundaries between contacting objects. The performance of the algorithm is verified using mathematically defined shapes. It is then applied to the visual neurons of the housefly, Musca domestica. Knowledge of the fly"s visual system may lead to improved machine visions systems. This effort has provided the impetus to develop this segmentation algorithm. The described segmentation method can be applied to any high contrast serial slice data set that is well aligned and registered. The medical field alone has many applications for rapid generation of 3D segmented models from MRI and other medical imaging modalities.

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

    PubMed

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

    2016-05-16

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

  1. LED uniform illumination system for DMD-based confocal microscopy

    NASA Astrophysics Data System (ADS)

    Xiao, Kaimin; Hou, Wenmei; Xu, Qixin; Peng, Bofang

    2013-10-01

    Due to the coherence of laser light source it could produce coherent noise in parallel confocal microscopy based on Digital Micromirror Device (DMD) and thus affect the resolution. LED light source instead of the laser light source can give full play because of its incoherence characterization. In this paper, free-form surface lens is used for LED secondary optics design. According to the LED characteristics and the law of refraction, we have established differential equations of free-form surface. We solved equations with the method of Runge-Kutta by Matlab and the model was built in Tracepro for optical simulation. The results show that the uniformity on the DMD is better than 90% and the lighting efficiency is higher than before. The measured data show us a much more uniform illumination on DMD and LED uniform illumination system successfully avoided the gray error which was caused by the uneven illumination. The LED driver circuit using DC power supply provides us a more stable light source. The axial optical tomography is more accurate and the reconstruction of three-dimensional image is more clearer.

  2. Confocal microscopy for intracellular co-localization of proteins.

    PubMed

    Miyashita, Toshiyuki

    2015-01-01

    Confocal laser scanning microscopy is the best method to visualize intracellular co-localization of proteins in intact cells. Because of the point scan/pinhole detection system, light contribution from the neighborhood of the scanning spot in the specimen can be eliminated, allowing high Z-axis resolution. Fluorescence detection by sensitive photomultiplier tubes allows the usage of filters with a narrow bandpath, resulting in minimal cross-talk (overlap) between two spectra. This is particularly important in demonstrating co-localization of proteins with multicolor labeling. Here, the methods outlining the detection of transiently expressed tagged proteins and the detection of endogenous proteins are described. Ideally, the intracellular co-localization of two endogenous proteins should be demonstrated. However, when antibodies raised against the protein of interest are unavailable for immunofluorescence or the available cell lines do not express the protein of interest sufficiently enough for immunofluorescence, an alternative method is to transfect cells with expression plasmids that encode tagged proteins and stain the cells with anti-tag antibodies. However, it should be noted that the tagging of proteins of interest or their overexpression could potentially alter the intracellular localization or the function of the target protein. PMID:25859973

  3. Rhinosporidium seeberi Nuclear Cycle Activities Using Confocal Microscopy.

    PubMed

    Delfino, Darly; Mendoza, Leonel; Vilela, Raquel

    2016-02-01

    Rhinosporidium seeberi is an uncultivated Ichthyosporean infecting animals, including humans. Recent studies suggested R. seeberi undergoes synchronized nuclear division without cytokinesis. We used confocal microscopy to investigate R. seeberi nuclear division cycles in formalin-fixed tissues stained with DAPI and phalloidin. We report that R. seeberi nuclei in juvenile and intermediary sporangia synchronously divided without cytokinesis. Intermediary sporangia display numerous 3-4 μm nuclei at different mitotic stages as well as a thick inner layer with strong affinity for phalloidin. Mature sporangia showed numerous 5-12 μm cell-walled endospores, each containing a 2-4 μm in diameter nucleus. Phalloidin did not bind to the inner layers of mature sporangia or endospores. The development of a "germinative zone" in the inner layer of mature sporangia containing hundreds of nuclei was also confirmed. This study establishes that during the R. seeberi life cycle synchronous nuclear divisions without cytokinesis takes place, resulting in the formation of thousands of nuclei. Cytokinesis, on the other hand, is a 1-time event and occurs in the latest stages of intermediate sporangia, after the formation of thousands of nuclei and just before mature sporangia development. PMID:26461427

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

    PubMed

    Patel, Dipika V; McGhee, Charles N

    2013-01-01

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

  5. Imaging liver biology in vivo using conventional confocal microscopy.

    PubMed

    Marques, Pedro E; Antunes, Maísa M; David, Bruna A; Pereira, Rafaela V; Teixeira, Mauro M; Menezes, Gustavo B

    2015-02-01

    Imaging of live animals using intravital microscopy (IVM) has provided a substantial advance in our understanding of cell biology. Here we describe how to adapt a conventional, relatively low-cost laser-scanning microscope to operate as a versatile imaging station. We present the surgical procedures needed to perform liver confocal IVM in mice, thereby allowing one to image different cells in their native environment, including hepatocytes, endothelial cells and leukocytes, as well as to analyze their morphology and function under physiological or pathological conditions. In addition, we propose a plethora of working doses of antibodies and probes to stain multiple cells and molecules simultaneously in vivo. Considering the central role of the liver in metabolism and immunity and the growing interest in the relationship between immune and parenchymal cells, this protocol, in which 20 min of preparation yields up to 4 h of imaging, provides useful insights for various research fields. In addition, the protocol can be easily adapted to investigate adipose tissue, mesentery, intestines, spleen and virtually any abdominal organ.

  6. Ellipsoid Segmentation Model for Analyzing Light-Attenuated 3D Confocal Image Stacks of Fluorescent Multi-Cellular Spheroids

    PubMed Central

    Barbier, Michaël; Jaensch, Steffen; Cornelissen, Frans; Vidic, Suzana; Gjerde, Kjersti; de Hoogt, Ronald; Graeser, Ralph; Gustin, Emmanuel; Chong, Yolanda T.

    2016-01-01

    In oncology, two-dimensional in-vitro culture models are the standard test beds for the discovery and development of cancer treatments, but in the last decades, evidence emerged that such models have low predictive value for clinical efficacy. Therefore they are increasingly complemented by more physiologically relevant 3D models, such as spheroid micro-tumor cultures. If suitable fluorescent labels are applied, confocal 3D image stacks can characterize the structure of such volumetric cultures and, for example, cell proliferation. However, several issues hamper accurate analysis. In particular, signal attenuation within the tissue of the spheroids prevents the acquisition of a complete image for spheroids over 100 micrometers in diameter. And quantitative analysis of large 3D image data sets is challenging, creating a need for methods which can be applied to large-scale experiments and account for impeding factors. We present a robust, computationally inexpensive 2.5D method for the segmentation of spheroid cultures and for counting proliferating cells within them. The spheroids are assumed to be approximately ellipsoid in shape. They are identified from information present in the Maximum Intensity Projection (MIP) and the corresponding height view, also known as Z-buffer. It alerts the user when potential bias-introducing factors cannot be compensated for and includes a compensation for signal attenuation. PMID:27303813

  7. Ellipsoid Segmentation Model for Analyzing Light-Attenuated 3D Confocal Image Stacks of Fluorescent Multi-Cellular Spheroids.

    PubMed

    Barbier, Michaël; Jaensch, Steffen; Cornelissen, Frans; Vidic, Suzana; Gjerde, Kjersti; de Hoogt, Ronald; Graeser, Ralph; Gustin, Emmanuel; Chong, Yolanda T

    2016-01-01

    In oncology, two-dimensional in-vitro culture models are the standard test beds for the discovery and development of cancer treatments, but in the last decades, evidence emerged that such models have low predictive value for clinical efficacy. Therefore they are increasingly complemented by more physiologically relevant 3D models, such as spheroid micro-tumor cultures. If suitable fluorescent labels are applied, confocal 3D image stacks can characterize the structure of such volumetric cultures and, for example, cell proliferation. However, several issues hamper accurate analysis. In particular, signal attenuation within the tissue of the spheroids prevents the acquisition of a complete image for spheroids over 100 micrometers in diameter. And quantitative analysis of large 3D image data sets is challenging, creating a need for methods which can be applied to large-scale experiments and account for impeding factors. We present a robust, computationally inexpensive 2.5D method for the segmentation of spheroid cultures and for counting proliferating cells within them. The spheroids are assumed to be approximately ellipsoid in shape. They are identified from information present in the Maximum Intensity Projection (MIP) and the corresponding height view, also known as Z-buffer. It alerts the user when potential bias-introducing factors cannot be compensated for and includes a compensation for signal attenuation. PMID:27303813

  8. Confocal (micro)-XRF for 3D anlaysis of elements distribution in hot environmental particles

    SciTech Connect

    Bielewski, M; Eriksson, M; Himbert, J; Simon, R; Betti, M; Hamilton, T F

    2007-11-27

    Studies on the fate and transport of radioactive contaminates in the environment are often constrained by a lack of knowledge on the elemental distribution and general behavior of particulate bound radionuclides contained in hot particles. A number of hot particles were previously isolated from soil samples collected at former U.S. nuclear test sites in the Marshall Islands and characterized using non-destructive techniques [1]. The present investigation at HASYLAB is a part of larger research program at ITU regarding the characterization of environmental radioactive particles different locations and source-terms. Radioactive particles in the environment are formed under a number of different release scenarios and, as such, their physicochemical properties may provide a basis for identifying source-term specific contamination regimes. Consequently, studies on hot particles are not only important in terms of studying the elemental composition and geochemical behavior of hot particles but may also lead to advances in assessing the long-term impacts of radioactive contamination on the environment. Six particles isolated from soil samples collected at the Marshall Islands were studied. The element distribution in the particles was determined by confocal {micro}-XRF analysis using the ANKA FLUO beam line. The CRL (compound refractive lens) was used to focus the exciting beam and the polycapillary half lens to collimate the detector. The dimensions of confocal spot were measured by 'knife edge scanning' method with thin gold structure placed at Si wafer. The values of 3.1 x 1.4 x 18.4 {micro}m were achieved if defined as FWHMs of measured L?intensity profiles and when the19.1 keV exciting radiation was used. The collected XRF spectra were analyzed offline with AXIL [2] software to obtain net intensities of element characteristic lines.Further data processing and reconstruction of element distribution was done with the software 'R' [3] dedicated for statistical

  9. Dual-color 3D superresolution microscopy by combined spectral-demixing and biplane imaging.

    PubMed

    Winterflood, Christian M; Platonova, Evgenia; Albrecht, David; Ewers, Helge

    2015-07-01

    Multicolor three-dimensional (3D) superresolution techniques allow important insight into the relative organization of cellular structures. While a number of innovative solutions have emerged, multicolor 3D techniques still face significant technical challenges. In this Letter we provide a straightforward approach to single-molecule localization microscopy imaging in three dimensions and two colors. We combine biplane imaging and spectral-demixing, which eliminates a number of problems, including color cross-talk, chromatic aberration effects, and problems with color registration. We present 3D dual-color images of nanoscopic structures in hippocampal neurons with a 3D compound resolution routinely achieved only in a single color.

  10. Visualization of calcium and zinc ions in Saccharomyces cerevisiae cells treated with PEFs (pulse electric fields) by laser confocal microscopy.

    PubMed

    Urszula, Pankiewicz; Jerzy, Jamroz; Sujka, Monika; Kowalski, Radosław

    2015-12-01

    The aim of the present work was to visualize the areas of increased concentration of calcium and zinc ions inside Saccharomyces cerevisiae cells with the use of confocal microscopy and to make an attempt to asses semi-quantitatively their concentration within the limits of the cells. Semi-quantitative analysis revealed that fluorescence inside cells from control samples was three-times lower than that observed for cells from the sample enriched with calcium. Differences in distribution of fluorescence intensity between cells originated from the samples enriched with zinc and control samples were also observed. On the basis of the optical sections, the 3D reconstructions of ion-rich areas distribution in the cell were made. The obtained results showed that confocal microscopy is a useful technique for visualization of the areas in S. cerevisiae cells which contain higher amount of calcium and zinc and it may be also used for semi-quantitative analysis.

  11. Lithographically-fabricated channel arrays for confocal x-ray fluorescence microscopy and XAFS

    NASA Astrophysics Data System (ADS)

    Woll, Arthur R.; Agyeman-Budu, David; Choudhury, Sanjukta; Coulthard, Ian; Finnefrock, Adam C.; Gordon, Robert; Hallin, Emil; Mass, Jennifer

    2014-03-01

    Confocal X-ray Fluorescence Microscopy (CXRF) employs overlapping focal regions of two x-ray optics—a condenser and collector—to directly probe a 3D volume. The minimum-achievable size of this probe volume is limited by the collector, for which polycapillaries are generally the optic of choice. Recently, we demonstrated an alternative collection optic for CXRF, consisting of an array of micron-scale collimating channels, etched in silicon, and arranged like spokes of a wheel directed towards a single source position. The optic, while successful, had a working distance of only 0.2 mm and exhibited relatively low total collection efficiency, limiting its practical application. Here, we describe a new design in which the collimating channels are formed by a staggered array of pillars whose side-walls taper away from the channel axis. This approach improves both collection efficiency and working distance, while maintaining excellent spatial resolution. We illustrate these improvements with confocal XRF data obtained at the Cornell High Energy Synchrotron Source (CHESS) and the Advanced Photon Source (APS) beamline 20-ID-B.

  12. Laser ablation of basal cell carcinomas guided by confocal microscopy

    NASA Astrophysics Data System (ADS)

    Sierra, Heidy; Cordova, Miguel; Nehal, Kishwer; Rossi, Anthony; Chen, Chih-Shan Jason; Rajadhyaksha, Milind

    2016-02-01

    Laser ablation offers precise and fast removal of superficial and early nodular types of basal cell carcinomas (BCCs). Nevertheless, the lack of histological confirmation has been a limitation. Reflectance confocal microscopy (RCM) imaging combined with a contrast agent can offer cellular-level histology-like feedback to detect the presence (or absence) of residual BCC directly on the patient. We conducted an ex vivo bench-top study to provide a set of effective ablation parameters (fluence, number of passes) to remove superficial BCCs while also controlling thermal coagulation post-ablation to allow uptake of contrast agent. The results for an Er:YAG laser (2.9 um and pulse duration 250us) show that with 6 passes of 25 J/cm2, thermal coagulation can be effectively controlled, to allow both the uptake of acetic acid (contrast agent) and detection of residual (or absence) BCCs. Confirmation was provided with histological examination. An initial in vivo study on 35 patients shows that the uptake of contrast agent aluminum chloride) and imaging quality is similar to that observed in the ex vivo study. The detection of the presence of residual tumor or complete clearance was confirmed in 10 wounds with (additional) histology and in 25 lesions with follow-up imaging. Our results indicate that resolution is sufficient but further development and use of appropriate contrast agent are necessary to improve sensitivity and specificity. Advances in RCM technology for imaging of lateral and deep margins directly on the patient may provide less invasive, faster and less expensive image-guided approaches for treatment of BCCs.

  13. Improving transverse resolution of confocal microscopy through spatiotemporal modulation

    NASA Astrophysics Data System (ADS)

    Wang, Baokai; Zou, Limin; Zhang, Su; Tan, Jiubin

    2015-11-01

    A new method is proposed in this paper to improve transverse resolution of a confocal microscope. By setting up the model of a confocal microscope system through spatiotemporal modulation with moving gratings or acousto-optical modulation without defocus distance under coherent light illumination and deducing two-dimensional coherent image formula and transfer function, simulation tests are run with or without spatiotemporal modulation to prove the effectiveness of the proposed method. Simulation results indicate the proposed method can be used to improve the transverse resolution of a confocal microscope system.

  14. Automatic Detection, Segmentation and Classification of Retinal Horizontal Neurons in Large-scale 3D Confocal Imagery

    SciTech Connect

    Karakaya, Mahmut; Kerekes, Ryan A; Gleason, Shaun Scott; Martins, Rodrigo; Dyer, Michael

    2011-01-01

    Automatic analysis of neuronal structure from wide-field-of-view 3D image stacks of retinal neurons is essential for statistically characterizing neuronal abnormalities that may be causally related to neural malfunctions or may be early indicators for a variety of neuropathies. In this paper, we study classification of neuron fields in large-scale 3D confocal image stacks, a challenging neurobiological problem because of the low spatial resolution imagery and presence of intertwined dendrites from different neurons. We present a fully automated, four-step processing approach for neuron classification with respect to the morphological structure of their dendrites. In our approach, we first localize each individual soma in the image by using morphological operators and active contours. By using each soma position as a seed point, we automatically determine an appropriate threshold to segment dendrites of each neuron. We then use skeletonization and network analysis to generate the morphological structures of segmented dendrites, and shape-based features are extracted from network representations of each neuron to characterize the neuron. Based on qualitative results and quantitative comparisons, we show that we are able to automatically compute relevant features that clearly distinguish between normal and abnormal cases for postnatal day 6 (P6) horizontal neurons.

  15. 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

  16. Measuring Corneal Haze by Using Scheimpflug Photography and Confocal Microscopy

    PubMed Central

    McLaren, Jay W.; Wacker, Katrin; Kane, Katrina M.; Patel, Sanjay V.

    2016-01-01

    Purpose We compared corneal backscatter estimated from a Scheimpflug camera with backscatter estimated from a clinical confocal microscope across a wide range of corneal haze. Methods A total of 59 corneas from 35 patients with a range of severity of Fuchs' endothelial corneal dystrophy and 15 corneas from 9 normal participants were examined using a Scheimpflug camera (Pentacam) and a confocal microscope (ConfoScan 4). The mean image brightness from the anterior 120 μm, midcornea, and posterior 60 μm of the cornea across the central 2 mm recorded by the Scheimpflug camera and analogous regions from the confocal microscope were measured and standardized. Differences between instruments and correlations between backscatter and disease severity were determined by using generalized estimating equation models. Results Backscatter measured by the two instruments in the anterior and midcornea were correlated (r = 0.67 and 0.43, respectively, P < 0.001), although in the posterior cornea they were not correlated (r = 0.13, P = 0.66). Measured with the Scheimpflug camera, mean backscatter from the anterior and midcornea were greater, whereas backscatter from the posterior cornea was lower (P < 0.001) than that measured by the confocal microscope. Backscatter from the anterior cornea was correlated with disease severity for both instruments (Scheimpflug, r = 0.55, P < 0.001; confocal, r = 0.49, P = 0.003). Conclusions The Scheimpflug camera and confocal microscope should not be used interchangeably to measure corneal haze. The ability to detect changes in backscatter with disease severity is superior with the Scheimpflug camera. However, the confocal microscope provides higher resolution of corneal structure. PMID:26803798

  17. Three-dimensional assessment of bone turnover using computed microtomography and laser-scanning confocal microscopy

    NASA Astrophysics Data System (ADS)

    Prevrhal, Sven; Jiang, Yebin; Zhao, Jenny; Genant, Harry K.

    2000-06-01

    Objective: Metabolic activity in trabecular bone is an important indicator in the therapy of bone diseases like osteoporosis. It is reflected by the amount of osteoid (young, not yet mineralized bone) and young calcified tissue (YCT). Our aim was to replace standard 2D histomorphometry with a 3D approach for osteoid and YCT measurement. Measurement Methods: Excised lumbar vertebrae of 5 ovariectomized (OVX) and 5 control rats were 3D-scanned with computed micro-tomography ((mu) CT, isotropic spatial resolution 20 micrometer3) and laser scanning confocal microscopy (LSCM, 20X magnification, 1X1X2 micrometer3 spatial resolution). (mu) CT shows trabecular bone structure; LSCM shows osteoid and YCT by fluorescent light. Image Processing Methods: The fraction of bone to tissue volume (BV/TV) and the number of trabeculae (Tb.N) were calculated from globally thresholded (mu) CT images. LSCM images were enhanced using top-hat transform, globally thresholded and morphologically closed. Separate regions were labeled by volume growing. We measured feature volume to background volume ratio and number of features per unit volume. Results and Conclusions: In the specimens obtained from the OVX rats, a significant increase in the volume fractions of osteoid and YCT could be seen. The (mu) CT-LSCM approach presents a significant improvement over time-consuming, standard histomorphometry. The image processing for both modalities could be achieved automatically.

  18. Digital holographic microscopy for imaging growth and treatment response in 3D tumor models

    NASA Astrophysics Data System (ADS)

    Li, Yuyu; Petrovic, Ljubica; Celli, Jonathan P.; Yelleswarapu, Chandra S.

    2014-03-01

    While three-dimensional tumor models have emerged as valuable tools in cancer research, the ability to longitudinally visualize the 3D tumor architecture restored by these systems is limited with microscopy techniques that provide only qualitative insight into sample depth, or which require terminal fixation for depth-resolved 3D imaging. Here we report the use of digital holographic microscopy (DHM) as a viable microscopy approach for quantitative, non-destructive longitudinal imaging of in vitro 3D tumor models. Following established methods we prepared 3D cultures of pancreatic cancer cells in overlay geometry on extracellular matrix beds and obtained digital holograms at multiple timepoints throughout the duration of growth. The holograms were digitally processed and the unwrapped phase images were obtained to quantify nodule thickness over time under normal growth, and in cultures subject to chemotherapy treatment. In this manner total nodule volumes are rapidly estimated and demonstrated here to show contrasting time dependent changes during growth and in response to treatment. This work suggests the utility of DHM to quantify changes in 3D structure over time and suggests the further development of this approach for time-lapse monitoring of 3D morphological changes during growth and in response to treatment that would otherwise be impractical to visualize.

  19. Resolution doubling using confocal microscopy via analogy with structured illumination microscopy

    NASA Astrophysics Data System (ADS)

    Hayashi, Shinichi

    2016-08-01

    Structured illumination microscopy (SIM) is a super-resolution fluorescence microscopy with a 2-fold higher lateral resolution than conventional wide-field fluorescence (WF) microscopy. Confocal fluorescence (CF) microscopy has approximately the same optical cutoff frequency as SIM; however, the maximum theoretical increase in lateral resolution over that of WF is 1.4-fold with an infinitesimal pinhole diameter. Quantitative comparisons based on an analytical imaging formula revealed that modulation transfer functions (MTFs) of SIM reconstructed images before postprocessing are nearly identical to those of CF images recorded with an infinitesimal pinhole diameter. Here, we propose a new method using an adequate pinhole diameter combined with the use of an apodized Fourier inverse filter to increase the lateral resolution of CF images to as much as that SIM images without significant noise degradation in practice. Furthermore, the proposed method does not require a posteriori parameterization and has reproducibility. This approach can be easily applied to conventional laser scanning CF, spinning disk CF, and multiphoton microscopies.

  20. Seeing a Mycobacterium-Infected Cell in Nanoscale 3D: Correlative Imaging by Light Microscopy and FIB/SEM Tomography

    PubMed Central

    Beckwith, Marianne Sandvold; Beckwith, Kai Sandvold; Sikorski, Pawel; Skogaker, Nan Tostrup

    2015-01-01

    Mycobacteria pose a threat to the world health today, with pathogenic and opportunistic bacteria causing tuberculosis and non-tuberculous disease in large parts of the population. Much is still unknown about the interplay between bacteria and host during infection and disease, and more research is needed to meet the challenge of drug resistance and inefficient vaccines. This work establishes a reliable and reproducible method for performing correlative imaging of human macrophages infected with mycobacteria at an ultra-high resolution and in 3D. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography is applied, together with confocal fluorescence microscopy for localization of appropriately infected cells. The method is based on an Aclar poly(chloro-tri-fluoro)ethylene substrate, micropatterned into an advantageous geometry by a simple thermomoulding process. The platform increases the throughput and quality of FIB/SEM tomography analyses, and was successfully applied to detail the intracellular environment of a whole mycobacterium-infected macrophage in 3D. PMID:26406896

  1. In vivo confocal microscopy of meibomian glands in primary blepharospasm

    PubMed Central

    Lin, Tong; Gong, Lan

    2016-01-01

    Abstract The aim of the study was to evaluate the morphological changes of meibomian glands (MGs) in primary blepharospasm (PBS) by in vivo laser scanning confocal microscopy (LSCM) and to investigate the correlations between clinical data of PBS and LSCM parameters of MGs. This prospective and case–control study recruited 30 consecutive PBS patients and 30 age- and gender-matched healthy controls. After questionnaire assessments of ocular surface disease index (OSDI), Jankovic rating scale, and blepharospasm disability index, all subjects underwent blink rate evaluation, tear film break-up time (TBUT), corneal fluorescein staining (CFS), Schirmer test, MG expressibility, meibum quality, MG dropout, and LSCM examination of the MGs. The main LSCM outcomes included the mean MG acinar area and density, orifice diameter, meibum secretion reflectivity, acinar irregularity, and inhomogeneity of interstice and acinar wall. The PBS patients had significantly higher blink rate, higher OSDI and CFS scores, lower TBUT and Schirmer test value, and worse MG expressibility than the controls (All P < 0.05), whereas meibum quality showed no difference (P > 0.05). The PBS patients showed lower values of MG acinar area, orifice diameter and meibum secretion reflectivity, and higher scores of acinar irregularity and inhomogeneity of interstices than the controls (All P < 0.05). For the PBS patients, the severity of blepharospasm evaluated by JCR scale was strong correlated with MG acinar area (P < 0.001), orifice diameter (P = 0.002), meibum secretion reflectivity (P = 0.002), and MG acinar irregularity (P = 0.013). The MG expressibility was significantly correlated to MG acinar area (P = 0.039), orifice diameter (P < 0.001), and MG acinar irregularity (P = 0.014). The OSDI score was moderate correlated with MG acinar irregularity (P = 0.016), whereas the TBUT value was positively correlated with MG acinar area (P = 0.045) and negatively correlated to MG acinar

  2. Heads-up 3D Microscopy: An Ergonomic and Educational Approach to Microsurgery.

    PubMed

    Mendez, Bernardino M; Chiodo, Michael V; Vandevender, Darl; Patel, Parit A

    2016-05-01

    Traditional microsurgery can lead surgeons to use postures that cause musculoskeletal fatigue, leaving them more prone to work-related injuries. A new technology from TrueVision transmits the microscopic image onto a 3-dimensional (3D) monitor, allowing surgeons to operate while sitting/standing in a heads-up position. The purpose of this study was to evaluate the feasibility of performing heads-up 3D microscopy as a more ergonomic alternative to traditional microsurgery. A feasibility study was conducted comparing heads-up 3D microscopy and traditional microscopy by performing femoral artery anastomoses on 8 Sprague-Dawley rats. Operative times and patency rates for each technology were compared. The 8 microsurgeons completed a questionnaire comparing image quality, comfort, technical feasibility, and educational value of the 2 technologies. Rat femoral artery anastomoses were successfully carried out by all 8 microsurgeons with each technology. There was no significant difference in anastomosis time between heads-up 3D and traditional microscopy (average times, 34.5 and 33.8 minutes, respectively; P = 0.66). Heads-up 3D microscopy was rated superior in neck and back comfort by 75% of participants. Image resolution, field of view, and technical feasibility were found to be superior or equivalent in 75% of participants, whereas 63% evaluated depth perception to be superior or equivalent. Heads-up 3D microscopy is a new technology that improves comfort for the microsurgeon without compromising image quality or technical feasibility. Its use has become prevalent in the field of ophthalmology and may also have utility in plastic and reconstructive surgery. PMID:27579241

  3. Heads-up 3D Microscopy: An Ergonomic and Educational Approach to Microsurgery.

    PubMed

    Mendez, Bernardino M; Chiodo, Michael V; Vandevender, Darl; Patel, Parit A

    2016-05-01

    Traditional microsurgery can lead surgeons to use postures that cause musculoskeletal fatigue, leaving them more prone to work-related injuries. A new technology from TrueVision transmits the microscopic image onto a 3-dimensional (3D) monitor, allowing surgeons to operate while sitting/standing in a heads-up position. The purpose of this study was to evaluate the feasibility of performing heads-up 3D microscopy as a more ergonomic alternative to traditional microsurgery. A feasibility study was conducted comparing heads-up 3D microscopy and traditional microscopy by performing femoral artery anastomoses on 8 Sprague-Dawley rats. Operative times and patency rates for each technology were compared. The 8 microsurgeons completed a questionnaire comparing image quality, comfort, technical feasibility, and educational value of the 2 technologies. Rat femoral artery anastomoses were successfully carried out by all 8 microsurgeons with each technology. There was no significant difference in anastomosis time between heads-up 3D and traditional microscopy (average times, 34.5 and 33.8 minutes, respectively; P = 0.66). Heads-up 3D microscopy was rated superior in neck and back comfort by 75% of participants. Image resolution, field of view, and technical feasibility were found to be superior or equivalent in 75% of participants, whereas 63% evaluated depth perception to be superior or equivalent. Heads-up 3D microscopy is a new technology that improves comfort for the microsurgeon without compromising image quality or technical feasibility. Its use has become prevalent in the field of ophthalmology and may also have utility in plastic and reconstructive surgery.

  4. High-resolution confocal Raman microscopy using pixel reassignment.

    PubMed

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

    2016-08-15

    We present a practical modification of fiber-coupled confocal Raman scanning microscopes that is able to provide high confocal resolution in conjunction with high light collection efficiency. For this purpose, the single detection fiber is replaced by a hexagonal lenslet array in combination with a hexagonally packed round-to-linear multimode fiber bundle. A multiline detector is used to collect individual Raman spectra for each fiber. Data post-processing based on pixel reassignment allows one to improve the lateral resolution by up to 41% compared to a single fiber of equal light collection efficiency. We present results from an experimental implementation featuring seven collection fibers, yielding a resolution improvement of about 30%. We believe that our implementation represents an attractive upgrade for existing confocal Raman microscopes that employ multi-line detectors. PMID:27519099

  5. Reflectance confocal microscopy of red blood cells: simulation and experiment

    PubMed Central

    Zeidan, Adel; Yelin, Dvir

    2015-01-01

    Measuring the morphology of red blood cells is important for clinical diagnosis, providing valuable indications on a patient’s health. In this work, we have simulated the appearance of normal red blood cells under a reflectance confocal microscope and discovered unique relations between the morphological parameters and the resulting characteristic interference patterns of the cell. The simulation results showed good agreement with in vitro reflectance confocal images of red blood cells, acquired using spectrally encoded flow cytometry that imaged the cells in a linear flow without artificial staining. By matching the simulated patterns to confocal images of the cells, this method could be used for measuring cell morphology in three dimensions and for studying their physiology. PMID:26600999

  6. 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

  7. 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...

  8. 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 ...

  9. Clinical usefulness of reflectance confocal microscopy in the management of facial lentigo maligna melanoma.

    PubMed

    Alarcón, I; Carrera, C; Puig, S; Malvehy, J

    2014-04-01

    Facial lentigo maligna melanoma can be a diagnostic challenge in daily clinical practice as it has similar clinical and morphological features to other lesions such as solar lentigines and pigmented actinic keratoses. Confocal microscopy is a noninvasive technique that provides real-time images of the epidermis and superficial dermis with cellular-level resolution. We describe 3 cases of suspected facial lentigo maligna that were assessed using dermoscopy and confocal microscopy before histopathology study. In the first case, diagnosed as lentigo maligna melanoma, presurgical mapping by confocal microscopy was performed to define the margins more accurately. In the second and third cases, with a clinical and dermoscopic suspicion of lentigo maligna melanoma, confocal microscopy was used to identify the optimal site for biopsy. PMID:24657027

  10. Confocal Raman microscopy for identification of bacterial species in biofilms

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

  11. Spectral confocal reflection microscopy using a white light source

    NASA Astrophysics Data System (ADS)

    Booth, M.; Juškaitis, R.; Wilson, T.

    2008-08-01

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

  12. Full-field interferometric confocal microscopy using a VCSEL array

    PubMed Central

    Redding, Brandon; Bromberg, Yaron; Choma, Michael A.; Cao, Hui

    2014-01-01

    We present an interferometric confocal microscope using an array of 1200 VCSELs coupled to a multimode fiber. Spatial coherence gating provides ~18,000 continuous virtual pinholes allowing an entire en face plane to be imaged in a snapshot. This approach maintains the same optical sectioning as a scanning confocal microscope without moving parts, while the high power of the VCSEL array (~5 mW per laser) enables high-speed image acquisition with integration times as short as 100 µs. Interferometric detection also recovers the phase of the image, enabling quantitative phase measurements and improving the contrast when imaging phase objects. PMID:25078199

  13. Multiplex fluorescence in situ hybridization (M-FISH) and confocal laser scanning microscopy (CLSM) to analyze multispecies oral biofilms.

    PubMed

    Karygianni, Lamprini; Hellwig, Elmar; Al-Ahmad, Ali

    2014-01-01

    Multiplex fluorescence in situ hybridization (M-FISH) constitutes a favorable microbiological method for the analysis of spatial distribution of highly variable phenotypes found in multispecies oral biofilms. The combined use of confocal laser scanning microscopy (CLSM) produces high-resolution three-dimensional (3D) images of individual bacteria in their natural environment. Here, we describe the application of M-FISH on early (Streptococcus spp., Actinomyces naeslundii) and late colonizers (Fusobacterium nucleatum, Veillonella spp.) of in situ-formed oral biofilms, the acquisition of CLSM images, as well as the qualitative and quantitative analysis of these digitally obtained and processed images.

  14. 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...

  15. 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...

  16. Analysis of Microstructure of the Cardiac Conduction System Based on Three-Dimensional Confocal Microscopy

    PubMed Central

    Romero, Daniel; Camara, Oscar; Sachse, Frank; Sebastian, Rafael

    2016-01-01

    The specialised conducting tissues present in the ventricles are responsible for the fast distribution of the electrical impulse from the atrio-ventricular node to regions in the subendocardial myocardium. Characterisation of anatomical features of the specialised conducting tissues in the ventricles is highly challenging, in particular its most distal section, which is connected to the working myocardium via Purkinje-myocardial junctions. The goal of this work is to characterise the architecture of the distal section of the Purkinje network by differentiating Purkinje cells from surrounding tissue, performing a segmentation of Purkinje fibres at cellular scale, and mathematically describing its morphology and interconnections. Purkinje cells from rabbit hearts were visualised by confocal microscopy using wheat germ agglutinin labelling. A total of 16 3D stacks including labeled Purkinje cells were collected, and semi-automatically segmented. State-of-the-art graph metrics were applied to estimate regional and global features of the Purkinje network complexity. Two types of cell types, tubular and star-like, were characterised from 3D segmentations. The analysis of 3D imaging data confirms the previously suggested presence of two types of Purkinje-myocardium connections, a 2D interconnection sheet and a funnel one, in which the narrow side of a Purkinje fibre connect progressively to muscle fibres. The complex network analysis of interconnected Purkinje cells showed no small-world connectivity or assortativity properties. These results might help building more realistic computational PK systems at high resolution levels including different cell configurations and shapes. Better knowledge on the organisation of the network might help in understanding the effects that several treatments such as radio-frequency ablation might have when the PK system is disrupted locally. PMID:27716829

  17. Resolution and signal-to-noise ratio improvement in confocal fluorescence microscopy using array detection and maximum-likelihood processing

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

    Confocal fluorescence microscopy (CFM) is widely used in biological sciences because of its enhanced 3D resolution that allows image sectioning and removal of out-of-focus blur. This is achieved by rejection of the light outside a detection pinhole in a plane confocal with the illuminated object. In this paper, an alternative detection arrangement is examined in which the entire detection/image plane is recorded using an array detector rather than a pinhole detector. Using this recorded data an attempt is then made to recover the object from the whole set of recorded photon array data; in this paper maximum-likelihood estimation has been applied. The recovered object estimates are shown (through computer simulation) to have good resolution, image sectioning and signal-to-noise ratio compared with conventional pinhole CFM images.

  18. 3D image reconstruction algorithms for cryo-electron-microscopy images of virus particles

    NASA Astrophysics Data System (ADS)

    Doerschuk, Peter C.; Johnson, John E.

    2000-11-01

    A statistical model for the object and the complete image formation process in cryo electron microscopy of viruses is presented. Using this model, maximum likelihood reconstructions of the 3D structure of viruses are computed using the expectation maximization algorithm and an example based on Cowpea mosaic virus is provided.

  19. Nanoparticle imaging. 3D structure of individual nanocrystals in solution by electron microscopy.

    PubMed

    Park, Jungwon; Elmlund, Hans; Ercius, Peter; Yuk, Jong Min; Limmer, David T; Chen, Qian; Kim, Kwanpyo; Han, Sang Hoon; Weitz, David A; Zettl, A; Alivisatos, A Paul

    2015-07-17

    Knowledge about the synthesis, growth mechanisms, and physical properties of colloidal nanoparticles has been limited by technical impediments. We introduce a method for determining three-dimensional (3D) structures of individual nanoparticles in solution. We combine a graphene liquid cell, high-resolution transmission electron microscopy, a direct electron detector, and an algorithm for single-particle 3D reconstruction originally developed for analysis of biological molecules. This method yielded two 3D structures of individual platinum nanocrystals at near-atomic resolution. Because our method derives the 3D structure from images of individual nanoparticles rotating freely in solution, it enables the analysis of heterogeneous populations of potentially unordered nanoparticles that are synthesized in solution, thereby providing a means to understand the structure and stability of defects at the nanoscale. PMID:26185247

  20. 3D structure of individual nanocrystals in solution by electron microscopy

    NASA Astrophysics Data System (ADS)

    Park, Jungwon; Elmlund, Hans; Ercius, Peter; Yuk, Jong Min; Limmer, David T.; Chen, Qian; Kim, Kwanpyo; Han, Sang Hoon; Weitz, David A.; Zettl, A.; Alivisatos, A. Paul

    2015-07-01

    Knowledge about the synthesis, growth mechanisms, and physical properties of colloidal nanoparticles has been limited by technical impediments. We introduce a method for determining three-dimensional (3D) structures of individual nanoparticles in solution. We combine a graphene liquid cell, high-resolution transmission electron microscopy, a direct electron detector, and an algorithm for single-particle 3D reconstruction originally developed for analysis of biological molecules. This method yielded two 3D structures of individual platinum nanocrystals at near-atomic resolution. Because our method derives the 3D structure from images of individual nanoparticles rotating freely in solution, it enables the analysis of heterogeneous populations of potentially unordered nanoparticles that are synthesized in solution, thereby providing a means to understand the structure and stability of defects at the nanoscale.

  1. 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

  2. Full-field interferometric confocal microscopy using a VCSEL array.

    PubMed

    Redding, Brandon; Bromberg, Yaron; Choma, Michael A; Cao, Hui

    2014-08-01

    We present an interferometric confocal microscope using an array of 1200 vertical cavity surface emitting lasers (VCSELs) coupled to a multimode fiber. Spatial coherence gating provides ~18,000 continuous virtual pinholes, allowing an entire en face plane to be imaged in a snapshot. This approach maintains the same optical sectioning as a scanning confocal microscope without moving parts, while the high power of the VCSEL array (∼5  mW per laser) enables high-speed image acquisition with integration times as short as 100 μs. Interferometric detection also recovers the phase of the image, enabling quantitative phase measurements and improving the contrast when imaging phase objects. PMID:25078199

  3. 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

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

    PubMed Central

    Chen, Guannan; Lui, Harvey

    2015-01-01

    Background Non-invasive cellular imaging of the skin in vivo can be achieved in reflectance confocal microscopy (RCM) and multiphoton microscopy (MPM) modalities to yield complementary images of the skin based on different optical properties. One of the challenges of in vivo microscopy is the delineation (i.e., segmentation) of cellular and subcellular architectural features. Methods In this work we present a method for combining watershed and level-set models for segmentation of multimodality images obtained by an integrated MPM and RCM imaging system from human skin in vivo. Results Firstly, a segmentation model based on watershed is introduced for obtaining the accurate structure of cell borders from the RCM image. Secondly,, a global region based energy level-set model is constructed for extracting the nucleus of each cell from the MPM image. Thirdly, a local region-based Lagrange Continuous level-set approach is used for segmenting cytoplasm from the MPM image. Conclusions Experimental results demonstrated that cell borders from RCM image and boundaries of cytoplasm and nucleus from MPM image can be obtained by our segmentation method with better accuracy and effectiveness. We are planning to use this method to perform quantitative analysis of MPM and RCM images of in vivo human skin to study the variations of cellular parameters such as cell size, nucleus size and other mophormetric features with skin pathologies. PMID:25694949

  5. Imaging of small particles using wide-field confocal microscopy

    NASA Astrophysics Data System (ADS)

    Morgan, Stephen P.; Sawyer, N. B. E.; Somekh, Michael G.; See, Chung Wah; Shekunov, B. Y.; Astrakharchik, E.

    2003-10-01

    Particle measurement is important in many applications such as the manufacture of drugs and paints, and aerosols. In bioimaging there is interest understanding the imaging of nanoparticles and subcellular scatterers. We present in this paper a wide field, phase measuring confocal microscope that can be used for such measurements. The wide field confocal response is obtained by illuminating both sample and reference arms of an interferometric microscope with nominally identical speckle patterns. When the speckle patterns are highly correlated the interference is significant. Contributions from out of focus planes result in uncorrelated speckle patterns and no interference. This provides a wide field confocal response. High speed measurements are enabled by parallel phase stepping using polarization optics. We have also developed a vector diffraction microscope model, using Mie theory as a scattering function, to validate the images of small particles. Correctly scaling the amplitudes of the unscattered and scattered electric fields enables co-polar transmission imaging to be modeled. Finally it is demonstrated that the phase is a more sensitive measurement of particle size than the amplitude.

  6. 3D high-density localization microscopy using hybrid astigmatic/ biplane imaging and sparse image reconstruction.

    PubMed

    Min, Junhong; Holden, Seamus J; Carlini, Lina; Unser, Michael; Manley, Suliana; Ye, Jong Chul

    2014-11-01

    Localization microscopy achieves nanoscale spatial resolution by iterative localization of sparsely activated molecules, which generally leads to a long acquisition time. By implementing advanced algorithms to treat overlapping point spread functions (PSFs), imaging of densely activated molecules can improve the limited temporal resolution, as has been well demonstrated in two-dimensional imaging. However, three-dimensional (3D) localization of high-density data remains challenging since PSFs are far more similar along the axial dimension than the lateral dimensions. Here, we present a new, high-density 3D imaging system and algorithm. The hybrid system is implemented by combining astigmatic and biplane imaging. The proposed 3D reconstruction algorithm is extended from our state-of-the art 2D high-density localization algorithm. Using mutual coherence analysis of model PSFs, we validated that the hybrid system is more suitable than astigmatic or biplane imaging alone for 3D localization of high-density data. The efficacy of the proposed method was confirmed via simulation and real data of microtubules. Furthermore, we also successfully demonstrated fluorescent-protein-based live cell 3D localization microscopy with a temporal resolution of just 3 seconds, capturing fast dynamics of the endoplasmic recticulum.

  7. Computer-aided microtomography with true 3-D display in electron microscopy.

    PubMed

    Nelson, A C

    1986-01-01

    A novel research system has been designed to permit three-dimensional (3-D) viewing of high resolution image data from transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The system consists of front-end primary data acquisition devices, such as TEM and SEM machines, which are equipped with computer-controlled specimen tilt stages. The output from these machines is in analogue form, where a video camera attached to the TEM provides the sequential analogue image output while the SEM direct video output is utilized. A 10 MHz digitizer transforms the video image to a digital array of 512 X 512 pixel units of 8 bits deep-stored in a frame buffer. Digital images from multiple projections are reconstructed into 3-D image boxes in a dedicated computer. Attached to the computer is a powerful true 3-D display device which has hardware for graphic manipulations including tilt and rotate on any axis and for probing the image with a 3-D cursor. Data editing and automatic contouring functions are used to enhance areas of interest, and specialized software is available for measurement of numbers, distances, areas, and volumes. With proper archiving of reconstructed image sequences, a dynamic 3-D presentation is possible. The microtomography system is highly versatile and can process image data on-line or from remote sites from which data records would typically be transported on computer tape, video tape, or floppy disk. PMID:3753610

  8. Lipid and protein distribution in epithelial cells assessed with confocal microscopy

    NASA Astrophysics Data System (ADS)

    Peterson, Kajsa H.; Randen, Michael; Hays, Richard M.; Magnusson, Karl-Eric

    1992-06-01

    Confocal laser scanning microscopy, image processing, and volume visualization were used to characterize the 3-D distribution of lectin receptors, lipid probes, and actin cytoskeleton in epithelial cells. Small intestine-like cells were grown on glass or filter supports and apically labelled with different fluorescent lipid and lectin probes. The restriction of the probes by the tight junctions was studied in living cells. Series of confocal x-y sections were transferred to an image processing system for analysis. The fluorescence intensity within a specified area of all x-y sections was plotted as a function of the vertical position of the sections. The curve inclination was used to describe the degree of restriction to the probes. It was found that lectins were more confined to the apical part than the lipids, which showed varying degree of redistribution to the basolateral membrane. Volume rendering, and specifically animated sequences with varying viewpoint and opacity mapping, were used to visualize the structure of actin cytoskeleton and distribution of lipid and lectin probes. In toad bladder epithelial cells, actin was labelled before and after treatment with the antidiuretic hormone vasopressin. The hormone-induced redistribution of actin in the apical and lateral portion of the cells was measured on x-z scanned images. Ratios of apical-to-lateral intensity were calculated. It was found that the decrease in the ratios after vasopressin treatment was around 30%. The decrease was due to loss of actin apically. This is supposed to facilitate apical fusion of vesicles containing the water-channel forming proteins, being important in water homeostasis.

  9. Coherent Microscopy for 3-D Movement Monitoring and Super-Resolved Imaging

    NASA Astrophysics Data System (ADS)

    Beiderman, Yevgeny; Amsel, Avigail; Tzadka, Yaniv; Fixler, Dror; Teicher, Mina; Micó, Vicente; Garcí, Javier; Javidi, Bahram; DaneshPanah, Mehdi; Moon, Inkyu; Zalevsky, Zeev

    In this chapter we present three types of microscopy-related configurations while the first one is used for 3-D movement monitoring of the inspected samples, the second one is used for super-resolved 3-D imaging, and the last one presents an overview digital holographic microscopy applications. The first configuration is based on temporal tracking of secondary reflected speckles when imaged by properly defocused optics. We validate the proposed scheme by using it to monitor 3-D spontaneous contraction of rat's cardiac muscle cells while allowing nanometric tracking accuracy without interferometric recording. The second configuration includes projection of temporally varying speckle patterns on top of the sample and by proper decoding exceeding the diffraction as well as the geometrical-related lateral resolution limitation. In the final part of the chapter, we overview applications of digital holographic microscopy (DHM) for real-time non-invasive 3-D sensing, tracking, and recognition of living microorganisms such as single- or multiple-cell organisms and bacteria.

  10. Sample holder for axial rotation of specimens in 3D microscopy.

    PubMed

    Bruns, T; Schickinger, S; Schneckenburger, H

    2015-10-01

    In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three-dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three-dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three-dimensional specimens is essential. Fluorescence z-projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results.

  11. 3D imaging of the cleared intact murine colon with light sheet microscopy

    NASA Astrophysics Data System (ADS)

    Zufiria, B.; Bocancea, D. I.; Gómez-Gaviro, M. V.; Vaquero, J. J.; Desco, M.; Fresno, M.; Ripoll, J.; Arranz, A.

    2016-03-01

    We here show 3D light sheet microscopy images of fixed and cleared murine colon tissue in-toto, which offer relevant cellular information without the need for physically sectioning the tissue. We have applied the recently developed CUBIC protocol (Susaki et al. Cell 157:726, 2014) for colon tissues and have found that this clearing protocol enables imaging all the way to the central part of the lumen with cellular resolution, thus opening new ways for 3D imaging of colon samples.

  12. Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions

    NASA Astrophysics Data System (ADS)

    Shuang, Bo; Wang, Wenxiao; Shen, Hao; Tauzin, Lawrence J.; Flatebo, Charlotte; Chen, Jianbo; Moringo, Nicholas A.; Bishop, Logan D. C.; Kelly, Kevin F.; Landes, Christy F.

    2016-08-01

    Super-resolution microscopy with phase masks is a promising technique for 3D imaging and tracking. Due to the complexity of the resultant point spread functions, generalized recovery algorithms are still missing. We introduce a 3D super-resolution recovery algorithm that works for a variety of phase masks generating 3D point spread functions. A fast deconvolution process generates initial guesses, which are further refined by least squares fitting. Overfitting is suppressed using a machine learning determined threshold. Preliminary results on experimental data show that our algorithm can be used to super-localize 3D adsorption events within a porous polymer film and is useful for evaluating potential phase masks. Finally, we demonstrate that parallel computation on graphics processing units can reduce the processing time required for 3D recovery. Simulations reveal that, through desktop parallelization, the ultimate limit of real-time processing is possible. Our program is the first open source recovery program for generalized 3D recovery using rotating point spread functions.

  13. Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions

    PubMed Central

    Shuang, Bo; Wang, Wenxiao; Shen, Hao; Tauzin, Lawrence J.; Flatebo, Charlotte; Chen, Jianbo; Moringo, Nicholas A.; Bishop, Logan D. C.; Kelly, Kevin F.; Landes, Christy F.

    2016-01-01

    Super-resolution microscopy with phase masks is a promising technique for 3D imaging and tracking. Due to the complexity of the resultant point spread functions, generalized recovery algorithms are still missing. We introduce a 3D super-resolution recovery algorithm that works for a variety of phase masks generating 3D point spread functions. A fast deconvolution process generates initial guesses, which are further refined by least squares fitting. Overfitting is suppressed using a machine learning determined threshold. Preliminary results on experimental data show that our algorithm can be used to super-localize 3D adsorption events within a porous polymer film and is useful for evaluating potential phase masks. Finally, we demonstrate that parallel computation on graphics processing units can reduce the processing time required for 3D recovery. Simulations reveal that, through desktop parallelization, the ultimate limit of real-time processing is possible. Our program is the first open source recovery program for generalized 3D recovery using rotating point spread functions. PMID:27488312

  14. Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions.

    PubMed

    Shuang, Bo; Wang, Wenxiao; Shen, Hao; Tauzin, Lawrence J; Flatebo, Charlotte; Chen, Jianbo; Moringo, Nicholas A; Bishop, Logan D C; Kelly, Kevin F; Landes, Christy F

    2016-01-01

    Super-resolution microscopy with phase masks is a promising technique for 3D imaging and tracking. Due to the complexity of the resultant point spread functions, generalized recovery algorithms are still missing. We introduce a 3D super-resolution recovery algorithm that works for a variety of phase masks generating 3D point spread functions. A fast deconvolution process generates initial guesses, which are further refined by least squares fitting. Overfitting is suppressed using a machine learning determined threshold. Preliminary results on experimental data show that our algorithm can be used to super-localize 3D adsorption events within a porous polymer film and is useful for evaluating potential phase masks. Finally, we demonstrate that parallel computation on graphics processing units can reduce the processing time required for 3D recovery. Simulations reveal that, through desktop parallelization, the ultimate limit of real-time processing is possible. Our program is the first open source recovery program for generalized 3D recovery using rotating point spread functions.

  15. Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions.

    PubMed

    Shuang, Bo; Wang, Wenxiao; Shen, Hao; Tauzin, Lawrence J; Flatebo, Charlotte; Chen, Jianbo; Moringo, Nicholas A; Bishop, Logan D C; Kelly, Kevin F; Landes, Christy F

    2016-01-01

    Super-resolution microscopy with phase masks is a promising technique for 3D imaging and tracking. Due to the complexity of the resultant point spread functions, generalized recovery algorithms are still missing. We introduce a 3D super-resolution recovery algorithm that works for a variety of phase masks generating 3D point spread functions. A fast deconvolution process generates initial guesses, which are further refined by least squares fitting. Overfitting is suppressed using a machine learning determined threshold. Preliminary results on experimental data show that our algorithm can be used to super-localize 3D adsorption events within a porous polymer film and is useful for evaluating potential phase masks. Finally, we demonstrate that parallel computation on graphics processing units can reduce the processing time required for 3D recovery. Simulations reveal that, through desktop parallelization, the ultimate limit of real-time processing is possible. Our program is the first open source recovery program for generalized 3D recovery using rotating point spread functions. PMID:27488312

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

    PubMed

    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. PMID:26813872

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

    PubMed Central

    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. PMID:26813872

  18. 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.

  19. In-vivo multi-spectral confocal microscopy

    NASA Astrophysics Data System (ADS)

    Rouse, Andrew R.; Udovich, Joshua A.; Gmitro, Arthur F.

    2005-03-01

    A multi-spectral confocal microendoscope (MCME) for in-vivo imaging has been developed. The MCME employs a flexible fiber-optic catheter coupled to a slit-scan confocal microscope with an imaging spectrometer. The catheter consists of a fiber-optic imaging bundle linked to a miniature objective and focus assembly. The focus mechanism allows for imaging to a maximum tissue depth of 200 microns. The 3mm diameter catheter may be used on its own or routed though the instrument channel of a commercial endoscope. The confocal nature of the system provides optical sectioning with 3 micron lateral resolution and 30 micron axial resolution. The system incorporates two laser sources and is therefore capable of simultaneous acquisition of spectra from multiple dyes using dual excitation. The prism based multi-spectral detection assembly is typically configured to collect 30 spectral samples over the visible range. The spectral sampling rate varies from 4nm/pixel at 490nm to 8nm/pixel at 660nm and the minimum resolvable wavelength difference varies from 8nm to 16nm over the same spectral range. Each of these characteristics are primarily dictated by the dispersion characteristics of the prism. The MCME is designed to examine cellular structures during optical biopsy and to exploit the diagnostic information contained within the spectral domain. The primary applications for the system include diagnosis of disease in the gastro-intestinal tract and female reproductive system. In-vitro, and ex-vivo multi-spectral results are presented.

  20. A coherent model for turbid imaging with confocal microscopy

    PubMed Central

    Glazowski, Christopher E.; Zavislan, James

    2013-01-01

    We present an engineering model of coherent imaging within a turbid volume, such as human tissues, with a confocal microscope. The model is built to analyze the statistical effect of aberrations and multiply scattered light on the resulting image. Numerical modeling of theory is compared with experimental results. We describe the construction of a stable phantom that represents the statistical effect of object turbidity on the image recorded. The model and phantom can serve as basis for system optimization in turbid imaging. PMID:23577285

  1. Adaptive optics confocal microscopy using direct wavefront sensing.

    PubMed

    Tao, Xiaodong; Fernandez, Bautista; Azucena, Oscar; Fu, Min; Garcia, Denise; Zuo, Yi; Chen, Diana C; Kubby, Joel

    2011-04-01

    Optical aberrations due to the inhomogeneous refractive index of tissue degrade the resolution and brightness of images in deep-tissue imaging. We introduce a confocal fluorescence microscope with adaptive optics, which can correct aberrations based on direct wavefront measurements using a Shack-Hartmann wavefront sensor with a fluorescent bead used as a point source reference beacon. The results show a 4.3× improvement in the Strehl ratio and a 240% improvement in the signal intensity for fixed mouse tissues at depths of up to 100 μm.

  2. Confocal microscopy through a multimode fiber using optical correlation

    NASA Astrophysics Data System (ADS)

    Loterie, Damien; Goorden, Sebastianus A.; Psaltis, Demetri; Moser, Christophe

    2015-12-01

    We report on a method to obtain confocal imaging through multimode fibers using optical correlation. First, we measure the fiber's transmission matrix in a calibration step. This allows us to create focused spots at one end of the fiber by shaping the wavefront sent into it from the opposite end. These spots are scanned over a sample, and the light coming back from the sample via the fiber is optically correlated with the input pattern. We show that this achieves spatial selectivity in the detection. The technique is demonstrated on microbeads, a dried epithelial cell, and a cover glass.

  3. Visualizing the Tumor Microenvironment of Liver Metastasis by Spinning Disk Confocal Microscopy.

    PubMed

    Babes, Liane; Kubes, Paul

    2016-01-01

    Intravital microscopy has evolved into an invaluable technique to study the complexity of tumors by visualizing individual cells in live organisms. Here, we describe a method for employing intravital spinning disk confocal microscopy to picture high-resolution tumor-stroma interactions in real time. We depict in detail the surgical procedures to image various tumor microenvironments and different cellular components in the liver.

  4. Video lensfree microscopy of 2D and 3D culture of cells

    NASA Astrophysics Data System (ADS)

    Allier, C. P.; Vinjimore Kesavan, S.; Coutard, J.-G.; Cioni, O.; Momey, F.; Navarro, F.; Menneteau, M.; Chalmond, B.; Obeid, P.; Haguet, V.; David-Watine, B.; Dubrulle, N.; Shorte, S.; van der Sanden, B.; Di Natale, C.; Hamard, L.; Wion, D.; Dolega, M. E.; Picollet-D'hahan, N.; Gidrol, X.; Dinten, J.-M.

    2014-03-01

    Innovative imaging methods are continuously developed to investigate the function of biological systems at the microscopic scale. As an alternative to advanced cell microscopy techniques, we are developing lensfree video microscopy that opens new ranges of capabilities, in particular at the mesoscopic level. Lensfree video microscopy allows the observation of a cell culture in an incubator over a very large field of view (24 mm2) for extended periods of time. As a result, a large set of comprehensive data can be gathered with strong statistics, both in space and time. Video lensfree microscopy can capture images of cells cultured in various physical environments. We emphasize on two different case studies: the quantitative analysis of the spontaneous network formation of HUVEC endothelial cells, and by coupling lensfree microscopy with 3D cell culture in the study of epithelial tissue morphogenesis. In summary, we demonstrate that lensfree video microscopy is a powerful tool to conduct cell assays in 2D and 3D culture experiments. The applications are in the realms of fundamental biology, tissue regeneration, drug development and toxicology studies.

  5. Comparison of confocal microscopy and two-photon microscopy in mouse cornea in vivo.

    PubMed

    Lee, Jun Ho; Lee, Seunghun; Gho, Yong Song; Song, In Seok; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2015-03-01

    High-resolution imaging of the cornea is important for studying corneal diseases at cellular levels. Confocal microscopy (CM) has been widely used in the clinic, and two-photon microscopy (TPM) has recently been introduced in various pre-clinical studies. We compared the performance of CM and TPM in normal mouse corneas and neovascularized mouse corneas induced by suturing. Balb/C mice and C57BL/6 mice expressing green fluorescent protein (GFP) were used to compare modalities based on intrinsic contrast and extrinsic fluorescence contrast. CM based on reflection (CMR), CM based on fluorescence (CMF), and TPM based on intrinsic/extrinsic fluorescence and second harmonic generation (SHG) were compared by imaging the same sections of mouse corneas sequentially in vivo. In normal mouse corneas, CMR visualized corneal cell morphologies with some background noise, and CMF visualized GFP expressing corneal cells clearly. TPM visualized corneal cells and collagen in the stroma based on fluorescence and SHG, respectively. However, in neovascularized mouse corneas, CMR could not resolve cells deep inside the cornea due to high background noise from the effects of increased structural irregularity induced by suturing. CMF and TPM visualized cells and induced vasculature better than CMR because both collect signals from fluorescent cells only. Both CMF and TPM had signal decays with depth due to the structural irregularity, with CMF having faster signal decay than TPM. CMR, CMF, and TPM showed different degrees of image degradation in neovascularized mouse corneas.

  6. Variational attenuation correction in two-view confocal microscopy

    PubMed Central

    2013-01-01

    Background Absorption and refraction induced signal attenuation can seriously hinder the extraction of quantitative information from confocal microscopic data. This signal attenuation can be estimated and corrected by algorithms that use physical image formation models. Especially in thick heterogeneous samples, current single view based models are unable to solve the underdetermined problem of estimating the attenuation-free intensities. Results We present a variational approach to estimate both, the real intensities and the spatially variant attenuation from two views of the same sample from opposite sides. Assuming noise-free measurements throughout the whole volume and pure absorption, this would in theory allow a perfect reconstruction without further assumptions. To cope with real world data, our approach respects photon noise, estimates apparent bleaching between the two recordings, and constrains the attenuation field to be smooth and sparse to avoid spurious attenuation estimates in regions lacking valid measurements. Conclusions We quantify the reconstruction quality on simulated data and compare it to the state-of-the art two-view approach and commonly used one-factor-per-slice approaches like the exponential decay model. Additionally we show its real-world applicability on model organisms from zoology (zebrafish) and botany (Arabidopsis). The results from these experiments show that the proposed approach improves the quantification of confocal microscopic data of thick specimen. PMID:24350574

  7. Characterization of lased enamel organic matrix using confocal microscopy

    NASA Astrophysics Data System (ADS)

    Hsu, Chin-Ying S.; Girija, Veerappan

    2001-10-01

    In the past decade several studies have demonstrated the increased acid resistance in enamel demineralization after laser irradiation. However, the exact mechanism of action to this effect still remains a speculation. Recently, the role of organic matrix was revealed to be significant in the laser-induced inhibition of enamel demineralization. The aim of the present study was to characterize the lipid component of organic matrix in mature lazed enamel and unlazed enamel histochemically using a hydrophobic fluorescent probe with Confocal Laser Scanning Microscope (CLSM). Partial decalcification of thin enamel sections was carried out using 0.5 M of EDTA in a stainless steel grid for 5 hours, following fixation with 3.5% paraformaldehyde. Thereafter the sections were stained with Nile red coupled with CLSM. The intensity of the light reflection was analyzed under the same conditions for all specimens, ruling out the autofluorescence in the control sections. Confocal imaging revealed a diffuse and increased fluorescence of the lipid stain in the lazed areas suggesting that the swelling and coating of organic matrix on the surface of enamel crystals in the peri and interprismatic spaces is rendering the increased acid resistance. These findings will substantiate the proposed organic blocking theory in partially explaining the laser-induced prevention of enamel demineralization.

  8. Investigation on 3D morphological changes of in vitro cells through digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Memmolo, Pasquale; Miccio, Lisa; Merola, Francesco; Netti, Paolo A.; Coppola, Giuseppe; Ferraro, Pietro

    2013-04-01

    We report the investigation of the identification and measurement of region of interest (ROI) in quantitative phase-contrast maps (QPMs) of biological cells by digital holographic microscopy (DHM), with the aim to analyze the 3D positions and 3D morphology together. We consider as test case for our tool the in vitro bull sperm head morphometry analysis. Extraction and measurement of various morphological parameters are performed by using two methods: the anisotropic diffusion filter, that is based on the Gaussian diffusivity function which allows more accuracy of the edge position, and the simple thresholding filter. In particular we consider the calculation of area, ellipticity, perimeter, major axis, minor axis and shape factor as a morphological parameter, instead, for the estimation of 3D position, we compute the centroid, the weighted centroid and the maximum phase values. A statistical analysis on a data set composed by N = 14 holograms relative to bovine spermatozoa and its reference holograms is reported.

  9. Functional imaging of living Paramecium by means of confocal and two-photon excitation fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Diaspro, Alberto; Fronte, Paola; Raimondo, Marco; Fato, Marco; DeLeo, Gianluca; Beltrame, Francesco; Cannone, Fabio; Chirico, Giberto; Ramoino, Paola

    2002-05-01

    Confocal and Two-photon excitation laser scanning microscopy allow gathering three-dimensional and temporal information from biological systems exploiting fluorescence labeling and autofluorescence properties. In this work we study biological events linked to functionality in Paramecium primaurelia. The internalization of material in ciliated one-celled organisms (protozoa) occurs via different mechanisms, even if most of nutrients, particulate or not, is taken up by food vacuoles formed at the bottom of the oral cavity. The endocytosis of small-sized molecules occurs at the parasomal sacs, located next the ciliar basal bodies. Vital fluorescent dyes (BSA-FITC, WGA-FITC, dextran-Texas Red, cholesteryl-Bodipy) and autofluorescence were used to study formation, movement, and fusion of vesicles during endocytosis and phagocytosis of Paramecium primaurelia. By immobilizing living cells pulsed with food vacuole and endosome markers at successive times after chasing in unlabeled medium, the intracellular movement and fusion of food vacuoles and of endosomes were visualized. A temporal analysis of fluorescence images and the false-color technique were used. Starting from time series or 3D data sets composite images were generated by associating with each originally acquired image a different color corresponding to each sampling point in time and along the z-axis. Second Harmonic Generation Imaging attempts are also outlined.

  10. Application of Laser Scanning Confocal Microscopy to Heat and Mass Transport Modeling in Porous Microstructures

    NASA Technical Reports Server (NTRS)

    Marshall, Jochen; Milos, Frank; Fredrich, Joanne; Rasky, Daniel J. (Technical Monitor)

    1997-01-01

    Laser Scanning Confocal Microscopy (LSCM) has been used to obtain digital images of the complicated 3-D (three-dimensional) microstructures of rigid, fibrous thermal protection system (TPS) materials. These orthotropic materials are comprised of refractory ceramic fibers with diameters in the range of 1 to 10 microns and have open porosities of 0.8 or more. Algorithms are being constructed to extract quantitative microstructural information from the digital data so that it may be applied to specific heat and mass transport modeling efforts; such information includes, for example, the solid and pore volume fractions, the internal surface area per volume, fiber diameter distributions, and fiber orientation distributions. This type of information is difficult to obtain in general, yet it is directly relevant to many computational efforts which seek to model macroscopic thermophysical phenomena in terms of microscopic mechanisms or interactions. Two such computational efforts for fibrous TPS materials are: i) the calculation of radiative transport properties; ii) the modeling of gas permeabilities.

  11. Reflectance confocal microscopy: an overview of technology and advances in telepathology.

    PubMed

    Batta, Mari M; Kessler, Stephen E; White, Peter F; Zhu, Weijian; Fox, Christi A

    2015-05-01

    The value of in vivo reflectance confocal microscopy (RCM) as a noninvasive adjunctive tool in dermatology has steadily advanced since its inception. With RCM, dermatologists can view horizontal sections of lesions in a resolution comparable to histology, observe dynamic processes in living skin, and monitor lesion evolution longitudinally. This article will compare RCM to dermoscopy and histology, review the general principles of the microscope, describe the findings seen on confocal images, and discuss the clinical applications of this noninvasive tool. Additionally, we describe a telepathology network dedicated to the transfer of confocal images to remote dermatopathologists for interpretation. Finally, we will discuss the adoption of RCM and the telepathology network in clinical practice.

  12. Subtractive imaging in confocal scanning microscopy using a CCD camera as a detector.

    PubMed

    Sánchez-Ortiga, Emilio; Sheppard, Colin J R; Saavedra, Genaro; Martínez-Corral, Manuel; Doblas, Ana; Calatayud, Arnau

    2012-04-01

    We report a scheme for the detector system of confocal microscopes in which the pinhole and a large-area detector are substituted by a CCD camera. The numerical integration of the intensities acquired by the active pixels emulates the signal passing through the pinhole. We demonstrate the imaging capability and the optical sectioning of the system. Subtractive-imaging confocal microscopy can be implemented in a simple manner, providing superresolution and improving optical sectioning.

  13. 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

  14. Fast 3D visualization of endogenous brain signals with high-sensitivity laser scanning photothermal microscopy

    PubMed Central

    Miyazaki, Jun; Iida, Tadatsune; Tanaka, Shinji; Hayashi-Takagi, Akiko; Kasai, Haruo; Okabe, Shigeo; Kobayashi, Takayoshi

    2016-01-01

    A fast, high-sensitivity photothermal microscope was developed by implementing a spatially segmented balanced detection scheme into a laser scanning microscope. We confirmed a 4.9 times improvement in signal-to-noise ratio in the spatially segmented balanced detection compared with that of conventional detection. The system demonstrated simultaneous bi-modal photothermal and confocal fluorescence imaging of transgenic mouse brain tissue with a pixel dwell time of 20 μs. The fluorescence image visualized neurons expressing yellow fluorescence proteins, while the photothermal signal detected endogenous chromophores in the mouse brain, allowing 3D visualization of the distribution of various features such as blood cells and fine structures probably due to lipids. This imaging modality was constructed using compact and cost-effective laser diodes, and will thus be widely useful in the life and medical sciences. PMID:27231615

  15. 3D print customized sample holders for live light sheet microscopy.

    PubMed

    Jeandupeux, Emeric; Lobjois, Valérie; Ducommun, Bernard

    2015-08-01

    A major hurdle to the widespread application of light sheet microscopy is the lack of versatile and non-intrusive sample holders that are adaptable to a variety of biological samples for live imaging. To overcome this limitation, we present herein the application of 3D printing to the fabrication of a fully customizable casting kit. 3D printing enables facile preparation of hydrogel sample holders adaptable to any shape and number of specimen. As an example, we present the use of this device to produce a four-sample holder adapted to parallel live monitoring of multicellular tumor spheroid growth. To share our solution with the light sheet microscopy community, all files necessary to produce or customize sample holders are freely available online.

  16. 3D imaging of the early embryonic chicken heart with focused ion beam scanning electron microscopy.

    PubMed

    Rennie, Monique Y; Gahan, Curran G; López, Claudia S; Thornburg, Kent L; Rugonyi, Sandra

    2014-08-01

    Early embryonic heart development is a period of dynamic growth and remodeling, with rapid changes occurring at the tissue, cell, and subcellular levels. A detailed understanding of the events that establish the components of the heart wall has been hampered by a lack of methodologies for three-dimensional (3D), high-resolution imaging. Focused ion beam scanning electron microscopy (FIB-SEM) is a novel technology for imaging 3D tissue volumes at the subcellular level. FIB-SEM alternates between imaging the block face with a scanning electron beam and milling away thin sections of tissue with a FIB, allowing for collection and analysis of 3D data. FIB-SEM was used to image the three layers of the day 4 chicken embryo heart: myocardium, cardiac jelly, and endocardium. Individual images obtained with FIB-SEM were comparable in quality and resolution to those obtained with transmission electron microscopy. Up to 1,100 serial images were obtained in 4 nm increments at 4.88 nm resolution, and image stacks were aligned to create volumes 800-1,500 μm3 in size. Segmentation of organelles revealed their organization and distinct volume fractions between cardiac wall layers. We conclude that FIB-SEM is a powerful modality for 3D subcellular imaging of the embryonic heart wall.

  17. 3D single molecule tracking in thick cellular specimens using multifocal plane microscopy

    NASA Astrophysics Data System (ADS)

    Ram, Sripad; Ward, E. Sally; Ober, Raimund J.

    2011-03-01

    One of the major challenges in single molecule microscopy concerns 3D tracking of single molecules in cellular specimens. This has been a major impediment to study many fundamental cellular processes, such as protein transport across thick cellular specimens (e.g. a cell-monolayer). Here we show that multifocal plane microscopy (MUM), an imaging modality developed by our group, provides the much needed solution to this longstanding problem. While MUM was previously used for 3D single molecule tracking at shallow depths (~ 1 micron) in live-cells, the question arises if MUM can also live up to the significant challenge of tracking single molecules in thick samples. Here by substantially expanding the capabilities of MUM, we demonstrate 3D tracking of quantum-dot labeled molecules in a ~ 10 micron thick cell monolayer. In this way we have reconstructed the complete 3D intracellular trafficking itinerary of single molecules at high spatial and temporal precision in a thick cell-sample. Funding support: NIH and the National MS Society.

  18. Infrared differential interference contrast microscopy for 3D interconnect overlay metrology.

    PubMed

    Ku, Yi-sha; Shyu, Deh-Ming; Lin, Yeou-Sung; Cho, Chia-Hung

    2013-08-12

    One of the main challenges for 3D interconnect metrology of bonded wafers is measuring through opaque silicon wafers using conventional optical microscopy. We demonstrate here the use infrared microscopy, enhanced by implementing the differential interference contrast (DIC) technique, to measure the wafer bonding overlay. A pair of two dimensional symmetric overlay marks were processed at both the front and back sides of thinned wafers to evaluate the bonding overlay. A self-developed analysis algorithm and theoretical fitting model was used to map the overlay error between the bonded wafers and the interconnect structures. The measurement accuracy was found to be better than 1.0 micron.

  19. 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. PMID:27515076

  20. 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

  1. Virtual rough samples to test 3D nanometer-scale scanning electron microscopy stereo photogrammetry

    NASA Astrophysics Data System (ADS)

    Villarrubia, J. S.; Tondare, V. N.; Vladár, A. E.

    2016-03-01

    The combination of scanning electron microscopy for high spatial resolution, images from multiple angles to provide 3D information, and commercially available stereo photogrammetry software for 3D reconstruction offers promise for nanometer-scale dimensional metrology in 3D. A method is described to test 3D photogrammetry software by the use of virtual samples—mathematical samples from which simulated images are made for use as inputs to the software under test. The virtual sample is constructed by wrapping a rough skin with any desired power spectral density around a smooth near-trapezoidal line with rounded top corners. Reconstruction is performed with images simulated from different angular viewpoints. The software's reconstructed 3D model is then compared to the known geometry of the virtual sample. Three commercial photogrammetry software packages were tested. Two of them produced results for line height and width that were within close to 1 nm of the correct values. All of the packages exhibited some difficulty in reconstructing details of the surface roughness.

  2. Towards non-invasive 3D hepatotoxicity assays with optical coherence phase microscopy

    NASA Astrophysics Data System (ADS)

    Nelson, Leonard J.; Koulovasilopoulos, Andreas; Treskes, Philipp; Hayes, Peter C.; Plevris, John N.; Bagnaninchi, Pierre O.

    2015-03-01

    Three-dimensional tissue-engineered models are increasingly recognised as more physiologically-relevant than standard 2D cell culture for pre-clinical drug toxicity testing. However, many types of conventional toxicity assays are incompatible with dense 3D tissues. This study investigated the use of optical coherence phase microscopy (OCPM) as a novel approach to assess cell death in 3D tissue culture. For 3D micro-spheroid formation Human hepatic C3A cells were encapsulated in hyaluronic acid gels and cultured in 100μl MEME/10%FBS in 96-well plates. After spheroid formation the 3D liver constructs were exposed to acetaminophen on culture day 8. Acetaminophen hepatotoxicity in 3D cultures was evaluated using standard biochemical assays. An inverted OCPM in common path configuration was developed with a Callisto OCT engine (Thorlabs), centred at 930nm and a custom scanning head. Intensity data were used to perform in-depth microstructural imaging. In addition, phase fluctuations were measured by collecting several successive B scans at the same location, and statistics on the first time derivative of the phase, i.e. time fluctuations, were analysed over the acquisition time interval to retrieve overall cell viability. OCPM intensity (cell cluster size) and phase fluctuation statistics were directly compared with biochemical assays. In this study, we investigated optical coherence phase tomography to assess cell death in a 3d liver model after exposure to a prototypical hepatotoxin, acetaminophen. We showed that OCPM has the potential to assess noninvasively and label-free drug toxicity in 3D tissue models.

  3. Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy.

    PubMed

    Arnold, Jan; Mahamid, Julia; Lucic, Vladan; de Marco, Alex; Fernandez, Jose-Jesus; Laugks, Tim; Mayer, Tobias; Hyman, Anthony A; Baumeister, Wolfgang; Plitzko, Jürgen M

    2016-02-23

    The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process. In this study, we present the development of a cryo-stage allowing for spinning-disk confocal light microscopy at cryogenic temperatures and describe the incorporation of the new hardware into existing workflows for cellular sample preparation by cryo-FIB. Introduction of fiducial markers and subsequent computation of three-dimensional coordinate transformations provide correlation between light microscopy and scanning electron microscopy/FIB. The correlative approach is employed to guide the FIB milling process of vitrified cellular samples and to capture specific structures, namely fluorescently labeled lipid droplets, in lamellas that are 300 nm thick. The correlation procedure is then applied to localize the fluorescently labeled structures in the transmission electron microscopy image of the lamella. This approach can be employed to navigate the acquisition of cryo-ET data within FIB-lamellas at specific locations, unambiguously identified by fluorescence microscopy.

  4. 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.

  5. Corneal confocal microscopy: Recent progress in the evaluation of diabetic neuropathy

    PubMed Central

    Papanas, Nikolaos; Ziegler, Dan

    2015-01-01

    The present brief review discusses recent progress with corneal confocal microscopy for the evaluation of diabetic sensorimotor polyneuropathy. Corneal confocal microscopy is a new, non-invasive and reproducible diagnostic modality, and it can also be easily applied for patient follow up. It enables new perspectives of studying the natural history of diabetic sensorimotor polyneuropathy, severity of nerve fiber pathology and documenting early nerve fiber regeneration after therapeutic intervention. It shows moderate to high sensitivity and specificity for the timely diagnosis of diabetic sensorimotor polyneuropathy. Currently, corneal confocal microscopy is mainly used in specialized centers, but deserves more widespread application for the assessment of diabetic sensorimotor polyneuropathy. Finally, further progress is required in terms of technical improvements for automated nerve fiber quantification and for analysis of larger images. PMID:26221515

  6. 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

  7. A new 3D tracking method exploiting the capabilities of digital holography in microscopy

    NASA Astrophysics Data System (ADS)

    Miccio, L.; Memmolo, P.; Merola, F.; Fusco, S.; Embrione, V.; Netti, P. A.; Ferraro, P.

    2013-04-01

    A method for 3D tracking has been developed exploiting Digital Holographic Microscopy (DHM) features. In the framework of self-consistent platform for manipulation and measurement of biological specimen we use DHM for quantitative and completely label free analysis of specimen with low amplitude contrast. Tracking capability extend the potentiality of DHM allowing to monitor the motion of appropriate probes and correlate it with sample properties. Complete 3D tracking has been obtained for the probes avoiding the issue of amplitude refocusing in traditional tracking processing. Our technique belongs to the video tracking methods that, conversely from Quadrant Photo-Diode method, opens the possibility to track multiples probes. All the common used video tracking algorithms are based on the numerical analysis of amplitude images in the focus plane and the shift of the maxima in the image plane are measured after the application of an appropriate threshold. Our approach for video tracking uses different theoretical basis. A set of interferograms is recorded and the complex wavefields are managed numerically to obtain three dimensional displacements of the probes. The procedure works properly on an higher number of probes and independently from their size. This method overcomes the traditional video tracking issues as the inability to measure the axial movement and the choice of suitable threshold mask. The novel configuration allows 3D tracking of micro-particles and simultaneously can furnish Quantitative Phase-contrast maps of tracked micro-objects by interference microscopy, without changing the configuration. In this paper, we show a new concept for a compact interferometric microscope that can ensure the multifunctionality, accomplishing accurate 3D tracking and quantitative phase-contrast analysis. Experimental results are presented and discussed for in vitro cells. Through a very simple and compact optical arrangement we show how two different functionalities

  8. 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.

  9. Case Report: melanoma and melanocytic nevus differentiation with reflectance confocal microscopy.

    PubMed Central

    Łudzik, Joanna; Witkowski, Alexander M; Pellacani, Giovanni

    2015-01-01

    Historically, melanoma has been typically diagnosed by naked-eye examination and confirmed with invasive biopsy. However, recently the use of reflectance confocal microscopy enables non-invasive bedside diagnosis of clinically equivocal lesions. We present a case in which reflectance confocal microscopy was used to evaluate two skin lesions in the same patient confirming the diagnosis of a melanoma and potentially avoiding invasive biopsy in the second benign melanocytic lesion.  Clinicians should be aware of the availability of new non-invasive technologies that can aid in early diagnosis of malignant skin tumors and potentially reduce the number of benign lesion excisions. PMID:26236471

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  11. Imaging anisotropy using differential polarization laser scanning confocal microscopy.

    PubMed

    Steinbach, Gábor; Pomozi, István; Zsiros, Ottó; Menczel, László; Garab, Gyozo

    2009-01-01

    We have constructed differential polarization (DP) attachments to a laser scanning microscope (LSM) for imaging the main DP quantities of anisotropic microscopic objects. The DP-LSM operates with high-frequency modulation and subsequent demodulation and displays the main DP quantities pixel by pixel. These, for linearly polarized light, include: (i) linear birefringence (LB), which is exhibited by structurally and/or optically anisotropic material; (ii) linear dichroism (LD), which carries information on the anisotropic distribution of the molecules, i.e. of their absorbance transition dipole vectors, in the sample; (iii) fluorescence-detected LD (FDLD), which carries the same information for fluorescent dyes upon excitations with two orthogonally polarized light beams; (iv) anisotropy of the fluorescence emission (r), excited with non-polarized light, which is determined by the distribution of the emission transition dipole vectors in the sample and is analogous with LD and (v) the degree of polarization of the fluorescence emission (P), excited with polarized light, which depends on the depolarization of the emission e.g. due to the rotation of molecules during their excitation lifetimes. In fluorescence regimes, the DP images can be recorded in the confocal regime of the microscope, which thus warrants good spatial resolution and the possibility of mapping the anisotropy in three dimensions. In this paper, we outline the design and technical realization of our DP-LSM and give a few examples on DP imaging of different biological samples.

  12. 3D imaging and characterization of microlenses and microlens arrays using nonlinear microscopy

    NASA Astrophysics Data System (ADS)

    Krmpot, Aleksandar J.; Tserevelakis, George J.; Murić, Branka D.; Filippidis, George; Pantelić, Dejan V.

    2013-05-01

    In this work, nonlinear laser scanning microscopy was employed for the characterization and three-dimensional (3D) imaging of microlenses and microlens arrays. Third-harmonic generation and two-photon excitation fluorescence (TPEF) signals were recorded and the obtained data were further processed in order to generate 3D reconstructions of the examined samples. Femtosecond laser pulses (1028 nm) were utilized for excitation. Microlenses were manufactured on Tot'hema and eosin sensitized gelatin layers using a green (532 nm) continuous wave laser beam using the direct laser writing method. The profiles of the microlens surface were obtained from the radial cross-sections, using a triple-Gaussian fit. The analytical shapes of the profiles were also used for ray tracing. Furthermore, the volumes of the microlenses were determined with high precision. The TPEF signal arising from the volume of the material was recorded and the respective 3D spatial fluorescence distribution of the samples was mapped. Nonlinear microscopy modalities have been shown to be a powerful diagnostic tool for microlens characterization as they enable in-depth investigations of the structural properties of the samples, in a nondestructive manner.

  13. Ex vivo confocal microscopy imaging to identify tumor tissue on freshly removed brain sample.

    PubMed

    Forest, Fabien; Cinotti, Elisa; Yvorel, Violaine; Habougit, Cyril; Vassal, François; Nuti, Christophe; Perrot, Jean-Luc; Labeille, Bruno; Péoc'h, Michel

    2015-09-01

    Confocal microscopy is a technique able to realize "optic sections" of a tissue with increasing applications. We wondered if we could apply an ex vivo confocal microscope designed for dermatological purpose in a routine use for the most frequent brain tumors. The aim of this work was to identify tumor tissue and its histopathological hallmarks, and to assess grading criteria used in neuropathological practice without tissue loss on freshly removed brain tissue. Seven infiltrating gliomas, nine meningiomas and three metastases of carcinomas were included. We compared imaging results obtained with the confocal microscope to frozen sections, smears and tissue sections of formalin-fixed tissue. Our results show that ex vivo confocal microscopy imaging can be applied to brain tumors in order to quickly identify tumor tissue without tissue loss. It can differentiate tumors and can assess most of grading criteria. Confocal microscopy could represent a new tool to identify tumor tissue on freshly removed sample and could help in selecting areas for biobanking of tumor tissue.

  14. Gastric Tissue Damage Analysis Generated by Ischemia: Bioimpedance, Confocal Endomicroscopy, and Light Microscopy

    PubMed Central

    Beltran, Nohra E.; Garcia, Laura E.; Garcia-Lorenzana, Mario

    2013-01-01

    The gastric mucosa ischemic tissular damage plays an important role in critical care patients' outcome, because it is the first damaged tissue by compensatory mechanism during shock. The aim of the study is to relate bioimpedance changes with tissular damage level generated by ischemia by means of confocal endomicroscopy and light microscopy. Bioimpedance of the gastric mucosa and confocal images were obtained from Wistar male rats during basal and ischemia conditions. They were anesthetized, and stain was applied (fluorescein and/or acriflavine). The impedance spectroscopy catheter was inserted and then confocal endomicroscopy probe. After basal measurements and biopsy, hepatic and gastric arteries clamping induced ischemia. Finally, pyloric antrum tissue was preserved in buffered formaldehyde (10%) for histology processing using light microscopy. Confocal images were equalized, binarized, and boundary defined, and infiltrations were quantified. Impedance and infiltrations increased with ischemia showing significant changes between basal and ischemia conditions (P < 0.01). Light microscopy analysis allows detection of general alterations in cellular and tissular integrity, confirming gastric reactance and confocal images quantification increments obtained during ischemia. PMID:23841094

  15. Reflectance confocal microscopy for scarring and non-scarring alopecia real-time assessment.

    PubMed

    Ardigò, Marco; Agozzino, Marina; Franceschini, Chiara; Donadio, Carlo; Abraham, Leonardo Spagnol; Barbieri, Luca; Sperduti, Isabella; Berardesca, Enzo; González, Salvador

    2016-07-01

    Clinical management of alopecia represents one of the major issues in dermatology. Scalp biopsies are not easily accepted because of the high bleeding and sensitive anatomical area. Trichoscopy is routinely used for diagnosis of alopecia, but in several cases lack to provide sufficient information on the status of the disease. Recently, reflectance confocal microscopy demonstrated its usefulness for the evaluation of several inflammatory skin condition and preliminary reports about alopecia have been proposed in the literature. The aim was to identify the confocal features characterizing scarring and non-scarring alopecia. Reflectance confocal microscopy from 86 patients affected by scarring (28 lichen planopilaris and 9 lupus erythematosus) and non-scarring alopecia (30 androgenic alopecia and 19 alopecia areata), were retrospectively, blinded evaluated. Good concordance between different readers on the confocal criteria has been assessed. Statistical significant features, specific for scarring alopecia and non-scarring alopecia have been identified. In this study, data on reflectance confocal microscopy features useful for the differential diagnosis between scarring and non-scarring alopecia have been identified. Further studies focusing on the use of this non-invasive technique in the therapeutic follow-up and distinction of sub-entities of alopecia are still required.

  16. Computational optical-sectioning microscopy for 3D quantization of cell motion: results and challenges

    NASA Astrophysics Data System (ADS)

    McNally, James G.

    1994-09-01

    How cells move and navigate within a 3D tissue mass is of central importance in such diverse problems as embryonic development, wound healing and metastasis. This locomotion can now be visualized and quantified by using computation optical-sectioning microscopy. In this approach, a series of 2D images at different depths in a specimen are stacked to construct a 3D image, and then with a knowledge of the microscope's point-spread function, the actual distribution of fluorescent intensity in the specimen is estimated via computation. When coupled with wide-field optics and a cooled CCD camera, this approach permits non-destructive 3D imaging of living specimens over long time periods. With these techniques, we have observed a complex diversity of motile behaviors in a model embryonic system, the cellular slime mold Dictyostelium. To understand the mechanisms which control these various behaviors, we are examining motion in various Dictyostelium mutants with known defects in proteins thought to be essential for signal reception, cell-cell adhesion or locomotion. This application of computational techniques to analyze 3D cell locomotion raises several technical challenges. Image restoration techniques must be fast enough to process numerous 1 Gbyte time-lapse data sets (16 Mbytes per 3D image X 60 time points). Because some cells are weakly labeled and background intensity is often high due to unincorporated dye, the SNR in some of these images is poor. Currently, the images are processed by a regularized linear least- squares restoration method, and occasionally by a maximum-likelihood method. Also required for these studies are accurate automated- tracking procedures to generate both 3D trajectories for individual cells and 3D flows for a group of cells. Tracking is currently done independently for each cell, using a cell's image as a template to search for a similar image at the next time point. Finally, sophisticated visualization techniques are needed to view the

  17. Determination of Nanogram Microparticles from Explosives after Real Open-Air Explosions by Confocal Raman Microscopy.

    PubMed

    Zapata, Félix; García-Ruiz, Carmen

    2016-07-01

    Explosives are increasingly being used for terrorist attacks to cause devastating explosions. The detection of their postblast residues after an explosion is a high challenge, which has been barely investigated, particularly using spectroscopic techniques. In this research, a novel methodology using confocal Raman microscopy has been developed for the analysis of postblast residues from 10 open-air explosions caused by 10 different explosives (TNT, RDX, PETN, TATP, HMTD, dynamite, black powder, ANFO, chloratite, and ammonal) commonly used in improvised explosive devices. The methodology for the determination of postblast particles from explosives consisted of examining the samples surfaces with both the naked eye, first, and microscopically (10× and 50×), immediately afterward; and finally, analyzing the selected residues by confocal Raman spectroscopy in order to identify the postblast particles from explosives. Interestingly, confocal Raman microscopy has demonstrated to be highly suitable to rapidly, selectively, and noninvasively analyze postblast microscopic particles from explosives up to the nanogram range. PMID:27281604

  18. Determination of Nanogram Microparticles from Explosives after Real Open-Air Explosions by Confocal Raman Microscopy.

    PubMed

    Zapata, Félix; García-Ruiz, Carmen

    2016-07-01

    Explosives are increasingly being used for terrorist attacks to cause devastating explosions. The detection of their postblast residues after an explosion is a high challenge, which has been barely investigated, particularly using spectroscopic techniques. In this research, a novel methodology using confocal Raman microscopy has been developed for the analysis of postblast residues from 10 open-air explosions caused by 10 different explosives (TNT, RDX, PETN, TATP, HMTD, dynamite, black powder, ANFO, chloratite, and ammonal) commonly used in improvised explosive devices. The methodology for the determination of postblast particles from explosives consisted of examining the samples surfaces with both the naked eye, first, and microscopically (10× and 50×), immediately afterward; and finally, analyzing the selected residues by confocal Raman spectroscopy in order to identify the postblast particles from explosives. Interestingly, confocal Raman microscopy has demonstrated to be highly suitable to rapidly, selectively, and noninvasively analyze postblast microscopic particles from explosives up to the nanogram range.

  19. Effects of Fluorescein Staining on Laser In Vivo Confocal Microscopy Images of the Cornea

    PubMed Central

    Sindt, Christine W.; Critser, D. Brice; Grout, Trudy K.; Kern, Jami R.

    2012-01-01

    This study was designed to identify whether topical fluorescein, a common ophthalmic tool, affects laser in vivo confocal microscopy of the cornea, a tool with growing applications. Twenty-five eye care specialists were asked to identify presence or absence of fluorescein in 99 confocal micrographs of healthy corneas. Responses were statistically similar to guessing for the epithelium (48% ± 14% of respondents correct per image) and the subbasal nerve plexus (49% ± 11% correct), but results were less clear for the stroma. Dendritic immune cells were quantified in bilateral images from subjects who had been unilaterally stained with fluorescein. Density of dendritic immune cells was statistically similar between the unstained and contralateral stained eyes of 24 contact lens wearers (P = .72) and of 10 nonwearers (P = .53). Overall, the results indicated that fluorescein staining did not interfere with laser confocal microscopy of corneal epithelium, subbasal nerves, or dendritic immune cells. PMID:22363837

  20. Clinical applications of in vivo fluorescence confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

  1. Computational-optical microscopy for 3D biological imaging beyond the diffraction limit

    NASA Astrophysics Data System (ADS)

    Grover, Ginni

    In recent years, super-resolution imaging has become an important fluorescent microscopy tool. It has enabled imaging of structures smaller than the optical diffraction limit with resolution less than 50 nm. Extension to high-resolution volume imaging has been achieved by integration with various optical techniques. In this thesis, development of a fluorescent microscope to enable high resolution, extended depth, three dimensional (3D) imaging is discussed; which is achieved by integration of computational methods with optical systems. In the first part of the thesis, point spread function (PSF) engineering for volume imaging is discussed. A class of PSFs, referred to as double-helix (DH) PSFs, is generated. The PSFs exhibit two focused spots in the image plane which rotate about the optical axis, encoding depth in rotation of the image. These PSFs extend the depth-of-field up to a factor of ˜5. Precision performance of the DH-PSFs, based on an information theoretical analysis, is compared with other 3D methods with conclusion that the DH-PSFs provide the best precision and the longest depth-of-field. Out of various possible DH-PSFs, a suitable PSF is obtained for super-resolution microscopy. The DH-PSFs are implemented in imaging systems, such as a microscope, with a special phase modulation at the pupil plane. Surface-relief elements which are polarization-insensitive and ˜90% light efficient are developed for phase modulation. The photon-efficient DH-PSF microscopes thus developed are used, along with optimal position estimation algorithms, for tracking and super-resolution imaging in 3D. Imaging at depths-of-field of up to 2.5 microm is achieved without focus scanning. Microtubules were imaged with 3D resolution of (6, 9, 39) nm, which is in close agreement with the theoretical limit. A quantitative study of co-localization of two proteins in volume was conducted in live bacteria. In the last part of the thesis practical aspects of the DH-PSF microscope are

  2. 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...

  3. 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...

  4. 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...

  5. In vivo reflectance confocal microscopy features of a large cell acanthoma: report of a case

    PubMed Central

    Shahriari, Neda; Grant-Kels, Jane M.; Rabinovitz, Harold S.; Oliviero, Margaret; Scope, Alon

    2016-01-01

    Reflectance confocal microscopy (RCM) is an FDA approved noninvasive optical imaging technique that acquires cellular level-resolution skin images in vivo. Herein, we report a case of histopathologically proven large cell acanthoma (LCA) whose RCM features simulate those of squamous cell carcinoma in situ. PMID:27648388

  6. Optical coherence tomography combined with confocal microscopy for investigation of interfaces in class V cavities

    NASA Astrophysics Data System (ADS)

    Rominu, Mihai; Sinescu, Cosmin; Petrescu, Emanuela; Haiduc, Claudiu; Rominu, Roxana; Enescu, Marius; Hughes, Michael; Bradu, Adrian; Dobre, George; Podoleanu, Adrian G.

    2009-07-01

    Standardized class V cavities, prepared in human extracted teeth, were filled with Premise (Kerr) composite. The specimens were thermo cycled. The interfaces were examined using a system employing two simultaneous imaging channels, an en-face Optical Coherence Tomography channel and a confocal microscopy channel.

  7. In vivo reflectance confocal microscopy features of a large cell acanthoma: report of a case

    PubMed Central

    Shahriari, Neda; Grant-Kels, Jane M.; Rabinovitz, Harold S.; Oliviero, Margaret; Scope, Alon

    2016-01-01

    Reflectance confocal microscopy (RCM) is an FDA approved noninvasive optical imaging technique that acquires cellular level-resolution skin images in vivo. Herein, we report a case of histopathologically proven large cell acanthoma (LCA) whose RCM features simulate those of squamous cell carcinoma in situ.

  8. 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...

  9. In vivo reflectance confocal microscopy features of a large cell acanthoma: report of a case.

    PubMed

    Shahriari, Neda; Grant-Kels, Jane M; Rabinovitz, Harold S; Oliviero, Margaret; Scope, Alon

    2016-07-01

    Reflectance confocal microscopy (RCM) is an FDA approved noninvasive optical imaging technique that acquires cellular level-resolution skin images in vivo. Herein, we report a case of histopathologically proven large cell acanthoma (LCA) whose RCM features simulate those of squamous cell carcinoma in situ. PMID:27648388

  10. In vivo reflectance confocal microscopy features of a large cell acanthoma: report of a case.

    PubMed

    Shahriari, Neda; Grant-Kels, Jane M; Rabinovitz, Harold S; Oliviero, Margaret; Scope, Alon

    2016-07-01

    Reflectance confocal microscopy (RCM) is an FDA approved noninvasive optical imaging technique that acquires cellular level-resolution skin images in vivo. Herein, we report a case of histopathologically proven large cell acanthoma (LCA) whose RCM features simulate those of squamous cell carcinoma in situ.

  11. 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...

  12. Scipion: A software framework toward integration, reproducibility and validation in 3D electron microscopy.

    PubMed

    de la Rosa-Trevín, J M; Quintana, A; Del Cano, L; Zaldívar, A; Foche, I; Gutiérrez, J; Gómez-Blanco, J; Burguet-Castell, J; Cuenca-Alba, J; Abrishami, V; Vargas, J; Otón, J; Sharov, G; Vilas, J L; Navas, J; Conesa, P; Kazemi, M; Marabini, R; Sorzano, C O S; Carazo, J M

    2016-07-01

    In the past few years, 3D electron microscopy (3DEM) has undergone a revolution in instrumentation and methodology. One of the central players in this wide-reaching change is the continuous development of image processing software. Here we present Scipion, a software framework for integrating several 3DEM software packages through a workflow-based approach. Scipion allows the execution of reusable, standardized, traceable and reproducible image-processing protocols. These protocols incorporate tools from different programs while providing full interoperability among them. Scipion is an open-source project that can be downloaded from http://scipion.cnb.csic.es. PMID:27108186

  13. Scipion: A software framework toward integration, reproducibility and validation in 3D electron microscopy.

    PubMed

    de la Rosa-Trevín, J M; Quintana, A; Del Cano, L; Zaldívar, A; Foche, I; Gutiérrez, J; Gómez-Blanco, J; Burguet-Castell, J; Cuenca-Alba, J; Abrishami, V; Vargas, J; Otón, J; Sharov, G; Vilas, J L; Navas, J; Conesa, P; Kazemi, M; Marabini, R; Sorzano, C O S; Carazo, J M

    2016-07-01

    In the past few years, 3D electron microscopy (3DEM) has undergone a revolution in instrumentation and methodology. One of the central players in this wide-reaching change is the continuous development of image processing software. Here we present Scipion, a software framework for integrating several 3DEM software packages through a workflow-based approach. Scipion allows the execution of reusable, standardized, traceable and reproducible image-processing protocols. These protocols incorporate tools from different programs while providing full interoperability among them. Scipion is an open-source project that can be downloaded from http://scipion.cnb.csic.es.

  14. Visualizing the Tumor Microenvironment of Liver Metastasis by Spinning Disk Confocal Microscopy.

    PubMed

    Babes, Liane; Kubes, Paul

    2016-01-01

    Intravital microscopy has evolved into an invaluable technique to study the complexity of tumors by visualizing individual cells in live organisms. Here, we describe a method for employing intravital spinning disk confocal microscopy to picture high-resolution tumor-stroma interactions in real time. We depict in detail the surgical procedures to image various tumor microenvironments and different cellular components in the liver. PMID:27581024

  15. Filtering, reconstruction, and measurement of the geometry of nuclei from hippocampal neurons based on confocal microscopy data.

    PubMed

    Queisser, Gillian; Wittmann, Malte; Bading, Hilmar; Wittum, Gabriel

    2008-01-01

    The cell nucleus is often considered a spherical structure. However, the visualization of proteins associated with the nuclear envelope in rat hippocampal neurons indicates that the geometry of nuclei is far more complex. The shape of cell nuclei is likely to influence the nucleo-cytoplasmic exchange of macromolecules and ions, in particular calcium, a key regulator of neuronal gene expression. We developed a tool to retrieve the 3-D view of cell nuclei from laser scanning confocal microscopy data. By applying an inertia-based filter, based on a special structure detection mechanism, the signal-to-noise ratio of the image is enhanced, the signal is smoothed, gaps in the membrane are closed, while at the same time the geometric properties, such as diameters of the membrane, are preserved. After segmentation of the image data, the microscopy data are sufficiently processed to extract surface information of the membrane by creating an isosurface with a marching tetrahedra algorithm combined with a modified Dijkstra graph-search algorithm. All methods are tested on artificial data, as well as on real data, which are recorded with a laser scanning confocal microscope. Significant advantages of the inertia-based filter can be observed when comparing it to other state of the art nonlinear diffusion filters. An additional program is written to calculate surface and volume of cell nuclei. These results represent the first step toward establishing a geometry-based model of the-dynamics of cytoplasmic and nuclear calcium. PMID:18315367

  16. A correlative approach for combining microCT, light and transmission electron microscopy in a single 3D scenario

    PubMed Central

    2013-01-01

    Background In biomedical research, a huge variety of different techniques is currently available for the structural examination of small specimens, including conventional light microscopy (LM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), microscopic X-ray computed tomography (microCT), and many others. Since every imaging method is physically limited by certain parameters, a correlative use of complementary methods often yields a significant broader range of information. Here we demonstrate the advantages of the correlative use of microCT, light microscopy, and transmission electron microscopy for the analysis of small biological samples. Results We used a small juvenile bivalve mollusc (Mytilus galloprovincialis, approximately 0.8 mm length) to demonstrate the workflow of a correlative examination by microCT, LM serial section analysis, and TEM-re-sectioning. Initially these three datasets were analyzed separately, and subsequently they were fused in one 3D scene. This workflow is very straightforward. The specimen was processed as usual for transmission electron microscopy including post-fixation in osmium tetroxide and embedding in epoxy resin. Subsequently it was imaged with microCT. Post-fixation in osmium tetroxide yielded sufficient X-ray contrast for microCT imaging, since the X-ray absorption of epoxy resin is low. Thereafter, the same specimen was serially sectioned for LM investigation. The serial section images were aligned and specific organ systems were reconstructed based on manual segmentation and surface rendering. According to the region of interest (ROI), specific LM sections were detached from the slides, re-mounted on resin blocks and re-sectioned (ultrathin) for TEM. For analysis, image data from the three different modalities was co-registered into a single 3D scene using the software AMIRA®. We were able to register both the LM section series volume and TEM slices neatly to the microCT dataset, with

  17. Quantitative 3D molecular cutaneous absorption in human skin using label free nonlinear microscopy.

    PubMed

    Chen, Xueqin; Grégoire, Sébastien; Formanek, Florian; Galey, Jean-Baptiste; Rigneault, Hervé

    2015-02-28

    Understanding the penetration mechanisms of drugs into human skin is a key issue in pharmaceutical and cosmetics research. To date, the techniques available for percutaneous penetration of compounds fail to provide a quantitative 3D map of molecular concentration distribution in complex tissues as the detected microscopy images are an intricate combination of concentration distribution and laser beam attenuation upon deep penetration. Here we introduce and validate a novel framework for imaging and reconstructing molecular concentration within the depth of artificial and human skin samples. Our approach combines the use of deuterated molecular compounds together with coherent anti-Stokes Raman scattering spectroscopy and microscopy that permits targeted molecules to be unambiguously discriminated within skin layers. We demonstrate both intercellular and transcellular pathways for different active compounds, together with in-depth concentration profiles reflecting the detailed skin barrier architecture. This method provides an enabling platform for establishing functional activity of topically applied products. PMID:25550155

  18. Cellulose Nanocrystals as Chiral Inducers: Enantioselective Catalysis and Transmission Electron Microscopy 3D Characterization.

    PubMed

    Kaushik, Madhu; Basu, Kaustuv; Benoit, Charles; Cirtiu, Ciprian M; Vali, Hojatollah; Moores, Audrey

    2015-05-20

    Cellulose nanocrystals (CNCs), derived from cellulose, provide us with an opportunity to devise more sustainable solutions to current technological challenges. Enantioselective catalysis, especially heterogeneous, is the preferred method for the synthesis of pure chiral molecules in the fine chemical industries. Cellulose has been long sought as a chiral inducer in enantioselective catalysis. We report herein an unprecedentedly high enantiomeric excess (ee) for Pd patches deposited onto CNCs used as catalysts for the hydrogenation of prochiral ketones in water at room temperature and 4 bar H2. Our system, where CNCs acted as support and sole chiral source, achieved an ee of 65% with 100% conversions. Cryo-electron microscopy, high-resolution transmission electron microscopy, and tomography were used for the first time to study the 3D structure of a metal functionalized CNC hybrid. It established the presence of sub-nanometer-thick Pd patches at the surface of CNCs and provided insight into the chiral induction mechanism.

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

    PubMed

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

    2007-08-20

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

  20. Laser scanning confocal microscopy characterization of water repellent distribution in a sandstone pore network.

    PubMed

    Zoghlami, Karima; Gómez-Gras, David; Corbella, Mercè; Darragi, Fadila

    2008-11-01

    In the present work, we propose the use of the Laser Scanning Confocal Microscopy (LSCM) to determine the effect of water repellents on rock's pore-network configuration and interconnection. The rocks studied are sandstones of Miocene age, a building material that is commonly found in the architectural heritage of Tunisia. The porosity quantitative data of treated and untreated samples, obtained by mercury porosimetry tests, were compared. The results show a slight decrease in total porosity with the water repellent treatment, which reduced both microporosity and macroporosity. This reduction produced a modification in pore size distribution and a shift of the pore access size mode interval toward smaller pore diameters (from the 30-40 microm to the 20-30 microm intervals). The water repellent was observed in SEM images as a continuous film coating grain surfaces; moreover, it was easily visualized in LSCM, by staining the water repellent with Epodye fluorochrome, and the coating thickness was straightforwardly measured (1.5-2 microm). In fact, the combination of mercury intrusion porosimetry data and LSCM observations suggests that the porosity reduction and the shift of the pore diameter mode were mainly due to the general reduction of pore diameters, but also to the plugging of the smallest pores (less than 3-4 microm in diameter) by the water repellent film. Finally, the LSCM technique enabled the reconstruction of 3D views of the water repellent coating film in the pore network, indicating that its distribution was uniform and continuous over the 100 microm thick sample. The LSCM imaging facilitates the integration and interpretation of mercury porosimetry and SEM data. PMID:18767050

  1. X-ray microscopy for in situ characterization of 3D nanostructural evolution in the laboratory

    NASA Astrophysics Data System (ADS)

    Hornberger, Benjamin; Bale, Hrishikesh; Merkle, Arno; Feser, Michael; Harris, William; Etchin, Sergey; Leibowitz, Marty; Qiu, Wei; Tkachuk, Andrei; Gu, Allen; Bradley, Robert S.; Lu, Xuekun; Withers, Philip J.; Clarke, Amy; Henderson, Kevin; Cordes, Nikolaus; Patterson, Brian M.

    2015-09-01

    X-ray microscopy (XRM) has emerged as a powerful technique that reveals 3D images and quantitative information of interior structures. XRM executed both in the laboratory and at the synchrotron have demonstrated critical analysis and materials characterization on meso-, micro-, and nanoscales, with spatial resolution down to 50 nm in laboratory systems. The non-destructive nature of X-rays has made the technique widely appealing, with potential for "4D" characterization, delivering 3D micro- and nanostructural information on the same sample as a function of sequential processing or experimental conditions. Understanding volumetric and nanostructural changes, such as solid deformation, pore evolution, and crack propagation are fundamental to understanding how materials form, deform, and perform. We will present recent instrumentation developments in laboratory based XRM including a novel in situ nanomechanical testing stage. These developments bridge the gap between existing in situ stages for micro scale XRM, and SEM/TEM techniques that offer nanometer resolution but are limited to analysis of surfaces or extremely thin samples whose behavior is strongly influenced by surface effects. Several applications will be presented including 3D-characterization and in situ mechanical testing of polymers, metal alloys, composites and biomaterials. They span multiple length scales from the micro- to the nanoscale and different mechanical testing modes such as compression, indentation and tension.

  2. A one-piece 3D printed flexure translation stage for open-source microscopy.

    PubMed

    Sharkey, James P; Foo, Darryl C W; Kabla, Alexandre; Baumberg, Jeremy J; Bowman, Richard W

    2016-02-01

    Open source hardware has the potential to revolutionise the way we build scientific instruments; with the advent of readily available 3D printers, mechanical designs can now be shared, improved, and replicated faster and more easily than ever before. However, printed parts are typically plastic and often perform poorly compared to traditionally machined mechanisms. We have overcome many of the limitations of 3D printed mechanisms by exploiting the compliance of the plastic to produce a monolithic 3D printed flexure translation stage, capable of sub-micron-scale motion over a range of 8 × 8 × 4 mm. This requires minimal post-print clean-up and can be automated with readily available stepper motors. The resulting plastic composite structure is very stiff and exhibits remarkably low drift, moving less than 20 μm over the course of a week, without temperature stabilisation. This enables us to construct a miniature microscope with excellent mechanical stability, perfect for time-lapse measurements in situ in an incubator or fume hood. The ease of manufacture lends itself to use in containment facilities where disposability is advantageous and to experiments requiring many microscopes in parallel. High performance mechanisms based on printed flexures need not be limited to microscopy, and we anticipate their use in other devices both within the laboratory and beyond.

  3. A one-piece 3D printed flexure translation stage for open-source microscopy

    NASA Astrophysics Data System (ADS)

    Sharkey, James P.; Foo, Darryl C. W.; Kabla, Alexandre; Baumberg, Jeremy J.; Bowman, Richard W.

    2016-02-01

    Open source hardware has the potential to revolutionise the way we build scientific instruments; with the advent of readily available 3D printers, mechanical designs can now be shared, improved, and replicated faster and more easily than ever before. However, printed parts are typically plastic and often perform poorly compared to traditionally machined mechanisms. We have overcome many of the limitations of 3D printed mechanisms by exploiting the compliance of the plastic to produce a monolithic 3D printed flexure translation stage, capable of sub-micron-scale motion over a range of 8 × 8 × 4 mm. This requires minimal post-print clean-up and can be automated with readily available stepper motors. The resulting plastic composite structure is very stiff and exhibits remarkably low drift, moving less than 20 μm over the course of a week, without temperature stabilisation. This enables us to construct a miniature microscope with excellent mechanical stability, perfect for time-lapse measurements in situ in an incubator or fume hood. The ease of manufacture lends itself to use in containment facilities where disposability is advantageous and to experiments requiring many microscopes in parallel. High performance mechanisms based on printed flexures need not be limited to microscopy, and we anticipate their use in other devices both within the laboratory and beyond.

  4. A one-piece 3D printed flexure translation stage for open-source microscopy.

    PubMed

    Sharkey, James P; Foo, Darryl C W; Kabla, Alexandre; Baumberg, Jeremy J; Bowman, Richard W

    2016-02-01

    Open source hardware has the potential to revolutionise the way we build scientific instruments; with the advent of readily available 3D printers, mechanical designs can now be shared, improved, and replicated faster and more easily than ever before. However, printed parts are typically plastic and often perform poorly compared to traditionally machined mechanisms. We have overcome many of the limitations of 3D printed mechanisms by exploiting the compliance of the plastic to produce a monolithic 3D printed flexure translation stage, capable of sub-micron-scale motion over a range of 8 × 8 × 4 mm. This requires minimal post-print clean-up and can be automated with readily available stepper motors. The resulting plastic composite structure is very stiff and exhibits remarkably low drift, moving less than 20 μm over the course of a week, without temperature stabilisation. This enables us to construct a miniature microscope with excellent mechanical stability, perfect for time-lapse measurements in situ in an incubator or fume hood. The ease of manufacture lends itself to use in containment facilities where disposability is advantageous and to experiments requiring many microscopes in parallel. High performance mechanisms based on printed flexures need not be limited to microscopy, and we anticipate their use in other devices both within the laboratory and beyond. PMID:26931888

  5. 3D Imaging of Diatoms with Ion-abrasion Scanning Electron Microscopy

    PubMed Central

    Hildebrand, Mark; Kim, Sang; Shi, Dan; Scott, Keana; Subramaniam, Sriram

    2009-01-01

    Ion-abrasion scanning electron microscopy (IASEM) takes advantage of focused ion beams to abrade thin sections from the surface of bulk specimens, coupled with SEM to image the surface of each section, enabling 3D reconstructions of subcellular architecture at ~ 30 nm resolution. Here, we report the first application of IASEM for imaging a biomineralizing organism, the marine diatom Thalassiosira pseudonana. Diatoms have highly patterned silica-based cell wall structures that are unique models for the study and application of directed nanomaterials synthesis by biological systems. Our study provides new insights into the architecture and assembly principles of both the “hard” (siliceous) and “soft” (organic) components of the cell. From 3D reconstructions of developmentally synchronized diatoms captured at different stages, we show that both micro- and nanoscale siliceous structures can be visualized at specific stages in their formation. We show that not only are structures visualized in a whole-cell context, but demonstrate that fragile, early-stage structures are visible, and that this can be combined with elemental mapping in the exposed slice. We demonstrate that the 3D architectures of silica structures, and the cellular components that mediate their creation and positioning can be visualized simultaneously, providing new opportunities to study and manipulate mineral nanostructures in a genetically tractable system. PMID:19269330

  6. Optical clearing assisted confocal microscopy of ex vivo transgenic mouse skin

    NASA Astrophysics Data System (ADS)

    Song, Eunjoo; Ahn, YoonJoon; Ahn, Jinhyo; Ahn, Soyeon; Kim, Changhwan; Choi, Sanghoon; Boutilier, Richard Martin; Lee, Yongjoong; Kim, Pilhan; Lee, Ho

    2015-10-01

    We examined the optical clearing assisted confocal microscopy of the transgenic mouse skin. The pinna and dorsal skin were imaged with a confocal microscope after the application of glycerol and FocusClear. In case of the glycerol-treated pinna, the clearing was minimal due to the inefficient permeability. However, the imaging depth was improved when the pinna was treated with FocusClear. In case of dorsal skin, we were able to image deeply to the subcutaneous connective tissue with both agents. Various skin structures such as the vessel, epithelium cells, cartilage, dermal cells, and hair follicles were clearly imaged.

  7. 3D motion of DNA-Au nanoconjugates in graphene liquid cell electron microscopy.

    PubMed

    Chen, Qian; Smith, Jessica M; Park, Jungwon; Kim, Kwanpyo; Ho, Davy; Rasool, Haider I; Zettl, Alex; Alivisatos, A Paul

    2013-09-11

    Liquid-phase transmission electron microscopy (TEM) can probe and visualize dynamic events with structural or functional details at the nanoscale in a liquid medium. Earlier efforts have focused on the growth and transformation kinetics of hard material systems, relying on their stability under electron beam. Our recently developed graphene liquid cell technique pushed the spatial resolution of such imaging to the atomic scale but still focused on growth trajectories of metallic nanocrystals. Here, we adopt this technique to imaging three-dimensional (3D) dynamics of soft materials instead, double strand (dsDNA) connecting Au nanocrystals as one example, at nanometer resolution. We demonstrate first that a graphene liquid cell can seal an aqueous sample solution of a lower vapor pressure than previously investigated well against the high vacuum in TEM. Then, from quantitative analysis of real time nanocrystal trajectories, we show that the status and configuration of dsDNA dictate the motions of linked nanocrystals throughout the imaging time of minutes. This sustained connecting ability of dsDNA enables this unprecedented continuous imaging of its dynamics via TEM. Furthermore, the inert graphene surface minimizes sample-substrate interaction and allows the whole nanostructure to rotate freely in the liquid environment; we thus develop and implement the reconstruction of 3D configuration and motions of the nanostructure from the series of 2D projected TEM images captured while it rotates. In addition to further proving the nanoconjugate structural stability, this reconstruction demonstrates 3D dynamic imaging by TEM beyond its conventional use in seeing a flattened and dry sample. Altogether, we foresee the new and exciting use of graphene liquid cell TEM in imaging 3D biomolecular transformations or interaction dynamics at nanometer resolution. PMID:23944844

  8. Detection of Gold Nanoparticles Aggregation Growth Induced by Nucleic Acid through Laser Scanning Confocal Microscopy.

    PubMed

    Gary, Ramla; Carbone, Giovani; Petriashvili, Gia; De Santo, Maria Penelope; Barberi, Riccardo

    2016-01-01

    The gold nanoparticle (GNP) aggregation growth induced by deoxyribonucleic acid (DNA) is studied by laser scanning confocal and environmental scanning electron microscopies. As in the investigated case the direct light scattering analysis is not suitable, we observe the behavior of the fluorescence produced by a dye and we detect the aggregation by the shift and the broadening of the fluorescence peak. Results of laser scanning confocal microscopy images and the fluorescence emission spectra from lambda scan mode suggest, in fact, that the intruding of the hydrophobic moiety of the probe within the cationic surfactants bilayer film coating GNPs results in a Förster resonance energy transfer. The environmental scanning electron microscopy images show that DNA molecules act as template to assemble GNPs into three-dimensional structures which are reminiscent of the DNA helix. This study is useful to design better nanobiotechnological devices using GNPs and DNA. PMID:26907286

  9. Detection of Gold Nanoparticles Aggregation Growth Induced by Nucleic Acid through Laser Scanning Confocal Microscopy

    PubMed Central

    Gary, Ramla; Carbone, Giovani; Petriashvili, Gia; De Santo, Maria Penelope; Barberi, Riccardo

    2016-01-01

    The gold nanoparticle (GNP) aggregation growth induced by deoxyribonucleic acid (DNA) is studied by laser scanning confocal and environmental scanning electron microscopies. As in the investigated case the direct light scattering analysis is not suitable, we observe the behavior of the fluorescence produced by a dye and we detect the aggregation by the shift and the broadening of the fluorescence peak. Results of laser scanning confocal microscopy images and the fluorescence emission spectra from lambda scan mode suggest, in fact, that the intruding of the hydrophobic moiety of the probe within the cationic surfactants bilayer film coating GNPs results in a Förster resonance energy transfer. The environmental scanning electron microscopy images show that DNA molecules act as template to assemble GNPs into three-dimensional structures which are reminiscent of the DNA helix. This study is useful to design better nanobiotechnological devices using GNPs and DNA. PMID:26907286

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. Shrinking of ultrathin polyelectrolyte multilayer capsules upon annealing: A confocal laser scanning microscopy and scanning force microscopy study

    NASA Astrophysics Data System (ADS)

    Leporatti, S.; Gao, C.; Voigt, A.; Donath, E.; Möhwald, H.

    Heating-induced morphological changes of micrometer size capsules prepared by step-wise deposition of oppositely charged polyelectrolytes onto melamine formaldehyde (MF) latex particles and biological cells with subsequent dissolution of the core have been investigated by confocal laser scanning microscopy (CLSM) and scanning force microscopy (SFM). For poly(styrenesulfonate-Na salt)/poly(allylamine hydrochloride) polyelectrolyte capsules a remarkable heating-induced shrinking is observed. An increase of the wall thickness corresponding to the capsule diameter decrease is found. The morphology of these microcapsules after temperature treatment is characterized. The thickening of the polyelectrolyte multilayer is interpreted in terms of a configurational entropy increase via polyanion-polycation bond rearrangement.

  15. 3D elemental sensitive imaging using transmission X-ray microscopy.

    PubMed

    Liu, Yijin; Meirer, Florian; Wang, Junyue; Requena, Guillermo; Williams, Phillip; Nelson, Johanna; Mehta, Apurva; Andrews, Joy C; Pianetta, Piero

    2012-09-01

    Determination of the heterogeneous distribution of metals in alloy/battery/catalyst and biological materials is critical to fully characterize and/or evaluate the functionality of the materials. Using synchrotron-based transmission x-ray microscopy (TXM), it is now feasible to perform nanoscale-resolution imaging over a wide X-ray energy range covering the absorption edges of many elements; combining elemental sensitive imaging with determination of sample morphology. We present an efficient and reliable methodology to perform 3D elemental sensitive imaging with excellent sample penetration (tens of microns) using hard X-ray TXM. A sample of an Al-Si piston alloy is used to demonstrate the capability of the proposed method. PMID:22349401

  16. Electron Microscopy: From 2D to 3D Images with Special Reference to Muscle

    PubMed Central

    2015-01-01

    This is a brief and necessarily very sketchy presentation of the evolution in electron microscopy (EM) imaging that was driven by the necessity of extracting 3-D views from the essentially 2-D images produced by the electron beam. The lens design of standard transmission electron microscope has not been greatly altered since its inception. However, technical advances in specimen preparation, image collection and analysis gradually induced an astounding progression over a period of about 50 years. From the early images that redefined tissues, cell and cell organelles at the sub-micron level, to the current nano-resolution reconstructions of organelles and proteins the step is very large. The review is written by an investigator who has followed the field for many years, but often from the sidelines, and with great wonder. Her interest in muscle ultrastructure colors the writing. More specific detailed reviews are presented in this issue. PMID:26913146

  17. 3D elemental sensitive imaging using transmission X-ray microscopy.

    PubMed

    Liu, Yijin; Meirer, Florian; Wang, Junyue; Requena, Guillermo; Williams, Phillip; Nelson, Johanna; Mehta, Apurva; Andrews, Joy C; Pianetta, Piero

    2012-09-01

    Determination of the heterogeneous distribution of metals in alloy/battery/catalyst and biological materials is critical to fully characterize and/or evaluate the functionality of the materials. Using synchrotron-based transmission x-ray microscopy (TXM), it is now feasible to perform nanoscale-resolution imaging over a wide X-ray energy range covering the absorption edges of many elements; combining elemental sensitive imaging with determination of sample morphology. We present an efficient and reliable methodology to perform 3D elemental sensitive imaging with excellent sample penetration (tens of microns) using hard X-ray TXM. A sample of an Al-Si piston alloy is used to demonstrate the capability of the proposed method.

  18. Dynamic complex optical fields for optical manipulation, 3D microscopy, and photostimulation of neurotransmitters

    NASA Astrophysics Data System (ADS)

    Daria, Vincent R.; Stricker, Christian; Bekkers, John; Redman, Steve; Bachor, Hans

    2010-08-01

    We demonstrate a multi-functional system capable of multiple-site two-photon excitation of photo-sensitive compounds as well as transfer of optical mechanical properties on an array of mesoscopic particles. We use holographic projection of a single Ti:Sapphire laser operating in femtosecond pulse mode to show that the projected three-dimensional light patterns have sufficient spatiotemporal photon density for multi-site two-photon excitation of biological fluorescent markers and caged neurotransmitters. Using the same laser operating in continuous-wave mode, we can use the same light patterns for non-invasive transfer of both linear and orbital angular momentum on a variety of mesoscopic particles. The system also incorporates high-speed scanning using acousto-optic modulators to rapidly render 3D images of neuron samples via two-photon microscopy.

  19. The Use of Atomic Force Microscopy for 3D Analysis of Nucleic Acid Hybridization on Microarrays

    PubMed Central

    Dubrovin, E. V.; Presnova, G. V.; Rubtsova, M. Yu.; Egorov, A. M.; Grigorenko, V. G.; Yaminsky, I. V.

    2015-01-01

    Oligonucleotide microarrays are considered today to be one of the most efficient methods of gene diagnostics. The capability of atomic force microscopy (AFM) to characterize the three-dimensional morphology of single molecules on a surface allows one to use it as an effective tool for the 3D analysis of a microarray for the detection of nucleic acids. The high resolution of AFM offers ways to decrease the detection threshold of target DNA and increase the signal-to-noise ratio. In this work, we suggest an approach to the evaluation of the results of hybridization of gold nanoparticle-labeled nucleic acids on silicon microarrays based on an AFM analysis of the surface both in air and in liquid which takes into account of their three-dimensional structure. We suggest a quantitative measure of the hybridization results which is based on the fraction of the surface area occupied by the nanoparticles. PMID:26085952

  20. Electron Microscopy: From 2D to 3D Images with Special Reference to Muscle.

    PubMed

    Franzini-Armstrong, Clara

    2015-01-01

    This is a brief and necessarily very sketchy presentation of the evolution in electron microscopy (EM) imaging that was driven by the necessity of extracting 3-D views from the essentially 2-D images produced by the electron beam. The lens design of standard transmission electron microscope has not been greatly altered since its inception. However, technical advances in specimen preparation, image collection and analysis gradually induced an astounding progression over a period of about 50 years. From the early images that redefined tissues, cell and cell organelles at the sub-micron level, to the current nano-resolution reconstructions of organelles and proteins the step is very large. The review is written by an investigator who has followed the field for many years, but often from the sidelines, and with great wonder. Her interest in muscle ultrastructure colors the writing. More specific detailed reviews are presented in this issue. PMID:26913146

  1. 3D surface reconstruction and FIB microscopy of worn alumina hip prostheses

    NASA Astrophysics Data System (ADS)

    Zeng, P.; Inkson, B. J.; Rainforth, W. M.; Stewart, T.

    2008-08-01

    Interest in alumina-on-alumina total hip replacements (THR) continues to grow for the young and active patient due to their superior wear performance and biocompatibility compared to the alternative traditional polymer/metal prostheses. While alumina on alumina bearings offer an excellent solution, a region of high wear, known as stripe wear, is commonly observed on retrieved alumina hip components that poses concern. These in-vivo stripe wear mechanisms can be replicated in vitro by the introduction of micro-separation during the simulated walking cycle in hip joint simulation. However, the understanding of the mechanisms behind the stripe wear processes is relatively poor. 3D topographic reconstructions of titled SEM stereo pairs from different zones have been obtained to determine the local worn surface topography. Focused ion beam (FIB) microscopy was applied to examine the subsurface damage across the stripe wear. The paper presents novel images of sub-surface microcracks in alumina along with 3D reconstructions of the worn ceramic surfaces and a classification of four distinct wear zones following microseparation in hip prostheses.

  2. Local characterization of hindered Brownian motion by using digital video microscopy and 3D particle tracking

    SciTech Connect

    Dettmer, Simon L.; Keyser, Ulrich F.; Pagliara, Stefano

    2014-02-15

    In this article we present methods for measuring hindered Brownian motion in the confinement of complex 3D geometries using digital video microscopy. Here we discuss essential features of automated 3D particle tracking as well as diffusion data analysis. By introducing local mean squared displacement-vs-time curves, we are able to simultaneously measure the spatial dependence of diffusion coefficients, tracking accuracies and drift velocities. Such local measurements allow a more detailed and appropriate description of strongly heterogeneous systems as opposed to global measurements. Finite size effects of the tracking region on measuring mean squared displacements are also discussed. The use of these methods was crucial for the measurement of the diffusive behavior of spherical polystyrene particles (505 nm diameter) in a microfluidic chip. The particles explored an array of parallel channels with different cross sections as well as the bulk reservoirs. For this experiment we present the measurement of local tracking accuracies in all three axial directions as well as the diffusivity parallel to the channel axis while we observed no significant flow but purely Brownian motion. Finally, the presented algorithm is suitable also for tracking of fluorescently labeled particles and particles driven by an external force, e.g., electrokinetic or dielectrophoretic forces.

  3. Computational 3D reconstructions by optimization for cryo-electron microscopy

    NASA Astrophysics Data System (ADS)

    Yin, Zhye; Zheng, Yili; Doerschuk, Peter C.; Johnson, John E.

    2003-06-01

    An algorithm for the simultaneous 3-D reconstruction of several types of object, where each type of object may possibly have a rotational symmetry, from 2-D projection images, where for each image the type of object imaged, the projection orientation used to create the image, and the location of the object in the image are unknown, is described. The motivating application is the determination of the 3-D structure of small spherical viruses from cryo electron microscopy images. The algorithm is a maximum likelihood estimator which is computed by expectation maximization (EM). Due to the structure of the statistical model, the maximization step of EM can be easily computed but the expectation step requires 5-D numerical quadrature. The computational burden of the quadratures necessitates parallel computation and three different implementations of two different types of parallelism have been developed using pthreads (for shared memory processors) and MPI (for distributed memory processors). An example applying one of the MPI implementations, running on a 32 node PC cluster, to experimental images of Flock House Virus with comparison to the x-ray crystal diffraction structure of the virus is described.

  4. Dental pulp stem cells (DPSCs) differentiation study by confocal Raman microscopy

    NASA Astrophysics Data System (ADS)

    Salehi, H.; Collart-Dutilleul, P.-Y.; Gergely, C.; Cuisinier, F. J. G.

    2014-03-01

    Regenerative medicine brings a huge application for Mesenchymal stem cells such as Dental Pulp Stem Cells (DPSCs). Confocal Raman microscopy, a non-invasive, label free , real time and high spatial resolution imaging technique is used to study osteogenic differentiation of DPSCs. Integrated Raman intensities in the 2800-3000 cm-1 region (C-H stretching) and 960 cm-1 peak (phosphate PO4 3-) were collected. In Dental Pulp Stem Cells 21st day differentiated in buffer solution, phosphate peaks ν1 PO4 3- (first vibrational mode) at 960cm-1 and ν2 PO4 3- at 430cm-1 and ν4 PO4 3- at 585cm-1 are obviously present. Confocal Raman microscopy enables the detection of cell differentiation and it can be used to investigate clinical stem cell research.

  5. 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.

  6. Near-IR fluorescence and reflectance confocal microscopy for imaging of quantum dots in mammalian skin

    PubMed Central

    Mortensen, Luke J.; Glazowski, Christopher E.; Zavislan, James M.; DeLouise, Lisa A.

    2011-01-01

    Understanding the skin penetration of nanoparticles (NPs) is an important concern due to the increasing presence of NPs in consumer products, including cosmetics. Technical challenges have slowed progress in evaluating skin barrier and NP factors that contribute to skin penetration risk. To limit sampling error and other problems associated with histological processing, many researchers are implementing whole tissue confocal or multiphoton microscopies. This work introduces a fluorescence and reflectance confocal microscopy system that utilizes near-IR excitation and emission to detect near-IR lead sulfide quantum dots (QDs) through ex vivo human epidermis. We provide a detailed prediction and experimental analysis of QD detection sensitivity and demonstrate detection of QD skin penetration in a barrier disrupted model. The unique properties of near-IR lead-based QDs will enable future studies that examine the impact of further barrier-disrupting agents on skin penetration of QDs and elucidate mechanistic insight into QD tissue interactions at the cellular level. PMID:21698023

  7. OCT and in vivo confocal microscopy of a pigmented corneal tumor-like lesion.

    PubMed

    Szaflik, Jacek P; Oldak, Monika; Ulinska, Magdalena; Ulnska, Magdalena; Tesla, Piotr; Szaflik, Jerzy

    2009-01-01

    A 43-year-old woman presented with a pigmented flat tumor situated at the posterior surface of the cornea nasally in her left eye. Anterior-segment optical coherence tomography revealed that the lesion was similar to the iris leaf, was limited to the cornea, and did not communicate with the iridocorneal angle. In vivo scanning slit confocal microscopy imaged dense hyperreflective tissue behind the endothelium and bright spots dispersed on the adjacent endothelial surface. Multiple hyporeflective formations resembling cell nuclei were visualized within the hyperreflective mass and the cell borders were distinguished. The diagnosis of pigmented nevus or retrocorneal membrane was suspected. The authors conclude that anterior-segment optical coherence tomography and in vivo scanning slit confocal microscopy are useful in assessing the microstructure and penetration of pigmented corneal lesions.

  8. Velocity profile of thin film flows measured using a confocal microscopy particle image velocimetry system with simultaneous multi depth position

    NASA Astrophysics Data System (ADS)

    Kikuchi, K.; Mochizuki, O.

    2015-02-01

    In this paper, we report a technique for simultaneously visualizing flows near walls at nano-depth positions. To achieve such a high interval of depth gradient, we developed a tilted observation technique in a particle image velocimetry (PIV) system based on confocal microscopy. The focal plane along the bottom of the flow channel was tilted by tilting the micro-channel, enabling depth scanning in the microscopic field of view. Our system is suitable for measuring 3D two-component flow fields. The depth interval was approximately 220 nm over a depth range of 10 μm, depending on the tilt angle of the micro-channel. Applying the proposed system, we visualized the near-wall flow in a drainage film flow under laminar conditions to the depth of approximately 30 μm via vertical scanning from the bottom to the free surface. The velocity gradient was proportional to the distance from the wall, consistent with theoretical predictions. From the measured near-wall velocity gradient, we calculated the wall shear stress. The measurement accuracy was approximately 1.3 times higher in our proposed method than in the conventional confocal micro-PIV method.

  9. An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy.

    PubMed

    Bohórquez, Diego V; Samsa, Leigh A; Roholt, Andrew; Medicetty, Satish; Chandra, Rashmi; Liddle, Rodger A

    2014-01-01

    The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY--both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia--the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells. PMID:24587096

  10. An Enteroendocrine Cell – Enteric Glia Connection Revealed by 3D Electron Microscopy

    PubMed Central

    Bohórquez, Diego V.; Samsa, Leigh A.; Roholt, Andrew; Medicetty, Satish; Chandra, Rashmi; Liddle, Rodger A.

    2014-01-01

    The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY – both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia – the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells. PMID:24587096

  11. A 3D scanning confocal imaging method measures pit volume and captures the role of Rac in osteoclast function.

    PubMed

    Goldberg, Stephanie R; Georgiou, John; Glogauer, Michael; Grynpas, Marc D

    2012-07-01

    Modulation of Rho GTPases Rac1 and Rac2 impacts bone development, remodeling, and disease. In addition, GTPases are considered treatment targets for dysplastic and erosive bone diseases including Neurofibromatosis type 1. While it is important to understand the effects of Rac modulation on osteoclast function, two-dimensional resorption pit area measurements fall short in elucidating the volume aspect of bone resorption activity. Bone marrow from wild-type, Rac1 and Rac2 null mice was isolated from femora. Osteoclastogenesis was induced by adding M-CSF and RANKL in culture plates containing dentin slices and later stained with Picro Sirius Red to image resorption lacunae. Osteoclasts were also plated on glass cover slips and stained with phalloidin and DAPI to measure their surface area and the number of nuclei. Volumetric images were collected on a laser-scanning confocal system. Sirius Red confocal imaging provided an unambiguous, continuous definition of the pit boundary compared to reflected and transmitted light imaging. Rac1- and Rac2-deficient osteoclasts had fewer nuclei in comparison to wild-type counterparts. Rac1-deficient osteoclasts showed reduced resorption pit volume and surface area. Lacunae made by single Rac2 null osteoclasts had reduced volume but surprisingly surface area was unaffected. Surface area measures are deceiving since volume changed independently in resorption pits made by individual Rac2 null osteoclasts. Our innovative confocal imaging technique allows us to derive novel conclusions about Rac1 and Rac2 in osteoclast function. The data and method can be applied to study effects of genes and drugs including Rho GTPase modulators on osteoclast function and to develop pharmacotherapeutics to treat bone lytic disorders.

  12. Actin restructuring during Salmonella typhimurium infection investigated by confocal and super-resolution microscopy

    NASA Astrophysics Data System (ADS)

    Han, Jason J.; Kunde, Yuliya A.; Hong-Geller, Elizabeth; Werner, James H.

    2014-01-01

    We have used super-resolution optical microscopy and confocal microscopy to visualize the cytoskeletal restructuring of HeLa cells that accompanies and enables Salmonella typhimurium internalization. Herein, we report the use of confocal microscopy to verify and explore infection conditions that would be compatible with super-resolution optical microscopy, using Alexa-488 labeled phalloidin to stain the actin cytoskeletal network. While it is well known that actin restructuring and cytoskeletal rearrangements often accompany and assist in bacterial infection, most studies have employed conventional diffraction-limited fluorescence microscopy to explore these changes. Here we show that the superior spatial resolution provided by single-molecule localization methods (such as direct stochastic optical reconstruction microscopy) enables more precise visualization of the nanoscale changes in the actin cytoskeleton that accompany bacterial infection. In particular, we found that a thin (100-nm) ring of actin often surrounds an invading bacteria 10 to 20 min postinfection, with this ring being transitory in nature. We estimate that a few hundred monofilaments of actin surround the S. typhimurium in this heretofore unreported bacterial internalization intermediate.

  13. Observation of dendritic cell morphology under light, phase-contrast or confocal laser scanning microscopy.

    PubMed

    Tan, Yuen-Fen; Leong, Chooi-Fun; Cheong, Soon-Keng

    2010-12-01

    Dendritic cells (DCs) are professional antigen presenting cells of the immune system. They can be generated in vitro from peripheral blood monocytes supplemented with GM-CSF, IL-4 and TNF alpha. During induction, DCs will increase in size and acquire multiple cytoplasmic projections when compared to their precursor cells such as monocytes or haematopoietic stem cells which are usually round or spherical. Morphology of DCs can be visualized by conventional light microscopy after staining or phase-contrast inverted microscopy or confocal laser scanning microscopy. In this report, we described the morphological appearances of DCs captured using the above-mentioned techniques. We found that confocal laser scanning microscopy yielded DCs images with greater details but the operating cost for such a technique is high. On the other hand, the images obtained through light microscopy after appropriate staining or phase contrast microscopy were acceptable for identification purpose. Besides, these equipments are readily available in most laboratories and the cost of operation is affordable. Nevertheless, morphological identification is just one of the methods to characterise DCs. Other methods such as phenotypic expression markers and mixed leukocyte reactions are additional tools used in the characterisation of DCs. PMID:21329180

  14. Super-resolution imaging of the cytokinetic Z ring in live bacteria using fast 3D-structured illumination microscopy (f3D-SIM).

    PubMed

    Turnbull, Lynne; Strauss, Michael P; Liew, Andrew T F; Monahan, Leigh G; Whitchurch, Cynthia B; Harry, Elizabeth J

    2014-01-01

    Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques - stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

  15. Super-resolution imaging of the cytokinetic Z ring in live bacteria using fast 3D-structured illumination microscopy (f3D-SIM).

    PubMed

    Turnbull, Lynne; Strauss, Michael P; Liew, Andrew T F; Monahan, Leigh G; Whitchurch, Cynthia B; Harry, Elizabeth J

    2014-01-01

    Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques - stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide. PMID:25286090

  16. Improvements of axial resolution in confocal microscopy with fan-shaped apertures.

    PubMed

    Ma, Ye; Kuang, Cuifang; Gong, Wei; Xue, Li; Zheng, Yao; Wang, Yifan; Si, Ke; Liu, Xu

    2015-02-20

    Based on diffraction theory, this paper theoretically demonstrates improvements in axial resolution in confocal microscopy with fan-shaped apertures. It provides the optimal geometric parameters of the fan-shaped pupils to give the maximum axial resolution for a given pinhole size. To fully understand the overall performance, the signal-to-background ratio and the signal level with regard to the geometric parameters are discussed.

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

    PubMed

    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. PMID:23338938

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

    SciTech Connect

    Maity, Supratim; Roychowdhury, Tuhin; Chattopadhyay, Kalyan Kumar

    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.

  19. UNDERSTANDING THE EFFECTS OF SURFACTANT ADDITION ON RHEOLOGY USING LASER SCANNING CONFOCAL MICROSCOPY

    SciTech Connect

    White, T

    2007-05-08

    The effectiveness of three dispersants to modify rheology was examined using rheology measurements and laser scanning confocal microscopy (LSCM) in simulated waste solutions. All of the dispersants lowered the yield stress of the slurries below the baseline samples. The rheology curves were fitted reasonably to a Bingham Plastic model. The three-dimensional LSCM images of simulants showed distinct aggregates were greatly reduced after the addition of dispersants leading to a lowering of the yield stress of the simulated waste slurry solutions.

  20. 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.

  1. Combining confocal Raman microscopy and freeze-drying for quantification of substance penetration into human skin.

    PubMed

    Franzen, Lutz; Anderski, Juliane; Planz, Viktoria; Kostka, Karl-Heinz; Windbergs, Maike

    2014-12-01

    In the area of dermatological research, the knowledge of rate and extent of substance penetration into the human skin is essential not only for evaluation of therapeutics, but also for risk assessment of chemicals and cosmetic ingredients. Recently, confocal Raman microscopy emerged as a novel analytical technique for analysis of substance skin penetration. In contrast to destructive drug extraction and quantification, the technique is non-destructive and provides high spatial resolution in three dimensions. However, the generation of time-resolved concentration depth profiles is restrained by ongoing diffusion of the penetrating substance during analysis. To prevent that, substance diffusion in excised human skin can instantly be stopped at defined time points by freeze-drying the sample. Thus, combining sample preparation by freeze-drying with drug quantification by confocal Raman microscopy yields a novel analytical platform for non-invasive and quantitative in vitro analysis of substance skin penetration. This work presents the first proof-of-concept study for non-invasive quantitative substance depth profiling in freeze-dried excised human stratum corneum by confocal Raman microscopy. PMID:25219950

  2. In vivo Confocal Microscopy in Differentiating Ipilimumab-Induced Anterior Uveitis from Metastatic Uveal Melanoma

    PubMed Central

    Kiratli, Hayyam; Mocan, Mehmet C.; İrkeç, Murat

    2016-01-01

    This report aims to describe the facilitating role of in vivo confocal microscopy in differentiating inflammatory cells from a metastatic process in a patient with uveal melanoma and multiple systemic metastases who developed anterior uveitis while under ipilimumab treatment. A 43-year-old woman developed systemic metastases 11 months after treatment of amelanotic choroidal melanoma in her right eye with 30 Gy fractionated stereotactic radiotherapy. She first received temozolomide and then 4 cycles of ipilimumab 3 mg/kg/day. After the third cycle, severe anterior uveitis with coarse pigment clumps on the lens was seen in the left eye. Her left visual acuity declined from 20/20 to 20/80. Confocal microscopy revealed globular keratic precipitates with hyperreflective inclusions and endothelial blebs all suggestive of granulomatous uveitis. The uveitic reaction subsided after a 3-week course of topical corticosteroids, and her visual acuity was 20/20 again. Although uveal melanoma metastatic to the intraocular structures of the fellow eye is exceedingly rare and metastasis masquerading uveitis without any identifiable uveal lesion is even more unusual, it was still mandatory to rule out this distant possibility in our particular patient who already had widespread systemic metastases. Confocal microscopy was a useful complementary tool by identifying the inflammatory features of the keratic precipitates. PMID:27790127

  3. Fast segmentation of stained nuclei in terabyte-scale, time resolved 3D microscopy image stacks.

    PubMed

    Stegmaier, Johannes; Otte, Jens C; Kobitski, Andrei; Bartschat, Andreas; Garcia, Ariel; Nienhaus, G Ulrich; Strähle, Uwe; Mikut, Ralf

    2014-01-01

    Automated analysis of multi-dimensional microscopy images has become an integral part of modern research in life science. Most available algorithms that provide sufficient segmentation quality, however, are infeasible for a large amount of data due to their high complexity. In this contribution we present a fast parallelized segmentation method that is especially suited for the extraction of stained nuclei from microscopy images, e.g., of developing zebrafish embryos. The idea is to transform the input image based on gradient and normal directions in the proximity of detected seed points such that it can be handled by straightforward global thresholding like Otsu's method. We evaluate the quality of the obtained segmentation results on a set of real and simulated benchmark images in 2D and 3D and show the algorithm's superior performance compared to other state-of-the-art algorithms. We achieve an up to ten-fold decrease in processing times, allowing us to process large data sets while still providing reasonable segmentation results.

  4. Multi-beam confocal microscopy based on a custom image sensor with focal-plane pinhole array effect.

    PubMed

    Kagawa, Keiichiro; Seo, Min-Woong; Yasutomi, Keita; Terakawa, Susumu; Kawahito, Shoji

    2013-01-28

    Multi-beam confocal microscopy without any physical pinhole was demonstrated. As a key device, a custom CMOS image sensor realizing a focal-plane pinhole array effect by special pixel addressing and discarding of the unwanted photocarriers was developed. The axial resolution in the confocal mode measured by FWHM for a planar mirror was 8.9 μm, which showed that the confocality has been achieved with the proposed CMOS image sensor.

  5. 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. PMID:26786962

  6. Characterization of X-ray polycapillary optics by LiF crystal radiation detectors through confocal fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Bonfigli, Francesca; Hampai, Dariush; Dabagov, Sultan B.; Montereali, Rosa Maria

    2016-08-01

    Solid-state radiation imaging detectors based on photoluminescent colour centres in lithium fluoride (LiF) crystals have been successfully tested for both advanced 2D and 3D characterizations of X-ray polycapillary optics by a table-top laboratory system. Polycapillary optics can control X-ray beams propagation and allows obtaining quasi-parallel beam (half-lens) or focused beams (full-lens). The combination of a fine-focused micro X-ray tube and a polycapillary lens can provide the high intensity radiation fluxes that are necessary for high resolution X-ray imaging. In this paper we present novel results about advanced characterization of these complex optics by 2D as well as 3D confocal laser fluorescence microscopy of X-ray irradiated LiF crystal detectors. Two dimensional high spatial resolution images on a wide field of view of transmitted X-rays through a semi-lens and 3D direct inspection of the coloured volumes produced in LiF crystals by both focused and parallel X-ray beam transmitted by a full and a semi-lens, respectively, as well as their 3D reconstructions were obtained. The results show that the photoluminescent colour centres volume in LiF crystals combined with an optical sectioning reading system provide information about tomography of transmitted X-ray beams by policapillary optics in a single exposure process. For the first time, the use of LiF crystal plates as versatile radiation imaging luminescent detectors have been used to characterize the operation of polycapillary optics as X-ray lens, in focusing and parallel mode.

  7. The potential of 3D-FISH and super-resolution structured illumination microscopy for studies of 3D nuclear architecture: 3D structured illumination microscopy of defined chromosomal structures visualized by 3D (immuno)-FISH opens new perspectives for studies of nuclear architecture.

    PubMed

    Markaki, Yolanda; Smeets, Daniel; Fiedler, Susanne; Schmid, Volker J; Schermelleh, Lothar; Cremer, Thomas; Cremer, Marion

    2012-05-01

    Three-dimensional structured illumination microscopy (3D-SIM) has opened up new possibilities to study nuclear architecture at the ultrastructural level down to the ~100 nm range. We present first results and assess the potential using 3D-SIM in combination with 3D fluorescence in situ hybridization (3D-FISH) for the topographical analysis of defined nuclear targets. Our study also deals with the concern that artifacts produced by FISH may counteract the gain in resolution. We address the topography of DAPI-stained DNA in nuclei before and after 3D-FISH, nuclear pores and the lamina, chromosome territories, chromatin domains, and individual gene loci. We also look at the replication patterns of chromocenters and the topographical relationship of Xist-RNA within the inactive X-territory. These examples demonstrate that an appropriately adapted 3D-FISH/3D-SIM approach preserves key characteristics of the nuclear ultrastructure and that the gain in information obtained by 3D-SIM yields new insights into the functional nuclear organization. PMID:22508100

  8. 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

  9. TRAIL protein localization in human primary T cells by 3D microscopy using 3D interactive surface plot: a new method to visualize plasma membrane.

    PubMed

    Gras, Christophe; Smith, Nikaïa; Sengmanivong, Lucie; Gandini, Mariana; Kubelka, Claire Fernandes; Herbeuval, Jean-Philippe

    2013-01-31

    The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4(+) T cells is not known. Here we investigated whether primary CD4(+) T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4(+) T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images. PMID:23085529

  10. TRAIL protein localization in human primary T cells by 3D microscopy using 3D interactive surface plot: a new method to visualize plasma membrane.

    PubMed

    Gras, Christophe; Smith, Nikaïa; Sengmanivong, Lucie; Gandini, Mariana; Kubelka, Claire Fernandes; Herbeuval, Jean-Philippe

    2013-01-31

    The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4(+) T cells is not known. Here we investigated whether primary CD4(+) T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4(+) T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images.

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 3D measurements of live cells via digital holographic microscopy and terahertz spectroscopy

    NASA Astrophysics Data System (ADS)

    Park, Jun Yong; Oser, Dorian; Iapozzuto, Peter; Norbury, Sean; Mahajan, Supriya; Khmaladze, Alexander; Sharikova, Anna

    2016-03-01

    This is a study of the central nervous system (CNS) cells, including brain micro vascular endothelial cells (BMV) that constitute the blood brain barrier, and C6 glial cells that are the predominant cell in the brain. The cells are exposed to various chemicals by non-invasive, label-free methods. Digital holographic microscopy (DHM) is a technique that records an interference pattern between an object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information, and 3D images are obtained. The measurement of cell cultures by digital holographic microscopy yields information about cell death mechanisms, since these processes are correlated with individual cell volume. Our in-house DHM combines a visible (red) laser source with a conventional microscope base, and LabVIEW-run data processing. Terahertz spectral signatures are associated with structural changes in molecules and provide complementary information about cells. Both CNS cells BMV and C6 cells are treated with the drug "Methamphetamine" (METH), which induces apoptosis in neuronal cells and exhibits decrease in cell volume, a characteristic of cells undergoing apoptosis (induced cell death). METH can cause CNS cell death by cross-talk between mitochondria-, endoplasmic reticulum-, and receptor-mediated apoptotic events, all of which results in drug induced changes in neuroplasticity and significant neuropathology. Doxorubicin (DOX), a popular anticancer drug, is used as a control. We observe that METH treatment resulted in more pronounced cell volume shrinkage in both the BMV and C6 cells, as compared to DOX-induced cell apoptosis.

  17. Investigation of resins suitable for the preparation of biological sample for 3-D electron microscopy.

    PubMed

    Kizilyaprak, Caroline; Longo, Giovanni; Daraspe, Jean; Humbel, Bruno M

    2015-02-01

    In the last two decades, the third-dimension has become a focus of attention in electron microscopy to better understand the interactions within subcellular compartments. Initially, transmission electron tomography (TEM tomography) was introduced to image the cell volume in semi-thin sections (∼ 500 nm). With the introduction of the focused ion beam scanning electron microscope, a new tool, FIB-SEM tomography, became available to image much larger volumes. During TEM tomography and FIB-SEM tomography, the resin section is exposed to a high electron/ion dose such that the stability of the resin embedded biological sample becomes an important issue. The shrinkage of a resin section in each dimension, especially in depth, is a well-known phenomenon. To ensure the dimensional integrity of the final volume of the cell, it is important to assess the properties of the different resins and determine the formulation which has the best stability in the electron/ion beam. Here, eight different resin formulations were examined. The effects of radiation damage were evaluated after different times of TEM irradiation. To get additional information on mass-loss and the physical properties of the resins (stiffness and adhesion), the topography of the irradiated areas was analysed with atomic force microscopy (AFM). Further, the behaviour of the resins was analysed after ion milling of the surface of the sample with different ion currents. In conclusion, two resin formulations, Hard Plus and the mixture of Durcupan/Epon, emerged that were considerably less affected and reasonably stable in the electron/ion beam and thus suitable for the 3-D investigation of biological samples. PMID:25433274

  18. Confocal Raman microscopy for in depth analysis in the field of cultural heritage

    NASA Astrophysics Data System (ADS)

    Lorenzetti, G.; Striova, J.; Zoppi, A.; Castellucci, E. M.

    2011-05-01

    In the field of cultural heritage, the main concern when a sample is analyzed is its safeguard, and this means that non-destructive techniques are required. In this work, we show how confocal Raman microscopy (CRM) may be successfully applied in the study of works of art as a valuable alternative to other well established techniques. CRM with a metallurgical objective was tested for the in depth study of thin samples that are of interest in the field of cultural heritage. The sensitivity of the instrumentation was first evaluated by analyzing single layers of pure polyethylene terephthalate (PET) films having a thickness of 12, 25, and 50 μm, respectively, and a multilayer sample of polypropylene (PP) and polyethylene (PE). Subsequently, the technique was applied to the analysis of historical dyed cotton yarns in order to check whether it was possible to achieve a better discrimination of the fibres' signals for an easier identification. A substantial improvement of the signal to noise ratio was found in the confocal arrangement with respect to the non-confocal one, suggesting the use of this technique for this kind of analysis in the field of cultural heritage. Furthermore, Raman spectroscopy in confocal configuration was exploited in the evaluation of cleaning performed on the mural painting specimens, treated with acrylic resin (Paraloid B72). Confocal Raman experiments were performed before and after laser cleaning (at different conditions) in order to monitor the presence and to approximate the polymer thickness: the method proved to be a valid comparative tool in assessment of cleaning efficiencies.

  19. The use of reflectance confocal microscopy for examination of benign and malignant skin tumors

    PubMed Central

    Wielowieyska-Szybińska, Dorota; Białek-Galas, Kamila; Podolec, Katarzyna

    2014-01-01

    Reflectance confocal microscopy (RCM) is a modern, non-invasive diagnostic method that enables real-time imaging of epidermis and upper layers of the dermis with a nearly histological precision and high contrast. The application of this technology in skin imaging in the last few years has resulted in the progress of dermatological diagnosis, providing virtual access to the living skin erasing the need for conventional histopathology. The RCM has a potential of wide application in the dermatological diagnostic process with a particular reference to benign and malignant skin tumors. This article provides a summary of the latest reports and previous achievements in the field of RCM application in the diagnostic process of skin neoplasms. A range of dermatological indications and general characteristics of confocal images in various types of tumors are presented. PMID:25610353

  20. Segmentation of vascular structures and hematopoietic cells in 3D microscopy images and quantitative analysis

    NASA Astrophysics Data System (ADS)

    Mu, Jian; Yang, Lin; Kamocka, Malgorzata M.; Zollman, Amy L.; Carlesso, Nadia; Chen, Danny Z.

    2015-03-01

    In this paper, we present image processing methods for quantitative study of how the bone marrow microenvironment changes (characterized by altered vascular structure and hematopoietic cell distribution) caused by diseases or various factors. We develop algorithms that automatically segment vascular structures and hematopoietic cells in 3-D microscopy images, perform quantitative analysis of the properties of the segmented vascular structures and cells, and examine how such properties change. In processing images, we apply local thresholding to segment vessels, and add post-processing steps to deal with imaging artifacts. We propose an improved watershed algorithm that relies on both intensity and shape information and can separate multiple overlapping cells better than common watershed methods. We then quantitatively compute various features of the vascular structures and hematopoietic cells, such as the branches and sizes of vessels and the distribution of cells. In analyzing vascular properties, we provide algorithms for pruning fake vessel segments and branches based on vessel skeletons. Our algorithms can segment vascular structures and hematopoietic cells with good quality. We use our methods to quantitatively examine the changes in the bone marrow microenvironment caused by the deletion of Notch pathway. Our quantitative analysis reveals property changes in samples with deleted Notch pathway. Our tool is useful for biologists to quantitatively measure changes in the bone marrow microenvironment, for developing possible therapeutic strategies to help the bone marrow microenvironment recovery.

  1. Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells.

    PubMed

    Itano, Michelle S; Bleck, Marina; Johnson, Daniel S; Simon, Sanford M

    2016-02-01

    Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions. PMID:26567131

  2. Clean localization super-resolution microscopy for 3D biological imaging

    NASA Astrophysics Data System (ADS)

    Mondal, Partha P.; Curthoys, Nikki M.; Hess, Samuel T.

    2016-01-01

    We propose clean localization microscopy (a variant of fPALM) using a molecule filtering technique. Localization imaging involves acquiring a large number of images containing single molecule signatures followed by one-to-one mapping to render a super-resolution image. In principle, this process can be repeated for other z-planes to construct a 3D image. But, single molecules observed from off-focal planes result in false representation of their presence in the focal plane, resulting in incorrect quantification and analysis. We overcome this with a single molecule filtering technique that imposes constraints on the diffraction limited spot size of single molecules in the image plane. Calibration with sub-diffraction size beads puts a natural cutoff on the actual diffraction-limited size of single molecules in the focal plane. This helps in distinguishing beads present in the focal plane from those in the off-focal planes thereby providing an estimate of the single molecules in the focal plane. We study the distribution of actin (labeled with a photoactivatable CAGE 552 dye) in NIH 3T3 mouse fibroblast cells.

  3. Real Time Gabor-Domain Optical Coherence Microscopy for 3D Imaging.

    PubMed

    Rolland, Jannick P; Canavesi, Cristina; Tankam, Patrice; Cogliati, Andrea; Lanis, Mara; Santhanam, Anand P

    2016-01-01

    Fast, robust, nondestructive 3D imaging is needed for the characterization of microscopic tissue structures across various clinical applications. A custom microelectromechanical system (MEMS)-based 2D scanner was developed to achieve, together with a multi-level GPU architecture, 55 kHz fast-axis A-scan acquisition in a Gabor-domain optical coherence microscopy (GD-OCM) custom instrument. GD-OCM yields high-definition micrometer-class volumetric images. A dynamic depth of focusing capability through a bio-inspired liquid lens-based microscope design, as in whales' eyes, was developed to enable the high definition instrument throughout a large field of view of 1 mm3 volume of imaging. Developing this technology is prime to enable integration within the workflow of clinical environments. Imaging at an invariant resolution of 2 μm has been achieved throughout a volume of 1 × 1 × 0.6 mm3, acquired in less than 2 minutes. Volumetric scans of human skin in vivo and an excised human cornea are presented. PMID:27046601

  4. Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells.

    PubMed

    Itano, Michelle S; Bleck, Marina; Johnson, Daniel S; Simon, Sanford M

    2016-02-01

    Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions.

  5. Blind deconvolution of 3D fluorescence microscopy using depth-variant asymmetric PSF.

    PubMed

    Kim, Boyoung; Naemura, Takeshi

    2016-06-01

    The 3D wide-field fluorescence microscopy suffers from depth-variant asymmetric blur. The depth-variance and axial asymmetry are due to refractive index mismatch between the immersion and the specimen layer. The radial asymmetry is due to lens imperfections and local refractive index inhomogeneities in the specimen. To obtain the PSF that has these characteristics, there were PSF premeasurement trials. However, they are useless since imaging conditions such as camera position and refractive index of the specimen are changed between the premeasurement and actual imaging. In this article, we focus on removing unknown depth-variant asymmetric blur in such an optical system under the assumption of refractive index homogeneities in the specimen. We propose finding few parameters in the mathematical PSF model from observed images in which the PSF model has a depth-variant asymmetric shape. After generating an initial PSF from the analysis of intensities in the observed image, the parameters are estimated based on a maximum likelihood estimator. Using the estimated PSF, we implement an accelerated GEM algorithm for image deconvolution. Deconvolution result shows the superiority of our algorithm in terms of accuracy, which quantitatively evaluated by FWHM, relative contrast, standard deviation values of intensity peaks and FWHM. Microsc. Res. Tech. 79:480-494, 2016. © 2016 Wiley Periodicals, Inc. PMID:27062314

  6. In situ 3D characterization of historical coatings and wood using multimodal nonlinear optical microscopy.

    PubMed

    Latour, Gaël; Echard, Jean-Philippe; Didier, Marie; Schanne-Klein, Marie-Claire

    2012-10-22

    We demonstrate multimodal nonlinear optical imaging of historical artifacts by combining Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence (2PEF) microscopies. We first identify the nonlinear optical response of materials commonly encountered in coatings of cultural heritage artifacts by analyzing one- and multi-layered model samples. We observe 2PEF signals from cochineal lake and sandarac and show that pigments and varnish films can be discriminated by exploiting their different emission spectral ranges as in luminescence linear spectroscopy. We then demonstrate SHG imaging of a filler, plaster, composed of bassanite particles which exhibit a non centrosymmetric crystal structure. We also show that SHG/2PEF imaging enables the visualization of wood microstructure through typically 60 µm-thick coatings by revealing crystalline cellulose (SHG signal) and lignin (2PEF signal) in the wood cell walls. Finally, in situ multimodal nonlinear imaging is demonstrated in a historical violin. SHG/2PEF imaging thus appears as a promising non-destructive and contactless tool for in situ 3D investigation of historical coatings and more generally for wood characterization and coating analysis at micrometer scale. PMID:23187225

  7. Single particle cryo-electron microscopy and 3-D reconstruction of viruses.

    PubMed

    Guo, Fei; Jiang, Wen

    2014-01-01

    With fast progresses in instrumentation, image processing algorithms, and computational resources, single particle electron cryo-microscopy (cryo-EM) 3-D reconstruction of icosahedral viruses has now reached near-atomic resolutions (3-4 Å). With comparable resolutions and more predictable outcomes, cryo-EM is now considered a preferred method over X-ray crystallography for determination of atomic structure of icosahedral viruses. At near-atomic resolutions, all-atom models or backbone models can be reliably built that allow residue level understanding of viral assembly and conformational changes among different stages of viral life cycle. With the developments of asymmetric reconstruction, it is now possible to visualize the complete structure of a complex virus with not only its icosahedral shell but also its multiple non-icosahedral structural features. In this chapter, we will describe single particle cryo-EM experimental and computational procedures for both near-atomic resolution reconstruction of icosahedral viruses and asymmetric reconstruction of viruses with both icosahedral and non-icosahedral structure components. Procedures for rigorous validation of the reconstructions and resolution evaluations using truly independent de novo initial models and refinements are also introduced.

  8. A resource from 3D electron microscopy of hippocampal neuropil for user training and tool development

    PubMed Central

    Harris, Kristen M.; Spacek, Josef; Bell, Maria Elizabeth; Parker, Patrick H.; Lindsey, Laurence F.; Baden, Alexander D.; Vogelstein, Joshua T.; Burns, Randal

    2015-01-01

    Resurgent interest in synaptic circuitry and plasticity has emphasized the importance of 3D reconstruction from serial section electron microscopy (3DEM). Three volumes of hippocampal CA1 neuropil from adult rat were imaged at X-Y resolution of ~2 nm on serial sections of ~50–60 nm thickness. These are the first densely reconstructed hippocampal volumes. All axons, dendrites, glia, and synapses were reconstructed in a cube (~10 μm3) surrounding a large dendritic spine, a cylinder (~43 μm3) surrounding an oblique dendritic segment (3.4 μm long), and a parallelepiped (~178 μm3) surrounding an apical dendritic segment (4.9 μm long). The data provide standards for identifying ultrastructural objects in 3DEM, realistic reconstructions for modeling biophysical properties of synaptic transmission, and a test bed for enhancing reconstruction tools. Representative synapses are quantified from varying section planes, and microtubules, polyribosomes, smooth endoplasmic reticulum, and endosomes are identified and reconstructed in a subset of dendrites. The original images, traces, and Reconstruct software and files are freely available and visualized at the Open Connectome Project (Data Citation 1). PMID:26347348

  9. Single Particle Cryo-electron Microscopy and 3-D Reconstruction of Viruses

    PubMed Central

    Guo, Fei; Jiang, Wen

    2014-01-01

    With fast progresses in instrumentation, image processing algorithms, and computational resources, single particle electron cryo-microscopy (cryo-EM) 3-D reconstruction of icosahedral viruses has now reached near-atomic resolutions (3–4 Å). With comparable resolutions and more predictable outcomes, cryo-EM is now considered a preferred method over X-ray crystallography for determination of atomic structure of icosahedral viruses. At near-atomic resolutions, all-atom models or backbone models can be reliably built that allow residue level understanding of viral assembly and conformational changes among different stages of viral life cycle. With the developments of asymmetric reconstruction, it is now possible to visualize the complete structure of a complex virus with not only its icosahedral shell but also its multiple non-icosahedral structural features. In this chapter, we will describe single particle cryo-EM experimental and computational procedures for both near-atomic resolution reconstruction of icosahedral viruses and asymmetric reconstruction of viruses with both icosahedral and non-icosahedral structure components. Procedures for rigorous validation of the reconstructions and resolution evaluations using truly independent de novo initial models and refinements are also introduced. PMID:24357374

  10. Determination of sex by exfoliative cytology using acridine orange confocal microscopy: A short study

    PubMed Central

    Reddy, D Shyam Prasad; Sherlin, Herald J; Ramani, Pratibha; Prakash, P Ajay

    2012-01-01

    Context: Establishing individuality is an imperative aspect in any investigation procedure. Sometimes, in identifying an individual, it becomes necessary to determine the sex of that particular individual. Combining rapidity with reliability, an innovative idea has been put forward using a confocal microscope in exfoliative cytology. In the present study, we have determined the sex of the individual from buccal mucosal scrapings. The exfoliative cells were observed for Barr bodies under a confocal microscope, and the percentage of Barr-body-positive cells was determined. Aims: The main objective of this study is to assess confocal microscopy for the determination of sex by observing Barr bodies in the exfoliative cells of both men and women. Settings and Design: Samples of buccal mucosa smears were made followed by acridine orange staining. The stained slides were observed under a confocal microscope and the data obtained was subjected for statistical analysis, especially for mean and standard deviation. Materials and Methods: Samples of buccal mucosa smears from 20 men and 20 women were obtained by scraping with flat wooden sticks (exfoliative cytology). The smears were fixed in 100% alcohol for 15 min, followed by acridine orange (AO) staining as described by Von Bertalanffy et al. Smears stained with AO were examined under a confocal microscope and the percentage of Barr-body-positive cells was determined. Statistical Analysis Used: Data obtained was subjected for statistical analysis, especially for mean and standard deviation. Results: Two non-overlapping ranges for the percentage of Barr-body-positive cells have been obtained for men and women. It was observed that in the male samples, the percentage of Barr-body-positive cells ranged from 0-3%. In the female samples, the percentage of Barr-body-positive cells ranged from 18-72%, and all the females showed the presence of Barr bodies. Conclusion: The study showed that the presence of Barr body in buccal

  11. 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

  12. Photon efficient double-helix PSF microscopy with application to 3D photo-activation localization imaging

    PubMed Central

    Grover, Ginni; Quirin, Sean; Fiedler, Callie; Piestun, Rafael

    2011-01-01

    We present a double-helix point spread function (DH-PSF) based three-dimensional (3D) microscope with efficient photon collection using a phase mask fabricated by gray-level lithography. The system using the phase mask more than doubles the efficiency of current liquid crystal spatial light modulator implementations. We demonstrate the phase mask DH-PSF microscope for 3D photo-activation localization microscopy (PM-DH-PALM) over an extended axial range. PMID:22076263

  13. Extraction of Individual Filaments from 2D Confocal Microscopy Images of Flat Cells.

    PubMed

    Basu, Saurav; Chi Liu; Rohde, Gustavo Kunde

    2015-01-01

    A crucial step in understanding the architecture of cells and tissues from microscopy images, and consequently explain important biological events such as wound healing and cancer metastases, is the complete extraction and enumeration of individual filaments from the cellular cytoskeletal network. Current efforts at quantitative estimation of filament length distribution, architecture and orientation from microscopy images are predominantly limited to visual estimation and indirect experimental inference. Here we demonstrate the application of a new algorithm to reliably estimate centerlines of biological filament bundles and extract individual filaments from the centerlines by systematically disambiguating filament intersections. We utilize a filament enhancement step followed by reverse diffusion based filament localization and an integer programming based set combination to systematically extract accurate filaments automatically from microscopy images. Experiments on simulated and real confocal microscope images of flat cells (2D images) show efficacy of the new method.

  14. Physicochemical composition of osteoporotic bone in the trichothiodystrophy premature aging mouse determined by confocal Raman microscopy.

    PubMed

    van Apeldoorn, Aart A; de Boer, Jan; van Steeg, Harry; Hoeijmakers, Jan H J; Otto, Cees; van Blitterswijk, Clemens A

    2007-01-01

    Although it has been established that premature aging trichothiodystrophy (TTD) mice display typical signs of osteoporosis, exact changes in physicochemical properties of these mice have not been elucidated. We used confocal Raman microscopy and histology to study femora of TTD mice. We measured femora isolated from xeroderma pigmentosum group A (XPA)/TTD double mutant mice to establish that Raman microscopy can be applied to measure differences in bone composition. Raman data from XPA/TTD mice showed remarkable changes in bone mineral composition. Moreover, we observed a severe form of osteoporosis, with strongly reduced cortical bone thickness. We used Raman microscopy to analyze bone composition in eight wild-type and eight TTD animals, and observed decreased levels of phosphate and carbonate in the cortex of femora isolated from TTD mice. In contrast, the bands representing the bone protein matrix were not affected in these mice.

  15. Analytic 3D imaging of mammalian nucleus at nanoscale using coherent x-rays and optical fluorescence microscopy.

    PubMed

    Song, Changyong; Takagi, Masatoshi; Park, Jaehyun; Xu, Rui; Gallagher-Jones, Marcus; Imamoto, Naoko; Ishikawa, Tetsuya

    2014-09-01

    Despite the notable progress that has been made with nano-bio imaging probes, quantitative nanoscale imaging of multistructured specimens such as mammalian cells remains challenging due to their inherent structural complexity. Here, we successfully performed three-dimensional (3D) imaging of mammalian nuclei by combining coherent x-ray diffraction microscopy, explicitly visualizing nuclear substructures at several tens of nanometer resolution, and optical fluorescence microscopy, cross confirming the substructures with immunostaining. This demonstrates the successful application of coherent x-rays to obtain the 3D ultrastructure of mammalian nuclei and establishes a solid route to nanoscale imaging of complex specimens.

  16. Plastic casts and confocal laser scanning microscopy applied to the observation of enamel tubules in the red Kangaroo (Macropus rufus).

    PubMed

    Suzuki, Masatsugu; Ushijima, Natsumi; Kohno, Ayako; Sawa, Yoshihiko; Yoshida, Shigemitsu; Sekikawa, Mitsuo; Ohtaishi, Noriyuki

    2003-03-01

    Scanning electron microscopy for plastic casts and confocal laser scanning microscopy for Villanueva bone-stained ground sections were used together to observe enamel tubules in red kangaroo molars. Although the tubular structures such as terminals, bends, expansions, splits, divergences and rejoinings in this species were within the variations of marsupial species, their morphological characteristics were demonstrated with extremely clear and persuasive images. Thus, the combined observations of plastic casts by scanning electron microscopy and Villanueva bone-stain sections by confocal laser scanning microscopy were found to be of value for the investigation of enamel tubules and tubular structures in other hard tissues.

  17. Confocal and electron microscopy to characterize sialoglycoconjugates in mouse sublingual gland acinar cells.

    PubMed

    Menghi, G; Bondi, A M; Marchetti, L; Ballarini, P; Materazzi, G

    1998-08-01

    Double lectin labeling for confocal microscopy and lectin-protein A-gold binding for electron microscopy were applied to the mouse sublingual gland in order to study surface and cytoplasmic sialoglycoconjugates. For this purpose, serially cut sections were submitted to sialidase followed by incubation with lectins recognizing usually acceptor sugars for terminal sialic acids. At the electron microscope level, the residues subtended to sialic acid were individually identified on adjacent sections by an indirect technique of labeling, whereas with confocal microscopy the above sugars were simultaneously visualized on the same section by a double staining method using fluorescein isothiocyanate (FITC)- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated lectins. Acinar cells were found to contain the terminal sequence sialic acid-beta-galactose in abundance while the sequence sialic acid-alpha-N-acetylgalactosamine appeared to be present in modest amounts. Both sialoglycoconjugates were homogeneously codistributed inside acinar cells. The combination with a saponification method also allowed the occurrence of C4 acetylated sialic acids linked to beta-galactose to be discovered, at the electron microscope level, on acinar cell secretory products.

  18. Probing the compressibility of tumor cell nuclei by combined atomic force-confocal microscopy.

    PubMed

    Krause, Marina; Te Riet, Joost; Wolf, Katarina

    2013-12-01

    The cell nucleus is the largest and stiffest organelle rendering it the limiting compartment during migration of invasive tumor cells through dense connective tissue. We here describe a combined atomic force microscopy (AFM)-confocal microscopy approach for measurement of bulk nuclear stiffness together with simultaneous visualization of the cantilever-nucleus contact and the fate of the cell. Using cantilevers functionalized with either tips or beads and spring constants ranging from 0.06-10 N m(-1), force-deformation curves were generated from nuclear positions of adherent HT1080 fibrosarcoma cell populations at unchallenged integrity, and a nuclear stiffness range of 0.2 to 2.5 kPa was identified depending on cantilever type and the use of extended fitting models. Chromatin-decondensating agent trichostatin A (TSA) induced nuclear softening of up to 50%, demonstrating the feasibility of our approach. Finally, using a stiff bead-functionalized cantilever pushing at maximal system-intrinsic force, the nucleus was deformed to 20% of its original height which after TSA treatment reduced further to 5% remaining height confirming chromatin organization as an important determinant of nuclear stiffness. Thus, combined AFM-confocal microscopy is a feasible approach to study nuclear compressibility to complement concepts of limiting nuclear deformation in cancer cell invasion and other biological processes.

  19. Scanning a microhabitat: plant-microbe interactions revealed by confocal laser microscopy

    PubMed Central

    Cardinale, Massimiliano

    2014-01-01

    No plant or cryptogam exists in nature without microorganisms associated with its tissues. Plants as microbial hosts are puzzles of different microhabitats, each of them colonized by specifically adapted microbiomes. The interactions with such microorganisms have drastic effects on the host fitness. Since the last 20 years, the combination of microscopic tools and molecular approaches contributed to new insights into microbe-host interactions. Particularly, confocal laser scanning microscopy (CLSM) facilitated the exploration of microbial habitats and allowed the observation of host-associated microorganisms in situ with an unprecedented accuracy. Here I present an overview of the progresses made in the study of the interactions between microorganisms and plants or plant-like organisms, focusing on the role of CLSM for the understanding of their significance. I critically discuss risks of misinterpretation when procedures of CLSM are not properly optimized. I also review approaches for quantitative and statistical analyses of CLSM images, the combination with other molecular and microscopic methods, and suggest the re-evaluation of natural autofluorescence. In this review, technical aspects were coupled with scientific outcomes, to facilitate the readers in identifying possible CLSM applications in their research or to expand their existing potential. The scope of this review is to highlight the importance of confocal microscopy in the study of plant-microbe interactions and also to be an inspiration for integrating microscopy with molecular techniques in future researches of microbial ecology. PMID:24639675

  20. Probing the compressibility of tumor cell nuclei by combined atomic force-confocal microscopy

    NASA Astrophysics Data System (ADS)

    Krause, Marina; te Riet, Joost; Wolf, Katarina

    2013-12-01

    The cell nucleus is the largest and stiffest organelle rendering it the limiting compartment during migration of invasive tumor cells through dense connective tissue. We here describe a combined atomic force microscopy (AFM)-confocal microscopy approach for measurement of bulk nuclear stiffness together with simultaneous visualization of the cantilever-nucleus contact and the fate of the cell. Using cantilevers functionalized with either tips or beads and spring constants ranging from 0.06-10 N m-1, force-deformation curves were generated from nuclear positions of adherent HT1080 fibrosarcoma cell populations at unchallenged integrity, and a nuclear stiffness range of 0.2 to 2.5 kPa was identified depending on cantilever type and the use of extended fitting models. Chromatin-decondensating agent trichostatin A (TSA) induced nuclear softening of up to 50%, demonstrating the feasibility of our approach. Finally, using a stiff bead-functionalized cantilever pushing at maximal system-intrinsic force, the nucleus was deformed to 20% of its original height which after TSA treatment reduced further to 5% remaining height confirming chromatin organization as an important determinant of nuclear stiffness. Thus, combined AFM-confocal microscopy is a feasible approach to study nuclear compressibility to complement concepts of limiting nuclear deformation in cancer cell invasion and other biological processes.

  1. Sugarcane cell wall structure and lignin distribution investigated by confocal and electron microscopy.

    PubMed

    Sant'Anna, Celso; Costa, Lilian T; Abud, Yuri; Biancatto, Lucas; Miguens, Flávio Costa; de Souza, Wanderley

    2013-08-01

    Lignocellulosic plant cell wall is considered a potential source for second generation biofuels. The plant cell wall is a highly complex structure mainly composed of cellulose, hemicelluloses, and lignin that form a network of crosslinked fibers. The structural organization of the sugarcane cell wall has not been previously analyzed in detail, and this analysis is a prerequisite for further studies on the recalcitrance and deconstruction of its biomass. In this work, cellulose and lignin localization were investigated by confocal laser scanning microscopy. In addition, the internode sugarcane cell wall structural organization was analyzed by electron microscopy. Internode stem anatomy showed a typical monocot structure consisting of epidermis, hypoderm, and vascular bundles scattered throughout ground parenchyma tissue and surrounded by sclerenchyma fibers. Confocal images of safranin labeled sugarcane showed that lignin distribution was predominant in the vessel elements, cell wall corners (CC), and middle lamella (ML), while cellulose-rich cell walls were randomly distributed in the ML and organized in the other cell wall layers. KMnO4 cytochemistry revealed that lignin was predominantly distributed in secondary cell walls, ML and CC. Cell wall sublayers (S1, S2, and S3) were identified and measured by transmission electron microscopy. Our results provide insights that may help further understanding of sugarcane cell wall organization, which is crucial for the research and technology of plant-based biofuel production. PMID:23733560

  2. Quantitative 3D elemental analysis inside plant roots by means of synchrotron confocal micro X-ray fluorescence

    NASA Astrophysics Data System (ADS)

    Terzano, R.; Vekemans, B.; Tomasi, N.; Spagnuolo, M.; Schoonjans, T.; Vincze, L.; Pinton, R.; Cesco, S.; Ruggiero, P.

    2009-04-01

    The knowledge of the distribution and concentration of elements within plants is a fundamental step to better understand how these plants uptake specific elements from the medium of growth and how they manage acquisition and compartmentalisation of nutrients as well as toxic metals. For some elements, either nutrients or toxicants, it can be of relevance to know their concentration level within microscopic volumes in plant organs, where they are stored or accumulated. Usually, this type of microscopic analysis requires complex cutting procedures and extensive sample manipulations. In this research, the technique of synchrotron micro X-ray fluorescence in the confocal mode was applied to image the distribution of elements in selected key-planes of tomato roots without the need of any sample preparation, except washing and freeze-drying. Using this method, a first polycapillary lens focussed the X-ray beam with an energy of 12.4 keV down to a 20 µm beam that is penetrating the sample, and a second polycapillary half-lens, that was positioned at the detection side at 90 degrees to the first polycapillary, could then restrict further the view on this irradiated volume to a defined microscopic volume (typically 20x20x20 µm3) from which the induced fluorescent radiation is finally collected by the energy dispersive detector. In this way, it was possible to investigate the concentration levels of some elements such as K, Ca, Mn, Fe, Cu and Zn within the roots of tomato plants. The quantification was performed by means of a dedicated XRF Fundamental Parameter (FP) method in order to calculate the concentrations of trace elements within the analysed plants. Utilizing fundamental atomic parameters, the applied FP method is taking into account the influence of sample self-absorption and especially the specific detection processes by the polycapillary lens. Quantification was assessed and validated by using different standards: NIST SRM 1573a (trace elements in tomato leaves

  3. Characterization of microporous membranes using confocal scanning laser microscopy in fluorescence mode

    NASA Astrophysics Data System (ADS)

    Charcosset, C.; Bernengo, J.-C.

    2000-12-01

    Confocal Scanning Laser Microscopy (CSLM) in fluorescence mode was used to characterize microporous membranes. Two microfiltration membranes were investigated: a mixed ester (cellulose nitrate/cellulose acetate) 1.2 μm-rated membrane and a polycarbonate track-etched membrane with cylindrical pores of 2 μm diameter. Optical sections of the membranes stained with rhodamine and mounted in glycerol were performed at 1 μm intervals, from 0 to 10 μm. CSLM was found useful for microporous membrane characterization, as it gives some insight into bulk membrane morphology.

  4. Fluorescence spectroscopy and confocal microscopy of the mycotoxin citrinin in condensed phase and hydrogel films.

    PubMed

    Lauer, Milena H; Gehlen, Marcelo H; de Jesus, Karen; Berlinck, Roberto G S

    2014-05-01

    The emission spectra, quantum yields and fluorescence lifetimes of citrinin in organic solvents and hydrogel films have been determined. Citrinin shows complex fluorescence decays due to the presence of two tautomers in solution and interconversion from excited-state double proton transfer (ESDPT) process. The fluorescence decay times associated with the two tautomers have values near 1 and 5 ns depending on the medium. In hydrogel films of agarose and alginate, fluorescence imaging showed that citrinin is not homogeneously dispersed and highly emissive micrometer spots may be formed. Fluorescence spectrum and decay analysis are used to recognize the presence of citrinin in hydrogel films using confocal fluorescence microscopy and spectroscopy.

  5. Label-free detection of anticancer drug paclitaxel in living cells by confocal Raman microscopy

    NASA Astrophysics Data System (ADS)

    Salehi, H.; Derely, L.; Vegh, A.-G.; Durand, J.-C.; Gergely, C.; Larroque, C.; Fauroux, M.-A.; Cuisinier, F. J. G.

    2013-03-01

    Confocal Raman microscopy, a non-invasive, label-free, and high spatial resolution imaging technique is employed to trace the anticancer drug paclitaxel in living Michigan Cancer Foundation-7 (MCF-7) cells. The Raman images were treated by K-mean cluster analysis to detect the drug in cells. Distribution of paclitaxel in cells is verified by calculating the correlation coefficient between the reference spectrum of the drug and the whole Raman image spectra. A time dependent gradual diffusion of paclitaxel all over the cell is observed suggesting a complementary picture of the pharmaceutical action of this drug based on rapid binding of free tubulin to crystallized paclitaxel.

  6. 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.

  7. Spectrally encoded slit confocal microscopy using a wavelength-swept laser

    NASA Astrophysics Data System (ADS)

    Kim, Soocheol; Hwang, Jaehyun; Heo, Jung; Ryu, Suho; Lee, Donghak; Kim, Sang-Hoon; Oh, Seung Jae; Joo, Chulmin

    2015-03-01

    We present an implementation of spectrally encoded slit confocal microscopy. The method employs a rapid wavelength-swept laser as the light source and illuminates a specimen with a line focus that scans through the specimen as the wavelength sweeps. The reflected light from the specimen is imaged with a stationary line scan camera, in which the finite pixel height serves as a slit aperture. This scanner-free operation enables a simple and cost-effective implementation in a small form factor, while allowing for the three-dimensional imaging of biological samples.

  8. Amiodarone-Induced Multiorgan Toxicity with Ocular Findings on Confocal Microscopy

    PubMed Central

    Turk, Ugur; Turk, Bengu Gerceker; Yılmaz, Suzan Guven; Tuncer, Esref; Alioğlu, Emin; Dereli, Tugrul

    2015-01-01

    Amiodarone is an antiarrhythmic medication that can adversely effect various organs including lungs, thyroid gland, liver, eyes, skin, and nerves. The risk of adverse effects increases with high doses and prolonged use. We report a 54-year-old female who presented with multiorgan toxicity after 8 months of low dose (200 mg/day) amiodarone treatment. The findings of confocal microscopy due to amiodarone-induced keratopathy are described. Amiodarone may cause multiorgan toxicity even at lower doses and for shorter treatment periods. PMID:25949090

  9. The application of dermal papillary rings in dermatology by in vivo confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Xiang, W. Z.; Xu, A. E.; Xu, J.; Bi, Z. G.; Shang, Y. B.; Ren, Q. S.

    2010-08-01

    Confocal laser scanning microscopy (CLSM) allows noninvasive visualization of human skin in vivo, without needing to fix or section the tissue. Melanocytes and pigmented keratinocytes at the level of the basal layer form bright dermal papillary rings which are readily amenable to identify in confocal images. Our purpose was to explore the role of dermal papillary rings in assessment of lesion location, the diagnosis, differential diagnosis of lesions and assessment of therapeutic efficacy by in vivo CLSM. Seventy-one patients were imaged with the VivaScope 1500 reflectance confocal microscope provided by Lucid, Inc. The results indicate that dermal papillary rings can assess the location of lesion; the application of dermal papillary rings can provide diagnostic support and differential diagnosis for vitiligo, nevus depigmentosus, tinea versicolor, halo nevus, common nevi, and assess the therapeutic efficacy of NBUVB phototherapy plus topical 0.1 percent tacrolimus ointment for vitiligo. In conclusion, our findings indicate that the dermal papillary rings play an important role in the assessment the location of lesion, diagnosis, differential diagnosis of lesions and assessment of therapeutic efficacy by in vivo CLSM. CLSM may be a promising tool for noninvasive examination in dermatology. However, larger studies are needed to expand the application of dermal papillary rings in dermatology.

  10. Simultaneous confocal fluorescence microscopy and optical coherence tomography for drug distribution and tissue integrity assessment

    NASA Astrophysics Data System (ADS)

    Rinehart, Matthew T.; LaCroix, Jeffrey; Henderson, Marcus; Katz, David; Wax, Adam

    2011-03-01

    The effectiveness of microbicidal gels, topical products developed to prevent infection by sexually transmitted diseases including HIV/AIDS, is governed by extent of gel coverage, pharmacokinetics of active pharmaceutical ingredients (APIs), and integrity of vaginal epithelium. While biopsies provide localized information about drug delivery and tissue structure, in vivo measurements are preferable in providing objective data on API and gel coating distribution as well as tissue integrity. We are developing a system combining confocal fluorescence microscopy with optical coherence tomography (OCT) to simultaneously measure local concentrations and diffusion coefficients of APIs during transport from microbicidal gels into tissue, while assessing tissue integrity. The confocal module acquires 2-D images of fluorescent APIs multiple times per second allowing analysis of lateral diffusion kinetics. The custom Fourier domain OCT module has a maximum a-scan rate of 54 kHz and provides depth-resolved tissue integrity information coregistered with the confocal fluorescence measurements. The combined system is validated by imaging phantoms with a surrogate fluorophore. Time-resolved API concentration measured at fixed depths is analyzed for diffusion kinetics. This multimodal system will eventually be implemented in vivo for objective evaluation of microbicide product performance.

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

    PubMed Central

    Tilli, Maddalena T; Parrish, Angela R; Cotarla, Ion; Jones, Laundette P; Johnson, Michael D; Furth, Priscilla A

    2008-01-01

    Background Genetically engineered mouse models of mammary gland cancer enable the in vivo study of molecular mechanisms and signaling during development and cancer pathophysiology. However, traditional whole mount and histological imaging modalities are only applicable to non-viable tissue. Methods We evaluated three techniques that can be quickly applied to living tissue for imaging normal and cancerous mammary gland: reflectance confocal microscopy, green fluorescent protein imaging, and ultrasound imaging. Results In the current study, reflectance confocal imaging offered the highest resolution and was used to optically section mammary ductal structures in the whole mammary gland. Glands remained viable in mammary gland whole organ culture when 1% acetic acid was used as a contrast agent. Our application of using green fluorescent protein expressing transgenic mice in our study allowed for whole mammary gland ductal structures imaging and enabled straightforward serial imaging of mammary gland ducts in whole organ culture to visualize the growth and differentiation process. Ultrasound imaging showed the lowest resolution. However, ultrasound was able to detect mammary preneoplastic lesions 0.2 mm in size and was used to follow cancer growth with serial imaging in living mice. Conclusion In conclusion, each technique enabled serial imaging of living mammary tissue and visualization of growth and development, quickly and with minimal tissue preparation. The use of the higher resolution reflectance confocal and green fluorescent protein imaging techniques and lower resolution ultrasound were complementary. PMID:18215290

  12. Robust Adaptive 3-D Segmentation of Vessel Laminae From Fluorescence Confocal Microscope Images and Parallel GPU Implementation

    PubMed Central

    Narayanaswamy, Arunachalam; Dwarakapuram, Saritha; Bjornsson, Christopher S.; Cutler, Barbara M.; Shain, William

    2010-01-01

    This paper presents robust 3-D algorithms to segment vasculature that is imaged by labeling laminae, rather than the lumenal volume. The signal is weak, sparse, noisy, nonuniform, low-contrast, and exhibits gaps and spectral artifacts, so adaptive thresholding and Hessian filtering based methods are not effective. The structure deviates from a tubular geometry, so tracing algorithms are not effective. We propose a four step approach. The first step detects candidate voxels using a robust hypothesis test based on a model that assumes Poisson noise and locally planar geometry. The second step performs an adaptive region growth to extract weakly labeled and fine vessels while rejecting spectral artifacts. To enable interactive visualization and estimation of features such as statistical confidence, local curvature, local thickness, and local normal, we perform the third step. In the third step, we construct an accurate mesh representation using marching tetrahedra, volume-preserving smoothing, and adaptive decimation algorithms. To enable topological analysis and efficient validation, we describe a method to estimate vessel centerlines using a ray casting and vote accumulation algorithm which forms the final step of our algorithm. Our algorithm lends itself to parallel processing, and yielded an 8× speedup on a graphics processor (GPU). On synthetic data, our meshes had average error per face (EPF) values of (0.1–1.6) voxels per mesh face for peak signal-to-noise ratios from (110–28 dB). Separately, the error from decimating the mesh to less than 1% of its original size, the EPF was less than 1 voxel/face. When validated on real datasets, the average recall and precision values were found to be 94.66% and 94.84%, respectively. PMID:20199906

  13. Visualising the 3D Structure of Fine-Grained Estuarine Sediments; Preliminary Interpretations of a Novel Dataset Obtained via Volume Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Wheatland, Jonathan; Bushby, Andy; Spencer, Kate; Carr, Simon

    2014-05-01

    Accurate measurement of the physical characteristics of sediment are critical to determining sediment transport behaviour and the stability of settled deposits. The properties (e.g. particle size, density, and settling velocity) of coarse-grained sediments (> 63 μm φ) can be easily characterised, hence their behaviour is relatively simple to predict and model. However, due to their small size and tendency to interact with their surrounding medium, the characteristics of fine sediments (< 63 μm φ) and their behaviour during transportation, deposition and consolidation is poorly understood. Recent studies have used correlative microscopy, a multi-method technique combining scanning confocal laser microscopy (SCLM), conventional optical microscopy (COM), and transmission electron microscopy (TEM), to characterise fine sediments at both the gross (> 1 μm) and sub-micron scale (Droppo et al., 1996). Whilst this technique has proven insightful, the measurement of geometric properties (e.g. the shape of primary particles and their spatial arrangement) can only be achieved by three-dimensional (3D) analysis and the scale of observation for e.g. TEM does not overlap with those techniques used to characterise sediments at larger scales (100s to 1000s microns) (e.g. video analysis). Volume electron microscopy [or focused ion beam scanning electron microscopy (FIB-SEM)] provides 3D analysis at scales of 10s to 1000s microns and though widely used in cell biology, has not been used to observe sediment. FIB-SEM requires samples that are vacuum stable and a key challenge will be to capture fragile, hydrated sediment samples whilst preserving their structural integrity. The aims of this work are therefore: 1) to modify preparation techniques currently used in cell biology for the stabilization of sedimentary materials; 2) to acquire 3D datasets for both fragile suspended sediments (flocs) and consolidated bed sediments and 3) to interpret the 3D structure of these samples. In

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

    PubMed Central

    Martial, Franck P.; Hartell, Nicholas A.

    2012-01-01

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

  15. One shot confocal microscopy based on wavelength/space conversion by use of multichannel spectrometer

    NASA Astrophysics Data System (ADS)

    Miyamoto, Shuji; Hase, Eiji; Ichikawa, Ryuji; Mnamikawa, Takeo; Yasui, Takeshi; Yamamoto, Hirotugu

    2016-03-01

    Confocal laser microscope (CLM) has been widely used in the fields of the non-contact surface topography, biomedical imaging, and other applications, because of two-dimensional (2D) or three-dimensional (3D) imaging capability with the confocal effect and the stray light elimination. Although the conventional CLM has acquired the 2D image by mechanical scanning of the focused beam spot, further reduction of image acquisition time and the robustness to various disturbances are strongly required. To this end, it is essential to omit mechanical scanning for the image acquisition. In this article, we developed the scan-less, full-field CLM by combination of the line-focused CLM with the wavelength/1D-space conversion. This combination enables us to form the 2D focal array of a 2D rainbow beam on a sample and to encode the 2D image information of a sample on the 2D rainbow beam. The image-encoded 2D rainbow beam was decoded as a spectral line image by a multi-channel spectrometer equipped with a CMOS camera without the need for the mechanical scanning. The confocal full-field image was acquired during 0.23 ms with the lateral resolution of 26.3μm and 4.9μm for the horizontal and vertical directions, respectively, and the depth resolution of 34.9μm. We further applied this scan-less, full-field CLM for biomedical imaging of a sliced specimen and non-contact surface topography of an industry products. These demonstrations highlight a high potential of the proposed scan-less, full-field CLM.

  16. Examination of heterogeneous crossing sequences between toner and rollerball pen strokes by digital microscopy and 3-D laser profilometry.

    PubMed

    Montani, Isabelle; Mazzella, Williams; Guichard, Marion; Marquis, Raymond

    2012-07-01

    The determination of line crossing sequences between rollerball pens and laser printers presents difficulties that may not be overcome using traditional techniques. This research aimed to study the potential of digital microscopy and 3-D laser profilometry to determine line crossing sequences between a toner and an aqueous ink line. Different paper types, rollerball pens, and writing pressure were tested. Correct opinions of the sequence were given for all case scenarios, using both techniques. When the toner was printed before the ink, a light reflection was observed in all crossing specimens, while this was never observed in the other sequence types. The 3-D laser profilometry, more time-consuming, presented the main advantage of providing quantitative results. The findings confirm the potential of the 3-D laser profilometry and demonstrate the efficiency of digital microscopy as a new technique for determining the sequence of line crossings involving rollerball pen ink and toner. PMID:22390180

  17. Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy.

    PubMed

    Huang, Chao; Sachse, Frank B; Hitchcock, Robert W; Kaza, Aditya K

    2016-01-01

    Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000). We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81) and nodal tissue (n = 81). In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2 ± 0.3% and 98.0 ± 0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2 ± 0.3% and 94.0 ± 2.4%, respectively). Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease. PMID:26808149

  18. Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy.

    PubMed

    Huang, Chao; Sachse, Frank B; Hitchcock, Robert W; Kaza, Aditya K

    2016-01-01

    Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000). We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81) and nodal tissue (n = 81). In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2 ± 0.3% and 98.0 ± 0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2 ± 0.3% and 94.0 ± 2.4%, respectively). Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease.

  19. Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy

    PubMed Central

    Huang, Chao; Sachse, Frank B.; Hitchcock, Robert W.; Kaza, Aditya K.

    2016-01-01

    Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000). We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81) and nodal tissue (n = 81). In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2±0.3% and 98.0±0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2±0.3% and 94.0±2.4%, respectively). Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease. PMID:26808149

  20. Combined ion conductance and fluorescence confocal microscopy for biological cell membrane transport studies

    NASA Astrophysics Data System (ADS)

    Shevchuk, A. I.; Novak, P.; Velazquez, M. A.; Fleming, T. P.; Korchev, Y. E.

    2013-09-01

    Optical visualization of nanoscale morphological changes taking place in living biological cells during such important processes as endo- and exocytosis is challenging due to the low refractive index of lipid membranes. In this paper we summarize and discuss advances in the powerful combination of two complementary live imaging techniques, ion conductance and fluorescence confocal microscopy, that allows cell membrane topography to be related with molecular-specific fluorescence at high spatial and temporal resolution. We demonstrate the feasibility of the use of ion conductance microscopy to image apical plasma membrane of mouse embryo trophoblast outgrowth cells at a resolution sufficient to depict single endocytic pits. This opens the possibility to study individual endocytic events in embryo trophoblast outgrowth cells where endocytosis plays a crucial role during early stages of embryo development.

  1. Dye-enhanced multimodal confocal microscopy for noninvasive detection of skin cancers in mouse models

    NASA Astrophysics Data System (ADS)

    Park, Jesung; Mroz, Pawel; Hamblin, Michael R.; Yaroslavsky, Anna N.

    2010-03-01

    Skin cancer is the most common form of human cancer. Its early diagnosis and timely treatment is of paramount importance for dermatology and surgical oncology. In this study, we evaluate the use of reflectance and fluorescence confocal microscopy for detecting skin cancers in an in-vivo trial with B16F10 melanoma and SCCVII squamous cell carcinoma in mice. For the experiments, the mice are anesthetized, then the tumors are infiltrated with aqueous solution of methylene blue and imaged. Reflectance images are acquired at 658 nm. Fluorescence is excited at 658 nm and registered in the range between 690 and 710 nm. After imaging, the mice are sacrificed. The tumors are excised and processed for hematoxylin and eosin histopathology, which is compared to the optical images. The results of the study indicate that in-vivo reflectance images provide valuable information on vascularization of the tumor, whereas the fluorescence images mimic the structural features seen in histopathology. Simultaneous dye-enhanced reflectance and fluorescence confocal microscopy shows promise for the detection, demarcation, and noninvasive monitoring of skin cancer development.

  2. 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.

  3. Confocal Raman microscopy and fluorescent in situ hybridization - A complementary approach for biofilm analysis.

    PubMed

    Kniggendorf, Ann-Kathrin; Nogueira, Regina; Kelb, Christian; Schadzek, Patrik; Meinhardt-Wollweber, Merve; Ngezahayo, Anaclet; Roth, Bernhard

    2016-10-01

    We combine confocal Raman microscopy (CRM) of wet samples with subsequent Fluorescent in situ hybridization (FISH) without significant limitations to either technique for analyzing the same sample of a microbial community on a cell-to-cell basis. This combination of techniques allows a much deeper, more complete understanding of complex environmental samples than provided by either technique alone. The minimalistic approach is based on laboratory glassware with micro-engravings for reproducible localization of the sample at cell scale combined with a fixation and de- and rehydration protocol for the respective techniques. As proof of concept, we analyzed a floc of nitrifying activated sludge, demonstrating that the sample can be tracked with cell-scale precision over different measurements and instruments. The collected information includes the microbial content, spatial shape, variant chemical compositions of the floc matrix and the mineral microparticles embedded within. In addition, the direct comparison of CRM and FISH revealed a difference in reported cell size due to the different cell components targeted by the respective technique. To the best of our knowledge, this is the first report of a direct cell-to-cell comparison of confocal Raman microscopy and Fluorescent in situ hybridization analysis performed on the same sample. An adaptation of the method to include native samples as a starting point is planned for the near future. The micro-engraving approach itself also opens up the possibility of combining other, functionally incompatible techniques as required for further in-depth investigations of low-volume samples. PMID:27423128

  4. In vivo fluorescence confocal microscopy: indocyanine green enhances the contrast of epidermal and dermal structures

    NASA Astrophysics Data System (ADS)

    Skvara, Hans; Kittler, Harald; Schmid, Johannes A.; Plut, Ulrike; Jonak, Constanze

    2011-09-01

    In recent years, in vivo skin imaging devices have been successfully implemented in skin research as well as in clinical routine. Of particular importance is the use of reflectance confocal microscopy (RCM) and fluorescence confocal microscopy (FCM) that enable visualization of the tissue with a resolution comparable to histology. A newly developed commercially available multi-laser device in which both technologies are integrated now offers the possibility to directly compare RCM with FCM. The fluorophore indocyanine green (ICG) was intradermally injected into healthy forearm skin of 10 volunteers followed by in vivo imaging at various time points. In the epidermis, accurate assessment of cell morphology with FCM was supplemented by identification of pigmented cells and structures with RCM. In dermal layers, only with FCM connective tissue fibers were clearly contoured down to a depth of more than 100 μm. The fluorescent signal still provided a favorable image contrast 24 and 48 hours after injection. Subsequently, ICG was applied to different types of skin diseases (basal cell carcinoma, actinic keratosis, seborrhoeic keratosis, and psoriasis) in order to demonstrate the diagnostic benefit of FCM when directly compared with RCM. Our data suggest a great impact of FCM in combination with ICG on clinical and experimental dermatology in the future.

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

    PubMed

    Alves da Rocha, Roney; Paiva, Igor Moura; Anjos, Virgílio; Furtado, Marco Antônio Moreira; Bell, Maria José Valenzuela

    2015-06-01

    In this work, we assessed the use of confocal Raman microscopy and artificial neural network as a practical method to assess and quantify adulteration of fluid milk by addition of whey. Milk samples with added whey (from 0 to 100%) were prepared, simulating different levels of fraudulent adulteration. All analyses were carried out by direct inspection at the light microscope after depositing drops from each sample on a microscope slide and drying them at room temperature. No pre- or posttreatment (e.g., sample preparation or spectral correction) was required in the analyses. Quantitative determination of adulteration was performed through a feed-forward artificial neural network (ANN). Different ANN configurations were evaluated based on their coefficient of determination (R2) and root mean square error values, which were criteria for selecting the best predictor model. In the selected model, we observed that data from both training and validation subsets presented R2>99.99%, indicating that the combination of confocal Raman microscopy and ANN is a rapid, simple, and efficient method to quantify milk adulteration by whey. Because sample preparation and postprocessing of spectra were not required, the method has potential applications in health surveillance and food quality monitoring.

  6. Determination of nitric oxide mediating intracellular Ca2+ release on neurons based on confocal microscopy imaging

    NASA Astrophysics Data System (ADS)

    Zheng, Liqin; Wang, Yuhua; He, Yipeng; Zeng, Yixiu; Zhang, Yanding; Xie, Shusen

    2014-09-01

    The gas NO is a ubiquitous intercellular messenger that modulates a wide range of physiological and pathophysiological functions. But few studies were made to study the role of NO in the Ca2+ release in dorsal root ganglion (DRG) neurons by confocal microscopy. Thus the objective of this study was to assess if NO has a role in Ca2+ signaling in DRG neurons using confocal microscopy combined with special fluorescence probe Fluo-3/AM. A 100 μM concentration of the NO donors (Sodium Nitroprusside, Dihydrate, SNP) and NO synthase inhibitor (NG-Monomethyl-L-arginine, Monoacetate salt, L-NMMA) was used in the study. Results showed that the fluorescence intensity increased rapidly after injecting SNP, which indicated that SNP could enhance intracellular Ca2+ release. And the fluorescence intensity shrank gradually with time and kept at a low level for quite a long period after loading with L-NMMA which indicated that L-NMMA could block intracellular Ca2+ release. All these results demonstrated that NO was involved in the regulation of intracellular Ca2+ release in the DRG neurons.

  7. Three-dimensional simultaneous optical coherence tomography and confocal fluorescence microscopy for investigation of lung tissue

    NASA Astrophysics Data System (ADS)

    Gaertner, Maria; Cimalla, Peter; Meissner, Sven; Kuebler, Wolfgang M.; Koch, Edmund

    2012-07-01

    Although several strategies exist for a minimal-invasive treatment of patients with lung failure, the mortality rate of acute respiratory distress syndrome still reaches 30% at minimum. This striking number indicates the necessity of understanding lung dynamics on an alveolar level. To investigate the dynamical behavior on a microscale, we used three-dimensional geometrical and functional imaging to observe tissue parameters including alveolar size and length of embedded elastic fibers during ventilation. We established a combined optical coherence tomography (OCT) and confocal fluorescence microscopy system that is able to monitor the distension of alveolar tissue and elastin fibers simultaneously within three dimensions. The OCT system can laterally resolve a 4.9 μm line pair feature and has an approximately 11 μm full-width-half-maximum axial resolution in air. confocal fluorescence microscopy visualizes molecular properties of the tissue with a resolution of 0.75 μm (laterally), and 5.9 μm (axially) via fluorescence detection of the dye sulforhodamine B specifically binding to elastin. For system evaluation, we used a mouse model in situ to perform lung distension by application of different constant pressure values within the physiological regime. Our method enables the investigation of alveolar dynamics by helping to reveal basic processes emerging during artificial ventilation and breathing.

  8. In vivo corneal confocal microscopy in diabetes: Where we are and where we can get.

    PubMed

    Maddaloni, Ernesto; Sabatino, Francesco

    2016-09-15

    In vivo corneal confocal microscopy (IVCCM) is a novel, reproducible, easy and noninvasive technique that allows the study of the different layers of the cornea at a cellular level. As cornea is the most innervated organ of human body, several studies investigated the use of corneal confocal microscopy to detect diabetic neuropathies, which are invalidating and deadly complications of diabetes mellitus. Corneal nerve innervation has been shown impaired in subjects with diabetes and a close association between damages of peripheral nerves due to the diabetes and alterations in corneal sub-basal nerve plexus detected by IVCCM has been widely demonstrated. Interestingly, these alterations seem to precede the clinical onset of diabetic neuropathies, paving the path for prevention studies. However, some concerns still prevent the full implementation of this technique in clinical practice. In this review we summarize the most recent and relevant evidences about the use of IVCCM for the diagnosis of peripheral sensorimotor polyneuropathy and of autonomic neuropathy in diabetes. New perspectives and current limitations are also discussed. PMID:27660697

  9. In vivo corneal confocal microscopy in diabetes: Where we are and where we can get

    PubMed Central

    Maddaloni, Ernesto; Sabatino, Francesco

    2016-01-01

    In vivo corneal confocal microscopy (IVCCM) is a novel, reproducible, easy and noninvasive technique that allows the study of the different layers of the cornea at a cellular level. As cornea is the most innervated organ of human body, several studies investigated the use of corneal confocal microscopy to detect diabetic neuropathies, which are invalidating and deadly complications of diabetes mellitus. Corneal nerve innervation has been shown impaired in subjects with diabetes and a close association between damages of peripheral nerves due to the diabetes and alterations in corneal sub-basal nerve plexus detected by IVCCM has been widely demonstrated. Interestingly, these alterations seem to precede the clinical onset of diabetic neuropathies, paving the path for prevention studies. However, some concerns still prevent the full implementation of this technique in clinical practice. In this review we summarize the most recent and relevant evidences about the use of IVCCM for the diagnosis of peripheral sensorimotor polyneuropathy and of autonomic neuropathy in diabetes. New perspectives and current limitations are also discussed.

  10. Adaptive and Background-Aware GAL4 Expression Enhancement of Co-registered Confocal Microscopy Images.

    PubMed

    Trapp, Martin; Schulze, Florian; Novikov, Alexey A; Tirian, Laszlo; J Dickson, Barry; Bühler, Katja

    2016-04-01

    GAL4 gene expression imaging using confocal microscopy is a common and powerful technique used to study the nervous system of a model organism such as Drosophila melanogaster. Recent research projects focused on high throughput screenings of thousands of different driver lines, resulting in large image databases. The amount of data generated makes manual assessment tedious or even impossible. The first and most important step in any automatic image processing and data extraction pipeline is to enhance areas with relevant signal. However, data acquired via high throughput imaging tends to be less then ideal for this task, often showing high amounts of background signal. Furthermore, neuronal structures and in particular thin and elongated projections with a weak staining signal are easily lost. In this paper we present a method for enhancing the relevant signal by utilizing a Hessian-based filter to augment thin and weak tube-like structures in the image. To get optimal results, we present a novel adaptive background-aware enhancement filter parametrized with the local background intensity, which is estimated based on a common background model. We also integrate recent research on adaptive image enhancement into our approach, allowing us to propose an effective solution for known problems present in confocal microscopy images. We provide an evaluation based on annotated image data and compare our results against current state-of-the-art algorithms. The results show that our algorithm clearly outperforms the existing solutions. PMID:26743993

  11. Highly versatile confocal microscopy system based on a tunable femtosecond Er:fiber source.

    PubMed

    Träutlein, D; Adler, F; Moutzouris, K; Jeromin, A; Leitenstorfer, A; Ferrando-May, E

    2008-03-01

    The performance of a confocal microscopy setup based on a single femtosecond fiber system is explored over a broad range of pump wavelengths for both linear and nonlinear imaging techniques. First, the benefits of a laser source in linear fluorescence excitation that is continuously tunable over most of the visible spectrum are demonstrated. The influences of subpicosecond pulse durations on the bleaching behavior of typical fluorophores are discussed. We then utilize the tunable near-infrared output of the femtosecond system in connection with a specially designed prism compressor for dispersion control. Pulses as short as 33 fs are measured in the confocal region. As a consequence, 2 mW of average power are sufficient for two-photon microscopy in an organotypic sample from the mouse brain. This result shows great prospect for deep-tissue imaging in the optimum transparency window around 1100 nm. In a third experiment, we prove that our compact setup is powerful enough to exploit even higher-order nonlinearities such as three-photon absorption that we use to induce spatially localized photodamage in DNA.

  12. In vivo corneal confocal microscopy in diabetes: Where we are and where we can get

    PubMed Central

    Maddaloni, Ernesto; Sabatino, Francesco

    2016-01-01

    In vivo corneal confocal microscopy (IVCCM) is a novel, reproducible, easy and noninvasive technique that allows the study of the different layers of the cornea at a cellular level. As cornea is the most innervated organ of human body, several studies investigated the use of corneal confocal microscopy to detect diabetic neuropathies, which are invalidating and deadly complications of diabetes mellitus. Corneal nerve innervation has been shown impaired in subjects with diabetes and a close association between damages of peripheral nerves due to the diabetes and alterations in corneal sub-basal nerve plexus detected by IVCCM has been widely demonstrated. Interestingly, these alterations seem to precede the clinical onset of diabetic neuropathies, paving the path for prevention studies. However, some concerns still prevent the full implementation of this technique in clinical practice. In this review we summarize the most recent and relevant evidences about the use of IVCCM for the diagnosis of peripheral sensorimotor polyneuropathy and of autonomic neuropathy in diabetes. New perspectives and current limitations are also discussed. PMID:27660697

  13. In vivo corneal confocal microscopy in diabetes: Where we are and where we can get.

    PubMed

    Maddaloni, Ernesto; Sabatino, Francesco

    2016-09-15

    In vivo corneal confocal microscopy (IVCCM) is a novel, reproducible, easy and noninvasive technique that allows the study of the different layers of the cornea at a cellular level. As cornea is the most innervated organ of human body, several studies investigated the use of corneal confocal microscopy to detect diabetic neuropathies, which are invalidating and deadly complications of diabetes mellitus. Corneal nerve innervation has been shown impaired in subjects with diabetes and a close association between damages of peripheral nerves due to the diabetes and alterations in corneal sub-basal nerve plexus detected by IVCCM has been widely demonstrated. Interestingly, these alterations seem to precede the clinical onset of diabetic neuropathies, paving the path for prevention studies. However, some concerns still prevent the full implementation of this technique in clinical practice. In this review we summarize the most recent and relevant evidences about the use of IVCCM for the diagnosis of peripheral sensorimotor polyneuropathy and of autonomic neuropathy in diabetes. New perspectives and current limitations are also discussed.

  14. Corneal confocal microscopy reveals trigeminal small sensory fiber neuropathy in amyotrophic lateral sclerosis

    PubMed Central

    Ferrari, Giulio; Grisan, Enrico; Scarpa, Fabio; Fazio, Raffaella; Comola, Mauro; Quattrini, Angelo; Comi, Giancarlo; Rama, Paolo; Riva, Nilo

    2014-01-01

    Although subclinical involvement of sensory neurons in amyotrophic lateral sclerosis (ALS) has been previously demonstrated, corneal small fiber sensory neuropathy has not been reported to-date. We examined a group of sporadic ALS patients with corneal confocal microscopy, a recently developed imaging technique allowing in vivo observation of corneal small sensory fibers. Corneal confocal microscopy (CCM) examination revealed a reduction of corneal small fiber sensory nerve number and branching in ALS patients. Quantitative analysis demonstrated an increase in tortuosity and reduction in length and fractal dimension of ALS patients’ corneal nerve fibers compared to age-matched controls. Moreover, bulbar function disability scores were significantly related to measures of corneal nerve fibers anatomical damage. Our study demonstrates for the first time a corneal small fiber sensory neuropathy in ALS patients. This finding further suggests a link between sporadic ALS and facial-onset sensory and motor neuronopathy (FOSMN) syndrome, a rare condition characterized by early sensory symptoms (with trigeminal nerve distribution), followed by wasting and weakness of bulbar and upper limb muscles. In addition, the finding supports a model of neurodegeneration in ALS as a focally advancing process. PMID:25360111

  15. Laser Scanning In Vivo Confocal Microscopy of Clear Grafts after Penetrating Keratoplasty

    PubMed Central

    Wang, Dai; Song, Peng; Wang, Shuting; Sun, Dapeng; Wang, Yuexin; Zhang, Yangyang

    2016-01-01

    Purpose. To evaluate the changes of keratocytes and dendritic cells in the central clear graft by laser scanning in vivo confocal microscopy after penetrating keratoplasty (PK). Methods. Thirty adult subjects receiving PK at Shandong Eye Institute and with clear grafts and no sign of immune rejection after surgery were recruited into this study, and 10 healthy adults were controls. The keratocytes and dendritic cells in the central graft were evaluated by laser scanning confocal microscopy, as well as epithelium cells, keratocytes, corneal endothelium cells, and corneal nerves (especially subepithelial plexus nerves). Results. Median density of subepithelial plexus nerves, keratocyte density in each layer of the stroma, and density of corneal endothelium cells were all lower in clear grafts than in controls. The dendritic cells of five (16.7%) patients were active in Bowman's membrane and stromal membrane of the graft after PK. Conclusions. Activated dendritic cells and Langerhans cells could be detected in some of the clear grafts, which indicated that the subclinical stress of immune reaction took part in the chronic injury of the clear graft after PK, even when there was no clinical rejection episode. PMID:27034940

  16. The use of Interferometric Microscopy to assess 3D modifications of deteriorated medieval glass.

    NASA Astrophysics Data System (ADS)

    Gentaz, L.; Lombardo, T.; Chabas, A.

    2012-04-01

    Due to low durability, Northern European medieval glass undergoes the action of the atmospheric environment leading in some cases to a state of dramatic deterioration. Modification features varies from a simple loss of transparency to a severe material loss. In order to understand the underlying mechanisms and preserve this heritage, fundamental research is necessary too. In this optic, field exposure of analogues and original stained glass was carried out to study the early stages of the glass weathering. Model glass and original stained glass (after removal of deterioration products) were exposed in real conditions in an urban site (Paris) for 48 months. A regular withdrawal of samples allowed a follow-up of short-term glass evolution. Morphological modifications of the exposed samples were investigated through conventional and non destructive microscopy, using respectively a Scanning Electron Microscope (SEM) and an Interferometric Microscope (IM). This latter allows a 3D quantification of the object with no sample preparation. For all glasses, both surface recession and build-up of deposit were observed as a consequence of a leaching process (interdiffusion of protons and glass cations). The build-up of a deposit comes from the reaction between the extracted glass cations and atmospheric gases. Instead, surface recession is due mainly to the formation of brittle layer of altered glass at the sub-surface, where a fracture network can appear, leading to the scaling of parts of this modified glass. Finally, dissolution of the glass takes place, inducing the formation of pits and craters. The arithmetic roughness (Ra) was used as an indicator of weathering increase, in order to evaluate the deterioration state. For instance, the Ra grew from few tens of nm for pristine glass to thousands of nm for scaled areas. This technique also allowed a precise quantification of dimensions (height, depth and width) of deposits and pits, and the estimation of their overall

  17. Virtual pinhole confocal microscope

    SciTech Connect

    George, J.S.; Rector, D.M.; Ranken, D.M.; Peterson, B.; Kesteron, J.

    1999-06-01

    Scanned confocal microscopes enhance imaging capabilities, providing improved contrast and image resolution in 3-D, but existing systems have significant technical shortcomings and are expensive. Researchers at Los Alamos National Laboratory have developed a novel approach--virtual pinhole confocal microscopy--that uses state of the art illumination, detection, and data processing technologies to produce an imager with a number of advantages: reduced cost, faster imaging, improved efficiency and sensitivity, improved reliability and much greater flexibility. Work at Los Alamos demonstrated proof of principle; prototype hardware and software have been used to demonstrate technical feasibility of several implementation strategies. The system uses high performance illumination, patterned in time and space. The authors have built functional confocal imagers using video display technologies (LCD or DLP) and novel scanner based on a micro-lens array. They have developed a prototype system for high performance data acquisition and processing, designed to support realtime confocal imaging. They have developed algorithms to reconstruct confocal images from a time series of spatially sub-sampled images; software development remains an area of active development. These advances allow the collection of high quality confocal images (in fluorescence, reflectance and transmission modes) with equipment that can inexpensively retrofit to existing microscopes. Planned future extensions to these technologies will significantly enhance capabilities for microscopic imaging in a variety of applications, including confocal endoscopy, and confocal spectral imaging.

  18. 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.

  19. Prototype study on a miniaturized dual-modality imaging system for photoacoustic microscopy and confocal fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Sung-Liang; Xie, Zhixing; Guo, L. Jay; Wang, Xueding

    2014-03-01

    It is beneficial to study tumor angiogenesis and microenvironments by imaging the microvasculature and cells at the same time. Photoacoustic microscopy (PAM) is capable of sensitive three-dimensional mapping of microvasculature, while fluorescence microscopy may be applied to assessment of tissue pathology. In this work, a fiber-optic based PAM and confocal fluorescence microscopy (CFM) dual-modality imaging system was designed and built, serving as a prototype of a miniaturized dual-modality imaging probe for endoscopic applications. As for the design, we employed miniature components, including a microelectromechanical systems (MEMS) scanner, a miniature objective lens, and a small size optical microring resonator as an acoustic detector. The system resolutions were calibrated as 8.8 μm in the lateral directions for both PAM and CFM, and 19 μm and 53 μm in the axial direction for PAM and CFM, respectively. Images of the animal bladders ex vivo were demonstrated to show the ability of the system in imaging not only microvasculature but also cellular structure.

  20. Comparison of reflectance confocal microscopy and two-photon second harmonic generation microscopy in fungal keratitis rabbit model ex vivo.

    PubMed

    Lee, Jun Ho; Lee, Seunghun; Yoon, Calvin J; Park, Jin Hyoung; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2016-02-01

    Fungal keratitis is an infection of the cornea by fungal pathogens. Diagnosis methods based on optical microscopy could be beneficial over the conventional microbiology method by allowing rapid and non-invasive examination. Reflectance confocal microscopy (RCM) and two-photon second harmonic generation microscopy (TPSHGM) have been applied to pre-clinical or clinical studies of fungal keratitis. In this report, RCM and TPSHGM were characterized and compared in the imaging of a fungal keratitis rabbit model ex vivo. Fungal infection was induced by using two strains of fungi: aspergillus fumigatus and candida albicans. The infected corneas were imaged in fresh condition by both modalities sequentially and their images were analyzed. Both RCM and TPSHGM could detect both fungal strains within the cornea based on morphology: aspergillus fumigatus had distinctive filamentous structures, and candida albicans had round structures superficially and elongated structures in the corneal stroma. These imaging results were confirmed by histology. Comparison between RCM and TPSHGM showed several characteristics. Although RCM and TPSHGM images had good correlation each other, their images were slightly different due to difference in contrast mechanism. RCM had relatively low image contrast with the infected turbid corneas due to high background signal. TPSHGM visualized cells and collagen in the cornea clearly compared to RCM, but used higher laser power to compensate low autofluorescence. Since these two modalities provide complementary information, combination of RCM and TPSHGM would be useful for fungal keratitis detection by compensating their weaknesses each other. PMID:26977371

  1. Comparison of reflectance confocal microscopy and two-photon second harmonic generation microscopy in fungal keratitis rabbit model ex vivo

    PubMed Central

    Lee, Jun Ho; Lee, Seunghun; Yoon, Calvin J.; Park, Jin Hyoung; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2016-01-01

    Fungal keratitis is an infection of the cornea by fungal pathogens. Diagnosis methods based on optical microscopy could be beneficial over the conventional microbiology method by allowing rapid and non-invasive examination. Reflectance confocal microscopy (RCM) and two-photon second harmonic generation microscopy (TPSHGM) have been applied to pre-clinical or clinical studies of fungal keratitis. In this report, RCM and TPSHGM were characterized and compared in the imaging of a fungal keratitis rabbit model ex vivo. Fungal infection was induced by using two strains of fungi: aspergillus fumigatus and candida albicans. The infected corneas were imaged in fresh condition by both modalities sequentially and their images were analyzed. Both RCM and TPSHGM could detect both fungal strains within the cornea based on morphology: aspergillus fumigatus had distinctive filamentous structures, and candida albicans had round structures superficially and elongated structures in the corneal stroma. These imaging results were confirmed by histology. Comparison between RCM and TPSHGM showed several characteristics. Although RCM and TPSHGM images had good correlation each other, their images were slightly different due to difference in contrast mechanism. RCM had relatively low image contrast with the infected turbid corneas due to high background signal. TPSHGM visualized cells and collagen in the cornea clearly compared to RCM, but used higher laser power to compensate low autofluorescence. Since these two modalities provide complementary information, combination of RCM and TPSHGM would be useful for fungal keratitis detection by compensating their weaknesses each other. PMID:26977371

  2. Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart.

    PubMed

    Huang, Chao; Kaza, Aditya K; Hitchcock, Robert W; Sachse, Frank B

    2014-01-01

    Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5-9 lines, which is comparable to 4-8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery.

  3. Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart

    PubMed Central

    Huang, Chao; Kaza, Aditya K.; Hitchcock, Robert W.; Sachse, Frank B.

    2014-01-01

    Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5–9 lines, which is comparable to 4–8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery. PMID:25309455

  4. [Studies on Effect of Alkali Pretreatment on Anaerobic Digestion of Rice Straw with Confocal Raman Microscopy].

    PubMed

    Xia, Yi-hua; Luo, Liu-bin; Li, Xiao-li; He, Yong; Sheng, Kui-chuan

    2015-03-01

    NaOH pretreatment is a convenient and effective method which is widely used in rice straw anaerobic digestion. But the mechanism of the alkaline (NaOH) hydrolysis of biopolymers compositions and polymeric cross-linked network structures of rice straw cell wall need further study. This paper firstly studied the effect and mechanism of alkali pretreatment on anaerobic digestion and biogas production of rice straw by using a combination of confocal Raman microscopy and transmission electron microscope. First, the original rice straw and the rice straw pretreated by NaOH were taken for mapping scanning by confocal Raman microscopy with micron-scale spatial resolution. Then principal component analysis was adopted to extract main information of Raman spectra, it could be found that the two types of samples were respectively presented with ray-like distribution in the first two principal component space, which were with cumulative contribution of 99%. And there was a clear boundary between the two types of samples without any overlapping, indicating that there was a significant difference of Raman spectral characteristic between original rice leaf and rice leaf pretreated by NaOH. Further analysis of the loading weights of the first two principal components showed that the Raman peaks at 1 739, 1 508 and 1 094 cm(-1) were the important bands, and these three Raman peaks were attributed to the scattering of hemicellulose, cellulose and lignin respectively. Following, chemical imaging analysis of hemicellulose, cellulose and lignin were achieved by combining these Raman peaks and microscopic image information. It could be found that the NaOH pretreatment resulted in a loss of dense spatial uniformity structure of tissue and great decreases of the contents of these three ingredients, particularly lignin. It can be concluded that it is feasible to non-destructively measure hemicellulose, lignin and cellulose in rice straw tissue by confocal Raman microscopy, and to achieve

  5. 3-D analysis of bacterial cell-(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate-reducing Acidovorax sp. strain BoFeN1 using complementary microscopy tomography approaches.

    PubMed

    Schmid, G; Zeitvogel, F; Hao, L; Ingino, P; Floetenmeyer, M; Stierhof, Y-D; Schroeppel, B; Burkhardt, C J; Kappler, A; Obst, M

    2014-07-01

    The formation of cell-(iron)mineral aggregates as a consequence of bacterial iron oxidation is an environmentally widespread process with a number of implications for processes such as sorption and coprecipitation of contaminants and nutrients. Whereas the overall appearance of such aggregates is easily accessible using 2-D microscopy techniques, the 3-D and internal structure remain obscure. In this study, we examined the 3-D structure of cell-(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate-reducing Acidovorax sp. strain BoFeN1 using a combination of advanced 3-D microscopy techniques. We obtained 3-D structural and chemical information on different cellular encrustation patterns at high spatial resolution (4-200 nm, depending on the method): more specifically, (1) cells free of iron minerals, (2) periplasm filled with iron minerals, (3) spike- or platelet-shaped iron mineral structures, (4) bulky structures on the cell surface, (5) extracellular iron mineral shell structures, (6) cells with iron mineral filled cytoplasm, and (7) agglomerations of extracellular globular structures. In addition to structural information, chemical nanotomography suggests a dominant role of extracellular polymeric substances (EPS) in controlling the formation of cell-(iron)mineral aggregates. Furthermore, samples in their hydrated state showed cell-(iron)mineral aggregates in pristine conditions free of preparation (i.e., drying/dehydration) artifacts. All these results were obtained using 3-D microscopy techniques such as focused ion beam (FIB)/scanning electron microscopy (SEM) tomography, transmission electron microscopy (TEM) tomography, scanning transmission (soft) X-ray microscopy (STXM) tomography, and confocal laser scanning microscopy (CLSM). It turned out that, due to the various different contrast mechanisms of the individual approaches, and due to the required sample preparation steps, only the combination of these techniques was able to provide a

  6. Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Krmpot, Aleksandar J.; Nikolić, Stanko N.; Vitali, Marco; Papadopoulos, Dimitrios K.; Oasa, Sho; Thyberg, Per; Tisa, Simone; Kinjo, Masataka; Nilsson, Lennart; Gehring, Walter J.; Terenius, Lars; Rigler, Rudolf; Vukojevic, Vladana

    2015-07-01

    Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.

  7. Impaired Intracellular Ca2+ Dynamics in Live Cardiomyocytes Revealed by Rapid Line Scan Confocal Microscopy

    NASA Astrophysics Data System (ADS)

    Plank, David M.; Sussman, Mark A.

    2005-06-01

    Altered intracellular Ca2+ dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca2+ handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca2+ chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca2+ concentration and has required the use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate Ca2+ concentrations. This study describes methodology to directly measure intracellular Ca2+ levels using inactivated, Fluo-3-AM-loaded cardiomyocytes equilibrated with Ca2+ concentration standards. Titration of Ca2+ concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca2+ concentrations. These standard plots can be used to convert transient Ca2+ fluorescence obtained with experimental cells to Ca2+ concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca2+ concentrations, simplifying data interpretation and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be useful to investigators requiring a straightforward method for measuring Ca2+ concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.

  8. Fault localization and analysis in semiconductor devices with optical-feedback infrared confocal microscopy

    SciTech Connect

    Sarmiento, Raymund; Cemine, Vernon Julius; Tagaca, Imee Rose; Salvador, Arnel; Mar Blanca, Carlo; Saloma, Caesar

    2007-11-01

    We report on a cost-effective optical setup for characterizing light-emitting semiconductor devices with optical-feedback confocal infrared microscopy and optical beam-induced resistance change.We utilize the focused beam from an infrared laser diode to induce local thermal resistance changes across the surface of a biased integrated circuit (IC) sample. Variations in the multiple current paths are mapped by scanning the IC across the focused beam. The high-contrast current maps allow accurate differentiation of the functional and defective sites, or the isolation of the surface-emittingp-i-n devices in the IC. Optical beam-induced current (OBIC) is not generated since the incident beam energy is lower than the bandgap energy of the p-i-n device. Inhomogeneous current distributions in the IC become apparent without the strong OBIC background. They are located at a diffraction-limited resolution by referencing the current maps against the confocal reflectance image that is simultaneously acquired via optical-feedback detection. Our technique permits the accurate identification of metal and semiconductor sites as well as the classification of different metallic structures according to thickness, composition, or spatial inhomogeneity.

  9. Adhesive improvement in optical coherence tomography combined with confocal microscopy for class V cavities investigations

    NASA Astrophysics Data System (ADS)

    Rominu, Mihai; Sinescu, Cosmin; Negrutiu, Meda L.; Rominu, Roxana O.; Pop, Daniela M.; Topala, Florin; Stoia, Adelina; Petrescu, Emanuela; Bradu, Adrian; Dobre, George; Podoleanu, Adrian G.

    2010-03-01

    The purpose of this study is to present a non invasive method for the marginal adaptation evaluation in class V composite restorations. Standardized class V cavities prepared in human extracted teeth were filled with composite resin (Premise, Kerr). The specimens were thermocycled. The interfaces were examined by Optical Coherence Tomography (OCT) combined with confocal microscopy and fluorescence. The optical configuration uses two single mode directional couplers with a superluminiscent diode as the source at 1300 nm. The scanning procedure is similar to that used in any confocal microscope, where the fast scanning is en-face (line rate) and the depth scanning is much slower (at the frame rate). Gaps at the interfaces as well as on the inside of the composite resin were identified. OCT has numerous advantages that justify its in vivo and in vitro use compared to conventional techniques. One of the main concerns was the fact that at the adhesive layer site it was very hard to tell the adhesive apart from material defects. For this reason the adhesive was optimized in order to be more scattering. This way we could make a difference between the adhesive layer and the material defects that could lead to microleakages.

  10. 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.

  11. The method of axial drift compensation of laser differential confocal microscopy based on zero-tracking

    NASA Astrophysics Data System (ADS)

    Wang, Yajie; Cui, Han; Wang, Yun; Qiu, Lirong; Zhao, Weiqian

    2015-08-01

    Laser differential confocal microscopy (DCM) has advantages of high axial resolution and strong ability of focus identification. However, the imaging mechanism of point scanning needs long measurement time, in the process due to itself mechanical instability and the influence of environment vibration the axial drift of object position is inevitable, which will reduce lateral resolution of the DCM. To ensure the lateral resolution we propose an axial drift compensation method based on zero-tracking in this paper. The method takes advantage of the linear region of differential confocal axial response curve, gets axial drift by detecting the laser intensity; uses grating sensor to monitor the real-time axial drift of lifting stage and realizes closed-loop control; uses capacitive sensor of objective driver to measure its position. After getting the axial drift of object, the lifting stage and objective driver will be driven to compensate position according to the axial drift. This method is realized by using Visual Studio 2010, and the experiment demonstrates that the compensation precision of the proposed method can reach 6 nm. It is not only easy to implement, but also can compensate the axial drift actively and real-timely. Above all, this method improves the system stability of DCM effectively.

  12. Alterations of filopodia by near infrared photoimmunotherapy: evaluation with 3D low-coherent quantitative phase microscopy

    PubMed Central

    Nakamura, Yuko; Nagaya, Tadanobu; Sato, Kazuhide; Harada, Toshiko; Okuyama, Shuhei; Choyke, Peter L.; Yamauchi, Toyohiko; Kobayashi, Hisataka

    2016-01-01

    Filopodia are highly organized cellular membrane structures that facilitate intercellular communication. Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that causes necrotic cell death. Three-dimensional low-coherent quantitative phase microscopy (3D LC-QPM) is based on a newly established low-coherent interference microscope designed to obtain serial topographic images of the cellular membrane. Herein, we report rapid involution of filopodia after NIR-PIT using 3D LC-QPM. For 3T3/HER2 cells, the number of filopodia decreased immediately after treatment with significant differences. Volume and relative height of 3T3/HER2 cells increased immediately after NIR light exposure, but significant differences were not observed. Thus, disappearance of filopodia, evaluated by 3D LC-QPM, is an early indicator of cell membrane damage after NIR-PIT. PMID:27446702

  13. Wide-field hyperspectral 3D imaging of functionalized gold nanoparticles targeting cancer cells by reflected light microscopy.

    PubMed

    Patskovsky, Sergiy; Bergeron, Eric; Rioux, David; Meunier, Michel

    2015-05-01

    We present a new hyperspectral reflected light microscopy system with a scanned broadband supercontinuum light source. This wide-field and low phototoxic hyperspectral imaging system has been successful for performing spectral three-dimensional (3D) localization and spectroscopic identification of CD44-targeted PEGylated AuNPs in fixed cell preparations. Such spatial and spectral information is essential for the improvement of nanoplasmonic-based imaging, disease detection and treatment in complex biological environment. The presented system can be used for real-time 3D NP tracking as spectral sensors, thus providing new avenues in the spatio-temporal characterization and detection of bioanalytes. 3D image of the distribution of functionalized AuNPs attached to CD44-expressing MDA-MB-231 human cancer cells. PMID:24961507

  14. Imaging bacterial 3D motion using digital in-line holographic microscopy and correlation-based de-noising algorithm

    PubMed Central

    Molaei, Mehdi; Sheng, Jian

    2014-01-01

    Abstract: Better understanding of bacteria environment interactions in the context of biofilm formation requires accurate 3-dimentional measurements of bacteria motility. Digital Holographic Microscopy (DHM) has demonstrated its capability in resolving 3D distribution and mobility of particulates in a dense suspension. Due to their low scattering efficiency, bacteria are substantially difficult to be imaged by DHM. In this paper, we introduce a novel correlation-based de-noising algorithm to remove the background noise and enhance the quality of the hologram. Implemented in conjunction with DHM, we demonstrate that the method allows DHM to resolve 3-D E. coli bacteria locations of a dense suspension (>107 cells/ml) with submicron resolutions (<0.5 µm) over substantial depth and to obtain thousands of 3D cell trajectories. PMID:25607177

  15. Alterations of filopodia by near infrared photoimmunotherapy: evaluation with 3D low-coherent quantitative phase microscopy.

    PubMed

    Nakamura, Yuko; Nagaya, Tadanobu; Sato, Kazuhide; Harada, Toshiko; Okuyama, Shuhei; Choyke, Peter L; Yamauchi, Toyohiko; Kobayashi, Hisataka

    2016-07-01

    Filopodia are highly organized cellular membrane structures that facilitate intercellular communication. Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that causes necrotic cell death. Three-dimensional low-coherent quantitative phase microscopy (3D LC-QPM) is based on a newly established low-coherent interference microscope designed to obtain serial topographic images of the cellular membrane. Herein, we report rapid involution of filopodia after NIR-PIT using 3D LC-QPM. For 3T3/HER2 cells, the number of filopodia decreased immediately after treatment with significant differences. Volume and relative height of 3T3/HER2 cells increased immediately after NIR light exposure, but significant differences were not observed. Thus, disappearance of filopodia, evaluated by 3D LC-QPM, is an early indicator of cell membrane damage after NIR-PIT. PMID:27446702

  16. Oral biofilm analysis of palatal expanders by fluorescence in-situ hybridization and confocal laser scanning microscopy.

    PubMed

    Klug, Barbara; Rodler, Claudia; Koller, Martin; Wimmer, Gernot; Kessler, Harald H; Grube, Martin; Santigli, Elisabeth

    2011-10-20

    microscopy. Well-known configurations could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

  17. Oral biofilm analysis of palatal expanders by fluorescence in-situ hybridization and confocal laser scanning microscopy.

    PubMed

    Klug, Barbara; Rodler, Claudia; Koller, Martin; Wimmer, Gernot; Kessler, Harald H; Grube, Martin; Santigli, Elisabeth

    2011-01-01

    microscopy. Well-known configurations could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created. PMID:22041974

  18. Mapping Li(+) Concentration and Transport via In Situ Confocal Raman Microscopy.

    PubMed

    Forster, Jason D; Harris, Stephen J; Urban, Jeffrey J

    2014-06-01

    We demonstrate confocal Raman microscopy as a general, nonperturbative tool to measure spatially resolved lithium ion concentrations in liquid electrolytes. By combining this high-spatial-resolution technique with a simple microfluidic device, we are able to measure the diffusion coefficient of lithium ions in dimethyl carbonate in two different concentration regimes. Because lithium ion transport plays a key role in the function of a variety of electrochemical devices, quantifying and visualizing this process is crucial for understanding device performance. This method for detecting lithium ions should be immediately useful in the study of lithium-ion-based devices, ion transport in porous media, and at electrode-electrolyte interfaces, and the analytical framework is useful for any system exhibiting a concentration-dependent Raman spectrum. PMID:26273887

  19. The use of reflectance confocal microscopy for monitoring response to therapy of skin malignancies

    PubMed Central

    Ulrich, Martina; Lange-Asschenfeldt, Susanne; Gonzalez, Salvador

    2012-01-01

    Summary Reflectance confocal microscopy (RCM) is a new non-invasive imaging technique that enables visualizing cells and structures in living skin in real-time with resolution close to that of histological analysis. RCM has been successfully implemented in the assessment of benign and malignant lesions. Most importantly, it also enables monitoring dynamic changes in the skin over time and in response to different therapies, e.g., imiquimod, photodynamic therapy, and others. Given the often traumatic nature of skin cancer that affects both the physiology and the psychology of the patients, it is crucial to have methods that enable monitoring the response to treatment but that minimize the distress and discomfort associated with such process. This article provides a very brief overview of the fundamentals of RCM and then focuses on its recent employment as a monitoring tool in skin cancer and other pathologies that may require frequent follow-up. PMID:23785598

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

    PubMed

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

    2014-01-01

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

  1. HIV detection by in-situ hybridization based on confocal reflected light microscopy

    NASA Astrophysics Data System (ADS)

    Smith, Louis C.; Jericevic, Zeljko; Cuellar, Roland; Paddock, Stephen W.; Lewis, Dorothy E.

    1991-05-01

    Elucidation of the pathogenesis of AIDS is confounded by the finding that few actively infected CD4+ cells (1 in 104-105) can be detected in the peripheral blood, even though there is dramatic depletion (often >90%) of CD4+ cells as the disease progresses. A sensitive, 35S-based human immunodeficiency virus (HIV) mRNA in situ hybridization technique was coupled with a new detection method, confocal laser scanning microscopy, to examine transcriptionally active HIV-infected cells from individuals at different disease stages. An algorithm for image segmentation and analysis has been developed to determine the proportion of HIV-positive cells. Data obtained using this improved detection method suggest that there are more HIV mRNA-producing cells in HIV-infected individuals than previously thought, based on other detection methods.

  2. Consistency and distribution of reflectance confocal microscopy features for diagnosis of cutaneous T cell lymphoma

    NASA Astrophysics Data System (ADS)

    Lange-Asschenfeldt, Susanne; Babilli, Jasmin; Beyer, Marc; Ríus-Diaz, Francisca; González, Salvador; Stockfleth, Eggert; Ulrich, Martina

    2012-01-01

    Reflectance confocal microscopy (RCM) represents a noninvasive imaging technique that has previously been used for characterization of mycosis fungoides (MF) in a pilot study. We aimed to test the applicability of RCM for diagnosis and differential diagnosis of MF in a clinical study. A total of 39 test sites of 15 patients with a biopsy-proven diagnosis of either MF, parapsoriasis, Sézary syndrome, or lymphomatoid papulosis were analyzed for presence and absence of RCM features of MF. Cochran and Chi2 analysis were applied to test the concordance between investigators and the distribution of RCM features, respectively. For selected parameters, the Cochran analysis showed good concordance between investigators. Inter-observer reproducibility was highest for junctional atypical lymphocytes, architectural disarray, and spongiosis. Similarly, Chi2 analysis demonstrated that selected features were present at particularly high frequency in individual skin diseases, with values ranging from 73% to 100% of all examined cases.

  3. An alternative method of promoter assessment by confocal laser scanning microscopy.

    PubMed

    Sahoo, Dipak K; Ranjan, Rajiv; Kumar, Deepak; Kumar, Alok; Sahoo, Bhabani S; Raha, Sumita; Maiti, Indu B; Dey, Nrisingha

    2009-10-01

    A rapid and useful method of promoter activity analysis using techniques of confocal laser scanning microscopy (CLSM) is described in the present study. The activities of some pararetroviral promoters such as CaMV35S (Cauliflower mosaic virus), FMVSgt3 (Figwort mosaic virus sub-genomic transcript) and MMVFLt12 (Mirabilis mosaic virus full-length transcript) coupled to GFP (green fluorescent protein) and GUS (beta-glucuronidase) reporter genes were determined simultaneously by the CLSM technique and other available conventional methods for reporter gene assay based on relevant biochemical and molecular approaches. Consistent and comparable results obtained by CLSM as well as by other conventional assay methods confirm the effectiveness of the CLSM approach for assessment of promoter activity. Hence the CLSM method can be suggested as an alternative way for promoter analysis on the basis of high throughput.

  4. Further study of trichosanthin's effect on mouse embryos with confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Xu, Hui; Zhang, Chunyang; Ma, Hui; Chen, Die Yan

    2001-09-01

    Trichosanthin(TCS), a ribosome inactivating protein extracted from the root tuber of a traditional Chinese medicine herb Tian Huo Fen(THF), possessed abortifacient, anti-tumor and anti-human immunodeficiency virus(HIV) activities. For centuries in China, THF has been used as an effective folk medicine to terminate early and midtrimester pregnancies and to treat ectopic pregnancies, hydatidiform moles and trophoblastic tumor. We observed the changes in reactive oxygen species and intracellular calcium in mouse embryos induced by TCS with confocal laser scanning microscopy in combination with the fluorescene diacetate (DCFHDA) and Fluo-3-AM. The results indicated that TCS induced increase in intracellular calcium and production of reactive oxygen species in mouse embryos , and TCS inhibited the development of mouse embryos effectively. Mouse embryos of different developmental stages before implantation are used in the experiments. This provides new insight into mechanism for abortifacient activity of TCS.

  5. Pharmaceutical applications of confocal laser scanning microscopy: the physical characterisation of pharmaceutical systems.

    PubMed

    Pygall, Samuel R; Whetstone, Joanne; Timmins, Peter; Melia, Colin D

    2007-12-10

    The application of confocal laser scanning microscopy (CLSM) to the physicochemical characterisation of pharmaceutical systems is not as widespread as its application within the field of cell biology. However, methods have been developed to exploit the imaging capabilities of CLSM to study a wide range of pharmaceutical systems, including phase-separated polymers, colloidal systems, microspheres, pellets, tablets, film coatings, hydrophilic matrices, and chromatographic stationary phases. Additionally, methods to measure diffusion in gels, bioadhesives, and for monitoring microenvironmental pH change within dosage forms have been utilised. CLSM has also been used in the study of the physical interaction of dosage forms with biological barriers such as the eye, skin and intestinal epithelia, and in particular, to determine the effectiveness of a plethora of pharmaceutical systems to deliver drugs through these barriers. In the future, there is continuing scope for wider exploitation of existing techniques, and continuing advancements in instrumentation.

  6. Use of Confocal Microscopy in the Search for Optical Counterparts to Gamma-Ray Bursts

    NASA Astrophysics Data System (ADS)

    Sharma, A.; Jacobson, R. E.

    1995-09-01

    A number of star-like images have been found on archival photographic plates, however there is debate as to whether these are optical emissions relating to Gamma-Ray bursts, or simply plate defects. Results from photographic science are summarized so that the characteristic shape of different types of images on photographic plates can be theoretically predicted. Explanation for the formation of the relief image and reasons for its continued study are described. New methods in the use of laser scanning confocal microscopy are presented to study the relief image and the depthwise disposition of silver so that a test procedure can be established to end the speculation surrounding the astrophysical truth of stellar-like images on photographic plates.

  7. In vivo amyloid aggregation kinetics tracked by time-lapse confocal microscopy in real-time.

    PubMed

    Villar-Piqué, Anna; Espargaró, Alba; Ventura, Salvador; Sabate, Raimon

    2016-01-01

    Amyloid polymerization underlies an increasing number of human diseases. Despite this process having been studied extensively in vitro, aggregation is a difficult process to track in vivo due to methodological limitations and the slow kinetics of aggregation reactions in cells and tissues. Herein we exploit the amyloid properties of the inclusions bodies (IBs) formed by amyloidogenic proteins in bacteria to address the kinetics of in vivo amyloid aggregation. To this aim we used time-lapse confocal microscopy and a fusion of the amyloid-beta peptide (A β42) with a fluorescent reporter. This strategy allowed us to follow the intracellular kinetics of amyloid-like aggregation in real-time and to discriminate between variants exhibiting different in vivo aggregation propensity. Overall, the approach opens the possibility to assess the impact of point mutations as well as potential anti-aggregation drugs in the process of amyloid formation in living cells.

  8. Separation of ballistic and diffusive fluorescence photons in confocal Light-Sheet Microscopy of Arabidopsis roots.

    PubMed

    Meinert, Tobias; Tietz, Olaf; Palme, Klaus J; Rohrbach, Alexander

    2016-01-01

    Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection. PMID:27553506

  9. Detection of apoptosis caused by anticancer drug paclitaxel in MCF-7 cells by confocal Raman microscopy

    NASA Astrophysics Data System (ADS)

    Salehi, H.; Middendorp, E.; Végh, A.-G.; Ramakrishnan, S.-K.; Gergely, C.; Cuisinier, F. J. G.

    2013-02-01

    Confocal Raman Microscopy, a non-invasive, label free imaging technique is used to study apoptosis in living MCF-7 cells. The images are based on Raman spectra of cells components. K-mean clustering was used to determine mitochondria position in cells and cytochrome c distribution inside the cells was based on correlation analysis. Cell apoptosis is defined as cytochrome c diffusion in cytoplasm. Co-localization of cytochrome c is found within mitochondria after three hours of incubation with 10 μM paclitaxel. Our results demonstrate that the presence of paclitaxel at this concentration in the culture media for 3 hours does not induce apoptosis of MCF7 cells via a caspase independent pathway.

  10. Separation of ballistic and diffusive fluorescence photons in confocal Light-Sheet Microscopy of Arabidopsis roots

    PubMed Central

    Meinert, Tobias; Tietz, Olaf; Palme, Klaus J.; Rohrbach, Alexander

    2016-01-01

    Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection. PMID:27553506

  11. Three-dimensional visualization of termite (Apicotermitinae) enteric valve using confocal laser scanning microscopy.

    PubMed

    Host, B; Twyffels, L; Roisin, Y; Vanderwinden, J-M

    2014-08-01

    Humivorous termites are dominant members of tropical rainforest soil communities. In the soil-feeding subfamily Apicotermitinae (Termitidae), the enteric valve connecting the first section of the hindgut to the paunch often displays a complex sclerotized armature everted towards the lumen of the paunch. This structure is central in termite taxonomy but its function remains hypothetical. Here, we evaluate the potential of confocal laser scanning microscopy to provide detailed imaging of the valve of Anoplotermes parvus, by comparison with bright-field microscopy and scanning electron microscopy. We detected a strong far-red emission of the enteric valve armature that sharply contrasted with the surrounding tissues, providing a convenient method to highlight minute structural elements of the valve and its three-dimensional structure. The method is easy to use and is applicable to standard archival material as demonstrated by images of enteric valves of four other Apicotermitinae species. It may represent a valuable asset for the study of termite enteric valves, for the purpose of taxonomy or functional morphology. PMID:24947115

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

    NASA Astrophysics Data System (ADS)

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

    2013-10-01

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

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

    PubMed

    Steinbach, Gábor; Kaňa, Radek

    2016-04-01

    Photosynthesis research employs several biophysical methods, including the detection of fluorescence. Even though fluorescence is a key method to detect photosynthetic efficiency, it has not been applied/adapted to single-cell confocal microscopy measurements to examine photosynthetic microorganisms. Experiments with photosynthetic cells may require automation to perform a large number of measurements with different parameters, especially concerning light conditions. However, commercial microscopes support custom protocols (through Time Controller offered by Olympus or Experiment Designer offered by Zeiss) that are often unable to provide special set-ups and connection to external devices (e.g., for irradiation). Our new system combining an Arduino microcontroller with the Cell⊕Finder software was developed for controlling Olympus FV1000 and FV1200 confocal microscopes and the attached hardware modules. Our software/hardware solution offers (1) a text file-based macro language to control the imaging functions of the microscope; (2) programmable control of several external hardware devices (light sources, thermal controllers, actuators) during imaging via the Arduino microcontroller; (3) the Cell⊕Finder software with ergonomic user environment, a fast selection method for the biologically important cells and precise positioning feature that reduces unwanted bleaching of the cells by the scanning laser. Cell⊕Finder can be downloaded from http://www.alga.cz/cellfinder. The system was applied to study changes in fluorescence intensity in Synechocystis sp. PCC6803 cells under long-term illumination. Thus, we were able to describe the kinetics of phycobilisome decoupling. Microscopy data showed that phycobilisome decoupling appears slowly after long-term (>1 h) exposure to high light. PMID:27050040

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

    PubMed

    Steinbach, Gábor; Kaňa, Radek

    2016-04-01

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

  15. Confocal microscopy and electrophysiological study of single patient corneal endothelium cell cultures

    NASA Astrophysics Data System (ADS)

    Tatini, Francesca; Rossi, Francesca; Coppi, Elisabetta; Magni, Giada; Fusco, Irene; Menabuoni, Luca; Pedata, Felicita; Pugliese, Anna Maria; Pini, Roberto

    2016-04-01

    The characterization of the ion channels in corneal endothelial cells and the elucidation of their involvement in corneal pathologies would lead to the identification of new molecular target for pharmacological treatments and to the clarification of corneal physiology. The corneal endothelium is an amitotic cell monolayer with a major role in preserving corneal transparency and in regulating the water and solute flux across the posterior surface of the cornea. Although endothelial cells are non-excitable, they express a range of ion channels, such as voltage-dependent Na+ channels and K+ channels, L-type Ca2 channels and many others. Interestingly, purinergic receptors have been linked to a variety of conditions within the eye but their presence in the endothelium and their role in its pathophysiology is still uncertain. In this study, we were able to extract endothelial cells from single human corneas, thus obtaining primary cultures that represent the peculiarity of each donor. Corneas were from tissues not suitable for transplant in patients. We characterized the endothelial cells by confocal microscopy, both within the intact cornea and in the primary endothelial cells cultures. We also studied the functional role of the purinergic system (adenosine, ATP and their receptors) by means of electrophysiological recordings. The experiments were performed by patch clamp recordings and confocal time-lapse microscopy and our results indicate that the application of purinergic compounds modulates the amplitude of outward currents in the isolated endothelial cells. These findings may lead to the proposal of new therapies for endothelium-related corneal diseases.

  16. Optical Coherence Tomography Angiography in Mice: Comparison with Confocal Scanning Laser Microscopy and Fluorescein Angiography

    PubMed Central

    Giannakaki-Zimmermann, Helena; Kokona, Despina; Wolf, Sebastian; Ebneter, Andreas; Zinkernagel, Martin S.

    2016-01-01

    Purpose Optical coherence tomography angiography (OCT-A) allows noninvasive visualization of retinal vessels in vivo. OCT-A was used to characterize the vascular network of the mouse retina and was compared with fluorescein angiography (FA) and histology. Methods In the present study, OCT-A based on a Heidelberg Engineering Spectralis system was used to investigate the vascular network in mice. Data was compared with FA and confocal microscopy of flat-mount histology stained with isolectin IB4. For quantitative analysis the National Cancer Institute's AngioTool software was used. Vessel density, the number of vessel junctions, and endpoints were measured and compared between the imaging modalities. Results The configuration of the superficial capillary network was comparable with OCT-A and flat-mount histology in BALBc mice. However, vessel density and the number of vessel junctions per region of interest (P = 0.0161 and P = 0.0015, respectively) in the deep vascular network of BALBc mice measured by OCT-A was significantly higher than with flat-mount histology. In C3A.Cg-Pde6b+Prph2Rd2/J mice, where the deep capillary plexus is absent, analysis of the superficial network provided similar results for all three imaging modalities. Conclusion OCT-A is a helpful imaging tool for noninvasive, in vivo imaging of the vascular plexus in mice. It may offer advantages over FA and confocal microscopy especially for imaging the deep vascular plexus. Translational Relevance The present study shows that OCT-A can be employed for small animal imaging to assess the vascular network and offers advantages over flat-mount histology and FA. PMID:27570710

  17. Spectrally encoded confocal microscopy of esophageal tissues at 100 kHz line rate

    PubMed Central

    Schlachter, Simon C.; Kang, DongKyun; Gora, Michalina J.; Vacas-Jacques, Paulino; Wu, Tao; Carruth, Robert W.; Wilsterman, Eric J.; Bouma, Brett E.; Woods, Kevin; Tearney, Guillermo J.

    2013-01-01

    Spectrally encoded confocal microscopy (SECM) is a reflectance confocal microscopy technology that uses a diffraction grating to illuminate different locations on the sample with distinct wavelengths. SECM can obtain line images without any beam scanning devices, which opens up the possibility of high-speed imaging with relatively simple probe optics. This feature makes SECM a promising technology for rapid endoscopic imaging of internal organs, such as the esophagus, at microscopic resolution. SECM imaging of the esophagus has been previously demonstrated at relatively low line rates (5 kHz). In this paper, we demonstrate SECM imaging of large regions of esophageal tissues at a high line imaging rate of 100 kHz. The SECM system comprises a wavelength-swept source with a fast sweep rate (100 kHz), high output power (80 mW), and a detector unit with a large bandwidth (100 MHz). The sensitivity of the 100-kHz SECM system was measured to be 60 dB and the transverse resolution was 1.6 µm. Excised swine and human esophageal tissues were imaged with the 100-kHz SECM system at a rate of 6.6 mm2/sec. Architectural and cellular features of esophageal tissues could be clearly visualized in the SECM images, including papillae, glands, and nuclei. These results demonstrate that large-area SECM imaging of esophageal tissues can be successfully conducted at a high line imaging rate of 100 kHz, which will enable whole-organ SECM imaging in vivo. PMID:24049684

  18. In Vivo Confocal Microscopy Demonstrates Bilateral Loss of Endothelial Cells in Unilateral Herpes Simplex Keratitis

    PubMed Central

    Müller, Rodrigo T.; Pourmirzaie, Roxanna; Pavan-Langston, Deborah; Cavalcanti, Bernardo M.; Aggarwal, Shruti; Colón, Clara; Jamali, Arsia; Cruzat, Andrea; Hamrah, Pedram

    2015-01-01

    Purpose To report bilateral corneal endothelial cell density (ECD), as well as its correlation with subbasal nerve changes, in patients with unilateral herpes simplex keratitis (HSK). Methods Thirty-six eyes of 36 patients with corneal scarring caused by HSK, as well as their respective contralateral clinically unaffected eyes, were prospectively studied and compared with 26 eyes of 26 healthy volunteers. In vivo confocal microscopy and corneal sensation of the central cornea were performed bilaterally in all patients and in one random eye of controls. The ECD and subbasal corneal nerve density, including the lengths of total nerves, main trunks, and branches were evaluated and correlated to central corneal sensation. Results The ECD was significantly lower in eyes affected with HSK than in controls (2304 ± 578 vs. 2940 ± 370 cells/mm2, P < 0.0001). Surprisingly, lower ECD was also detected in contralateral clinically unaffected eyes (2548 ± 423), compared to controls (P = 0.02). Both affected and contralateral eyes showed decrease in total nerve length, compared to controls (10.0 ± 6.3 vs. 17.6 ± 6.3 vs. 21.9 ± 4.3 mm/mm2, respectively; P < 0.05 for all). The ECD correlated positively with total nerve length (r = 0.39, P = 0.0009) and with corneal sensation (r = 0.31, P = 0.009). Conclusions In vivo confocal microscopy findings demonstrated alterations in corneal ECD in both affected and clinically unaffected contralateral eyes of patients with unilateral HSK. Moreover, the positive significant correlation between the ECD and the subbasal nerve density may suggest a potential link between corneal innervation and corneal endothelial cell homeostasis. PMID:26225629

  19. Effects of acids used in the microabrasion technique: Microhardness and confocal microscopy analysis

    PubMed Central

    Pini, Núbia-Inocencya-Pavesi; Ambrosano, Gláucia-Maria-Bovi; da Silva, Wander-José; Aguiar, Flávio-Henrique-Baggio; Lovadino, José-Roberto

    2015-01-01

    Background This study evaluated the effects of the acids used in the microabrasion on enamel. Material and Methods Seventy enamel/dentine blocks (25 mm2) of bovine incisors were divided into 7 groups (n=10). Experimental groups were treated by active/passive application of 35% H3PO4 (E1/E2) or 6.6% HCl (E3/E4). Control groups were treated by microabrasion with H3PO4+pumice (C5), HCl+silica (C6), or no treatment (C7). The superficial (SMH) and cross-sectional (CSMH; depths of 10, 25, 50, and 75 µm) microhardness of enamel were analyzed. Morphology was evaluated by confocal laser-scanning microscopy (CLSM). Data were analyzed by analysis of variance (Proc Mixed), Tukey, and Dunnet tests (α=5%). Results Active application (E1 and E3) resulted in higher microhardness than passive application (E2 and E4), with no difference between acids. For most groups, the CSMH decreased as the depth increased. All experimental groups and negative controls (C5 and C6) showed significantly reduced CSMH values compared to the control. A significantly higher mean CSMH result was obtained with the active application of H3PO4 (E1) compared to HCl (E3). Passive application did not result in CSMH differences between acids. CLSM revealed the conditioning pattern for each group. Conclusions Although the acids displayed an erosive action, use of microabrasive mixture led to less damage to the enamel layers. Key words:Enamel microabrasion, enamel microhardness, confocal laser scanning microscopy. PMID:26535098

  20. Spectrally encoded confocal microscopy (SECM) for rapid assessment of breast excision specimens (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Brachtel, Elena F.; Johnson, Nicole B.; Huck, Amelia E.; Rice-Stitt, Travis L.; Vangel, Mark G.; Smith, Barbara L.; Tearney, Guillermo J.; Kang, DongKyun

    2016-03-01

    Unacceptably large percentage (20-40%) of breast cancer lumpectomy patients are required to undergo multiple surgeries when positive margins are found upon post-operative histologic assessment. If the margin status can be determined during surgery, surgeon can resect additional tissues to achieve tumor-free margin, which will reduce the need for additional surgeries. Spectrally encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technology that has a potential to image the entire surgical margin within a short procedural time. Previously, SECM was shown to rapidly image a large area (10 mm by 10 mm) of human esophageal tissue within a short procedural time (15 seconds). When used in lumpectomy, SECM will be able to image the entire margin surface of ~30 cm2 in around 7.5 minutes. SECM images will then be used to determine margin status intra-operatively. In this paper, we present results from a study of testing accuracy of SECM for diagnosing malignant breast tissues. We have imaged freshly-excised breast specimens (N=46) with SECM. SECM images clearly visualized histomorphologic features associated with normal/benign and malignant breast tissues in a similar manner to histologic images. Diagnostic accuracy was tested by comparing SECM diagnoses made by three junior pathologists with corresponding histologic diagnoses made by a senior pathologist. SECM sensitivity and specificity were high, 0.91 and 0.93, respectively. Intra-observer agreement and inter-observer agreement were also high, 0.87 and 0.84, respectively. Results from this study showed that SECM has a potential to accurately determine margin status during breast cancer lumpectomy.

  1. Spectrally Encoded Confocal Microscopy (SECM) for Diagnosing of Breast Cancer in Excision and Margin Specimens

    PubMed Central

    Brachtel, Elena F.; Johnson, Nicole B.; Huck, Amelia E.; Rice-Stitt, Travis L.; Vangel, Mark G.; Smith, Barbara L.; Tearney, Guillermo J.; Kang, Dongkyun

    2016-01-01

    A large percentage of breast cancer patients treated with breast conserving surgery need to undergo multiple surgeries due to positive margins found during post-operative margin assessment. Carcinomas could be removed completely during the initial surgery and additional surgery avoided if positive margins can be determined intra-operatively. Spectrally-encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technology that has a potential to rapidly image the entire surgical margin at sub-cellular resolution and accurately determine margin status intra-operatively. In this paper, in order to test feasibility of using SECM for intra-operative margin assessment, we have evaluated the diagnostic accuracy of SECM for detecting various types of breast cancers. Forty-six surgically-removed breast specimens were imaged with a SECM system. Side-by-side comparison between SECM and histologic images showed that SECM images can visualize key histomorphologic patterns of normal/benign and malignant breast tissues. Small (500 µm × 500 µm) spatially-registered SECM and histologic images (n=124 for each) were diagnosed independently by three pathologists with expertise in breast pathology. Diagnostic accuracy of SECM for determining malignant tissues was high, average sensitivity of 0.91, specificity of 0.93, positive predictive value of 0.95, and negative predictive value of 0.87. Intra-observer agreement and inter-observer agreement for SECM were also high, 0.87 and 0.84, respectively. Results from this study suggest that SECM may be developed into an intra-operative margin assessment tool for guiding breast cancer excisions. PMID:26779830

  2. Web-based visualisation and analysis of 3D electron-microscopy data from EMDB and PDB☆

    PubMed Central

    Lagerstedt, Ingvar; Moore, William J.; Patwardhan, Ardan; Sanz-García, Eduardo; Best, Christoph; Swedlow, Jason R.; Kleywegt, Gerard J.

    2013-01-01

    The Protein Data Bank in Europe (PDBe) has developed web-based tools for the visualisation and analysis of 3D electron microscopy (3DEM) structures in the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB). The tools include: (1) a volume viewer for 3D visualisation of maps, tomograms and models, (2) a slice viewer for inspecting 2D slices of tomographic reconstructions, and (3) visual analysis pages to facilitate analysis and validation of maps, tomograms and models. These tools were designed to help non-experts and experts alike to get some insight into the content and assess the quality of 3DEM structures in EMDB and PDB without the need to install specialised software or to download large amounts of data from these archives. The technical challenges encountered in developing these tools, as well as the more general considerations when making archived data available to the user community through a web interface, are discussed. PMID:24113529

  3. Web-based visualisation and analysis of 3D electron-microscopy data from EMDB and PDB.

    PubMed

    Lagerstedt, Ingvar; Moore, William J; Patwardhan, Ardan; Sanz-García, Eduardo; Best, Christoph; Swedlow, Jason R; Kleywegt, Gerard J

    2013-11-01

    The Protein Data Bank in Europe (PDBe) has developed web-based tools for the visualisation and analysis of 3D electron microscopy (3DEM) structures in the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB). The tools include: (1) a volume viewer for 3D visualisation of maps, tomograms and models, (2) a slice viewer for inspecting 2D slices of tomographic reconstructions, and (3) visual analysis pages to facilitate analysis and validation of maps, tomograms and models. These tools were designed to help non-experts and experts alike to get some insight into the content and assess the quality of 3DEM structures in EMDB and PDB without the need to install specialised software or to download large amounts of data from these archives. The technical challenges encountered in developing these tools, as well as the more general considerations when making archived data available to the user community through a web interface, are discussed.

  4. Roughness of biopores and cracks in Bt-horizons by confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Leue, Martin; Gerke, Horst H.

    2016-04-01

    During preferential flow events in structured soils, the movement of water and reactive solutes is mostly restricted to larger inter-aggregate pores, cracks, and biopores. The micro-topography of such macropores in terms of pore shapes, geometry, and roughness is crucial for describing the exchange of water and solutes between macropores and the soil matrix. The objective of this study was to determine the surface roughness of intact structural surfaces from the Bt-horizon of Luvisols by confocal laser scanning microscopy. For this purpose, samples with the structural surface types including cracks with and without clay-organic coatings from Bt-horizons developed on loess and glacial till were compared. The surface roughness of these structures was calculated in terms of three parameters from selected surface regions of 0.36 mm² determined with a confocal laser scanning microscope of the type Keyence VK-X100K. These data were evaluated in terms of the root-mean-squared roughness, Rq, the curvature, Rku, and the ratio between surface area and base area, RA. Values of Rq and RA were smaller for coated as compared to uncoated cracks and earthworm burrows of the Bt-horizons from both parent materials. The results indicated that the illuviation of clayey material led to a "smoothing" of the crack surfaces, which was similar for the coarser textured till-Bt and the finer-textured loess-Bt surfaces. The roughness indicated by Rq and RA values was only slightly smaller and that indicated by Rku slightly higher for the structural surfaces from the loess as compared to those from the glacial till. These results suggest a minor importance of the parent material on the roughness of structural surfaces in the Bt-horizon. The similarity of Rq, RA, and Rku values between surfaces of earthworm burrows and uncoated cracks did not confirm an expected smoothing effect of the burrow walls by the earthworm. In contrast to burrow walls, root channels from the loess-Bt were smoother

  5. A multi-model approach to simultaneous segmentation and classification of heterogeneous populations of cell nuclei in 3D confocal microscope images.

    PubMed

    Lin, Gang; Chawla, Monica K; Olson, Kathy; Barnes, Carol A; Guzowski, John F; Bjornsson, Christopher; Shain, William; Roysam, Badrinath

    2007-09-01

    Automated segmentation and morphometry of fluorescently labeled cell nuclei in batches of 3D confocal stacks is essential for quantitative studies. Model-based segmentation algorithms are attractive due to their robustness. Previous methods incorporated a single nuclear model. This is a limitation for tissues containing multiple cell types with different nuclear features. Improved segmentation for such tissues requires algorithms that permit multiple models to be used simultaneously. This requires a tight integration of classification and segmentation algorithms. Two or more nuclear models are constructed semiautomatically from user-provided training examples. Starting with an initial over-segmentation produced by a gradient-weighted watershed algorithm, a hierarchical fragment merging tree rooted at each object is built. Linear discriminant analysis is used to classify each candidate using multiple object models. On the basis of the selected class, a Bayesian score is computed. Fragment merging decisions are made by comparing the score with that of other candidates, and the scores of constituent fragments of each candidate. The overall segmentation accuracy was 93.7% and classification accuracy was 93.5%, respectively, on a diverse collection of images drawn from five different regions of the rat brain. The multi-model method was found to achieve high accuracy on nuclear segmentation and classification by correctly resolving ambiguities in clustered regions containing heterogeneous cell populations.

  6. Monitoring UVR induced damage in single cells and isolated nuclei using SR-FTIR microspectroscopy and 3D confocal Raman imaging.

    PubMed

    Lipiec, Ewelina; Bambery, Keith R; Heraud, Philip; Kwiatek, Wojciech M; McNaughton, Don; Tobin, Mark J; Vogel, Christian; Wood, Bayden R

    2014-09-01

    SR-FTIR in combination with Principal Component Analysis (PCA) was applied to investigate macromolecular changes in a population of melanocytes and their extracted nuclei induced by environmentally relevant fluxes of UVR (Ultraviolet Radiation). Living cells and isolated cellular nuclei were investigated post-irradiation for three different irradiation dosages (130, 1505, 15,052 Jm(-2) UVR, weighted) after either 24 or 48 hours of incubation. DNA conformational changes were observed in cells exposed to an artificial UVR solar-simulator source as evidenced by a shift in the DNA asymmetric phosphodiester vibration from 1236 cm(-1) to 1242 cm(-1) in the case of the exposed cells and from 1225 cm(-1) to 1242 cm(-1) for irradiated nuclei. PCA Scores plots revealed distinct clustering of spectra from irradiated cells and nuclei from non-irradiated controls in response to the range of applied UVR radiation doses. 3D Raman confocal imaging in combination with k-means cluster analysis was applied to study the effect of the UVR radiation exposure on cellular nuclei. Chemical changes associated with apoptosis were detected and included intra-nuclear lipid deposition along with chromatin condensation. The results reported here demonstrate the utility of SR-FTIR and Raman spectroscopy to probe in situ DNA damage in cell nuclei resulting from UVR exposure. These results are in agreement with the increasing body of evidence that lipid accumulation is a characteristic of aggressive cancer cells, and are involved in the production of membranes for rapid cell proliferation.

  7. Feasibility study on 3-D shape analysis of high-aspect-ratio features using through-focus scanning optical microscopy

    PubMed Central

    Attota, Ravi Kiran; Weck, Peter; Kramar, John A.; Bunday, Benjamin; Vartanian, Victor

    2016-01-01

    In-line metrologies currently used in the semiconductor industry are being challenged by the aggressive pace of device scaling and the adoption of novel device architectures. Metrology and process control of three-dimensional (3-D) high-aspect-ratio (HAR) features are becoming increasingly important and also challenging. In this paper we present a feasibility study of through-focus scanning optical microscopy (TSOM) for 3-D shape analysis of HAR features. TSOM makes use of 3-D optical data collected using a conventional optical microscope for 3-D shape analysis. Simulation results of trenches and holes down to the 11 nm node are presented. The ability of TSOM to analyze an array of HAR features or a single isolated HAR feature is also presented. This allows for the use of targets with area over 100 times smaller than that of conventional gratings, saving valuable real estate on the wafers. Indications are that the sensitivity of TSOM may match or exceed the International Technology Roadmap for Semiconductors (ITRS) measurement requirements for the next several years. Both simulations and preliminary experimental results are presented. The simplicity, lowcost, high throughput, and nanometer scale 3-D shape sensitivity of TSOM make it an attractive inspection and process monitoring solution for nanomanufacturing. PMID:27464112

  8. Feasibility study on 3-D shape analysis of high-aspect-ratio features using through-focus scanning optical microscopy.

    PubMed

    Attota, Ravi Kiran; Weck, Peter; Kramar, John A; Bunday, Benjamin; Vartanian, Victor

    2016-07-25

    In-line metrologies currently used in the semiconductor industry are being challenged by the aggressive pace of device scaling and the adoption of novel device architectures. Metrology and process control of three-dimensional (3-D) high-aspect-ratio (HAR) features are becoming increasingly important and also challenging. In this paper we present a feasibility study of through-focus scanning optical microscopy (TSOM) for 3-D shape analysis of HAR features. TSOM makes use of 3-D optical data collected using a conventional optical microscope for 3-D shape analysis. Simulation results of trenches and holes down to the 11 nm node are presented. The ability of TSOM to analyze an array of HAR features or a single isolated HAR feature is also presented. This allows for the use of targets with area over 100 times smaller than that of conventional gratings, saving valuable real estate on the wafers. Indications are that the sensitivity of TSOM may match or exceed the International Technology Roadmap for Semiconductors (ITRS) measurement requirements for the next several years. Both simulations and preliminary experimental results are presented. The simplicity, lowcost, high throughput, and nanometer scale 3-D shape sensitivity of TSOM make it an attractive inspection and process monitoring solution for nanomanufacturing. PMID:27464112

  9. 3D scanning electron microscopy applied to surface characterization of fluorosed dental enamel.

    PubMed

    Limandri, Silvina; Galván Josa, Víctor; Valentinuzzi, María Cecilia; Chena, María Emilia; Castellano, Gustavo

    2016-05-01

    The enamel surfaces of fluorotic teeth were studied by scanning electron stereomicroscopy. Different whitening treatments were applied to 25 pieces to remove stains caused by fluorosis and their surfaces were characterized by stereomicroscopy in order to obtain functional and amplitude parameters. The topographic features resulting for each treatment were determined through these parameters. The results obtained show that the 3D reconstruction achieved from the SEM stereo pairs is a valuable potential alternative for the surface characterization of this kind of samples.

  10. Simple 3D images from fossil and recent micromaterial using light microscopy.

    PubMed

    Haug, J T; Haug, C; Maas, A; Fayers, S R; Trewin, N H; Waloszek, D

    2009-01-01

    Abstract We present a technique for extracting 3D information from small-scale fossil and Recent material and give a summary of other contemporary techniques for 3D methods of investigation. The only hardware needed for the here-presented technique is a microscope that can perform dark field and/or differential interference contrast with a mounted digital camera and a computer. Serial images are taken while the focus is successively shifted from the uppermost end of the specimen to the lowermost end, resulting in about 200 photographs. The data are then processed almost completely automatically by successive use of three freely available programs. Firstly, the stack of images is aligned by the use of CombineZM, which is used to produce a combined image with a high depth of field. Secondly, the aligned images are cropped and sharp edges extracted with the aid of ImageJ. Thirdly, although ImageJ is also capable of producing 3D representations, we preferred to process the image stack further using osirix as it has the facility to export various formats. One of the interesting export formats is a virtual Quicktime movie file (QTVR), which can be used for documentation, and stereo images can also be produced from this Quicktime VR. This method is easy to apply and can be used for documenting specimens in 3D (at least some aspects) without having to prepare them. Therefore, it is particularly useful as a safe method for documenting limited material, before using methods that may destroy the specimen of interest, or to investigate type material that cannot be treated with any preparatory technique. As light microscopes are available in most labs and free computer programs are easily accessible, this method can be readily applied. PMID:19196416

  11. The application of laser scanning confocal microscopy to the examination of hairs and textile fibers: an initial investigation.

    PubMed

    Kirkbride, K Paul; Tridico, Silvana R

    2010-02-25

    An initial investigation of the application of laser scanning confocal microscopy to the examination of hairs and fibers has been conducted. This technique allows the production of virtual transverse and longitudinal cross-sectional images of a wide range of hairs and fibers. Special mounting techniques are not required; specimens that have been mounted for conventional microscopy require no further treatment. Unlike physical cross-sectioning, in which it is difficult to produce multiple cross-sections from a single hair or fiber and the process is destructive, confocal microscopy allows the examiner to image the cross-section at any point in the field of view along the hair or fiber and it is non-destructive. Confocal microscopy is a fluorescence-based technique. The images described in this article were collected using only the autofluorescence exhibited by the specimen (i.e. fluorescence staining was not necessary). Colorless fibers generally and hairs required excitation at 405 nm in order to stimulate useful autofluorescence; longer wavelength excitation was suitable for dyed fibers. Although confocal microscopy was found to be generally applicable to the generation virtual transverse cross-sections from a wide range of hairs and fibers, on some occasions the autofluorescence signal was attenuated by heavy pigmentation or the presence of an opaque medulla in hairs, and by heavy delustering or the presence of air-filled voids in the case of fibers. In these situations only partial cross-sections were obtained.

  12. Improved volume rendering for the visualization of living cells examined with confocal microscopy

    NASA Astrophysics Data System (ADS)

    Enloe, L. Charity; Griffing, Lawrence R.

    2000-02-01

    This research applies recent advances in 3D isosurface reconstruction to images of test spheres and plant cells growing in suspension culture. Isosurfaces that represent object boundaries are constructed with a Marching Cubes algorithm applied to simple data sets, i.e., fluorescent test beads, and complex data sets, i.e., fluorescent plant cells, acquired with a Zeiss Confocal Laser Scanning Microscope (LSM). The marching cubes algorithm treats each pixel or voxel of the image as a separate entity when performing computations. To test the spatial accuracy of the reconstruction, control data representing the volume of a 25 micrometer test shaper was obtained with the LSM. This volume was then judged on the basis of uniformity and smoothness. Using polygon decimation and smoothing algorithms available through the visualization toolkit, 'voxellated' test spheres and cells were smoothed using several different smoothing algorithms after unessential polygons were eliminated. With these improvements, the shape of subcellular organelles could be modeled at various levels of accuracy. However, in order to accurately reconstruct these complex structures of interest to us, the subcellular organelles of the endosomal system or the endoplasmic reticulum of plant cells, measurements of the accuracy of connectedness of structures need to be developed.

  13. Automated Confocal Laser Scanning Microscopy and Semiautomated Image Processing for Analysis of Biofilms

    PubMed Central

    Kuehn, Martin; Hausner, Martina; Bungartz, Hans-Joachim; Wagner, Michael; Wilderer, Peter A.; Wuertz, Stefan

    1998-01-01

    The purpose of this study was to develop and apply a quantitative optical method suitable for routine measurements of biofilm structures under in situ conditions. A computer program was designed to perform automated investigations of biofilms by using image acquisition and image analysis techniques. To obtain a representative profile of a growing biofilm, a nondestructive procedure was created to study and quantify undisturbed microbial populations within the physical environment of a glass flow cell. Key components of the computer-controlled processing described in this paper are the on-line collection of confocal two-dimensional (2D) cross-sectional images from a preset 3D domain of interest followed by the off-line analysis of these 2D images. With the quantitative extraction of information contained in each image, a three-dimensional reconstruction of the principal biological events can be achieved. The program is convenient to handle and was generated to determine biovolumes and thus facilitate the examination of dynamic processes within biofilms. In the present study, Pseudomonas fluorescens or a green fluorescent protein-expressing Escherichia coli strain, EC12, was inoculated into glass flow cells and the respective monoculture biofilms were analyzed in three dimensions. In this paper we describe a method for the routine measurements of biofilms by using automated image acquisition and semiautomated image analysis. PMID:9797255

  14. Correlated Biofilm Imaging, Transport and Metabolism Measurements via Combined Nuclear Magnetic Resonance and Confocal Microscopy

    SciTech Connect

    Mclean, Jeffrey S.; Ona, Ositadinma; Majors, Paul D.

    2008-02-18

    Bacterial biofilms are complex, three-dimensional, communities that are found nearly everywhere in nature1 and are being recognized as the cause of treatment-resistant infections1 2. Advanced methods are required to characterize their collective and spatial patterns of metabolism however most techniques are invasive or destructive. Here we describe the use of a combined confocal laser scanning microscopy (CLSM) and nuclear magnetic resonance (NMR) microscopy system to monitor structure, mass transport, and metabolism in active biofilms. Non-invasive NMR methods provide macroscopic structure along with spatially-resolved metabolite profiles and diffusion measurements. CLSM enables monitoring of cells by fluorescent protein reporters to investigate biofilm structure and gene expression concurrently. A planar sample chamber design facilitates depth-resolved measurements on 140 nL sample volumes under laminar flow conditions. The techniques and approaches described here are applicable to environmental and medically relevant microbial communities, thus providing key metabolic information for promoting beneficial biofilms and treating associated diseases.

  15. Assessing strain mapping by electron backscatter diffraction and confocal Raman microscopy using wedge-indented Si.

    PubMed

    Friedman, Lawrence H; Vaudin, Mark D; Stranick, Stephan J; Stan, Gheorghe; Gerbig, Yvonne B; Osborn, William; Cook, Robert F

    2016-04-01

    The accuracy of electron backscatter diffraction (EBSD) and confocal Raman microscopy (CRM) for small-scale strain mapping are assessed using the multi-axial strain field surrounding a wedge indentation in Si as a test vehicle. The strain field is modeled using finite element analysis (FEA) that is adapted to the near-indentation surface profile measured by atomic force microscopy (AFM). The assessment consists of (1) direct experimental comparisons of strain and deformation and (2) comparisons in which the modeled strain field is used as an intermediate step. Direct experimental methods (1) consist of comparisons of surface elevation and gradient measured by AFM and EBSD and of Raman shifts measured and predicted by CRM and EBSD, respectively. Comparisons that utilize the combined FEA-AFM model (2) consist of predictions of distortion, strain, and rotation for comparison with EBSD measurements and predictions of Raman shift for comparison with CRM measurements. For both EBSD and CRM, convolution of measurements in depth-varying strain fields is considered. The interconnected comparisons suggest that EBSD was able to provide an accurate assessment of the wedge indentation deformation field to within the precision of the measurements, approximately 2×10(-4) in strain. CRM was similarly precise, but was limited in accuracy to several times this value. PMID:26939030

  16. Electrochemical preparation of silver and gold nanoparticles: Characterization by confocal and surface enhanced Raman microscopy

    NASA Astrophysics Data System (ADS)

    Plieth, W.; Dietz, H.; Anders, A.; Sandmann, G.; Meixner, A.; Weber, M.; Kneppe, H.

    2005-12-01

    Localized silver and gold nanoparticles, electrochemically prepared by means of the double-pulse technique, were investigated with respect to their optical and spectroscopic properties by scanning confocal microscopy combined with surface enhanced Raman spectroscopy (SERS) and subsequent comparison with the local image of scanning electron microscopy (SEM). Analogous to the silver cluster preparation technique, controlled electrodeposition of gold nanoparticles was demonstrated, varying size from 10 to 500 nm and particle density. The maximum SERS enhancement factors found in the measurements were: (i) 10 10 for silver particles and (ii) 10 8 for gold particles. The optical and spectroscopic data of the local nanoparticle structures investigated showed that SERS is a local phenomenon, because (i) only few particles are Raman active particles, (ii) strongest enhancements in SERS are obtained from particle agglomerates, (iii) typically the Raman radiation is emitted from irregular structures like the necks between two or more particles agglomerated. In the investigated range from 10 to 500 nm no significant influence of the particle size was observed.

  17. Shear bond strength, failure modes, and confocal microscopy of bonded amalgam restorations.

    PubMed

    Cianconi, Luigi; Conte, Gabriele; Mancini, Manuele

    2011-01-01

    This study evaluated the shear bond strength, failure modes, and confocal microscopy of two different amalgam alloy restorations lined with five adhesive systems. Two regular-set high-copper dental amalgam alloys, Amalcap Plus and Valiant Ph.D, and five commercially available adhesive systems were selected. One hundred and twenty freshly-extracted human third molars were used for the study. The results were statistically evaluated using two-factor analysis of variance (ANOVA). The shear bond strength (SBS) of amalgam to dentin was significantly affected by both the adhesive (p<0.0001) and amalgam alloy (p<0.0002). Regarding mode of failure (MF), among samples restored with Valiant Ph.D, 31 of 50 exhibited adhesive failure, and 19 displayed mixed failure. Laser optical microscopy (OM) of the bonded interface revealed the presence of a good hybrid layer was evident in all experimental groups. Higher bond strengths were measured for four of the five adhesives when used in combination with the spherical alloy. PMID:21383518

  18. Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

    PubMed

    Mas, Abraham; Amenós, Montse; Lois, L Maria

    2016-01-01

    Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living ce