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Optics Express Jul 2013In spiral phase contrast (SPC) microscopy the edge-enhancement is typically independent of the helicity of the phase vortex filter. Here we show that for layered...
In spiral phase contrast (SPC) microscopy the edge-enhancement is typically independent of the helicity of the phase vortex filter. Here we show that for layered specimens containing screw-dislocations, as are e.g. present in mica or some crystallized organic substances, the intensity distribution in the filtered image acquires a dependence on the rotational direction of the filter. This allows one to map the distribution of phase singularities in the topography of the sample, by taking the intensity difference between two images recorded with opposite handedness. For the demonstration of this feature in a microscopy set-up, we encode the vortex filter as a binary off-axis hologram displayed on a spatial light modulator (SLM) placed in a Fourier plane. Using a binary grating, the diffraction efficiencies for the plus and minus first diffraction orders are equal, giving rise to two image waves which travel in different directions and are Fourier filtered with opposite helicity. The corresponding two images can be recorded simultaneously in two separate regions of the camera chip. This enables mapping of dislocations in the sample in a single camera exposure, as was demonstrated for various transparent samples.
Topics: Equipment Design; Equipment Failure Analysis; Holography; Image Enhancement; Microscopy, Phase-Contrast; Refractometry
PubMed: 23938479
DOI: 10.1364/OE.21.016282 -
Journal of Anatomy Apr 1986Cultures of neurons and non-neuronal cells were prepared from adult mouse dorsal root ganglia and maintained for periods of up to six weeks upon uncoated petri dish...
Cultures of neurons and non-neuronal cells were prepared from adult mouse dorsal root ganglia and maintained for periods of up to six weeks upon uncoated petri dish substrates. Modifications were made to both existing isolation procedures and culture medium to ensure high cell survival. Cell structure was observed using phase contrast microscopy for living cells and standard light microscopy for Palmgren silver stained preparations. Scanning and transmission electron microscopy revealed the fine structure of the cells and showed the close relationship of satellite cells with the surviving dark neurons as culture establishment progressed. Satellite cells were associated with both the cell bodies and the regenerated neuritic processes. The use of the cultures for future cytotoxicity testing is assessed.
Topics: Animals; Cells, Cultured; Ganglia, Spinal; Mice; Mice, Inbred CBA; Microscopy, Electron; Microscopy, Phase-Contrast; Time Factors
PubMed: 3323151
DOI: No ID Found -
Applied Optics Jan 2006We describe a new technique for differential interference contrast (DIC) microscopy, which digitally generates phase gradient images independently of gradient...
We describe a new technique for differential interference contrast (DIC) microscopy, which digitally generates phase gradient images independently of gradient orientation. To prove the principle we investigated specimens recorded at different orientations on a microscope equipped with a precision rotating stage and using regular DIC optics. The digitally generated images successfully displayed and measured phase gradients, independently of gradient orientation. One could also generate images showing distribution of optical path differences or enhanced, regular DIC images with any shear direction. Using special DIC prisms, one can switch the bias and shear directions rapidly without mechanically rotating the specimen or the prisms and orientation-independent DIC images are obtained in a fraction of a second.
Topics: Algorithms; Animals; Cells, Cultured; Endothelial Cells; Image Enhancement; Image Interpretation, Computer-Assisted; Microscopy, Phase-Contrast; Refractometry; Reproducibility of Results; Sensitivity and Specificity; Swine
PubMed: 16463729
DOI: 10.1364/ao.45.000460 -
PloS One 2021We report the application of supervised machine learning to the automated classification of lipid droplets in label-free, quantitative-phase images. By comparing various...
We report the application of supervised machine learning to the automated classification of lipid droplets in label-free, quantitative-phase images. By comparing various machine learning methods commonly used in biomedical imaging and remote sensing, we found convolutional neural networks to outperform others, both quantitatively and qualitatively. We describe our imaging approach, all implemented machine learning methods, and their performance with respect to computational efficiency, required training resources, and relative method performance measured across multiple metrics. Overall, our results indicate that quantitative-phase imaging coupled to machine learning enables accurate lipid droplet classification in single living cells. As such, the present paradigm presents an excellent alternative of the more common fluorescent and Raman imaging modalities by enabling label-free, ultra-low phototoxicity, and deeper insight into the thermodynamics of metabolism of single cells.
Topics: Deep Learning; Image Processing, Computer-Assisted; Lipid Droplets; Microscopy, Phase-Contrast; Yarrowia
PubMed: 33819277
DOI: 10.1371/journal.pone.0249196 -
The Annals of Occupational Hygiene Jun 2015A Japanese round-robin study revealed that analysts who used a dark-medium (DM) objective lens reported higher fiber counts from American Industrial Hygiene Association...
A Japanese round-robin study revealed that analysts who used a dark-medium (DM) objective lens reported higher fiber counts from American Industrial Hygiene Association (AIHA) Proficiency Analytical Testing (PAT) chrysotile samples than those using a standard objective lens, but the cause of this difference was not investigated at that time. The purpose of this study is to determine any major source of this difference by performing two sets of round-robin studies. For the first round-robin study, 15 AIHA PAT samples (five each of chrysotile and amosite generated by water-suspended method, and five chrysotile generated by aerosolization method) were prepared with relocatable cover slips and examined by nine laboratories. A second round-robin study was then performed with six chrysotile field sample slides by six out of nine laboratories who participated in the first round-robin study. In addition, two phase-shift test slides to check analysts' visibility and an eight-form diatom test plate to compare resolution between the two objectives were examined. For the AIHA PAT chrysotile reference slides, use of the DM objective resulted in consistently higher fiber counts (1.45 times for all data) than the standard objective (P-value < 0.05), regardless of the filter generation (water-suspension or aerosol) method. For the AIHA PAT amosite reference and chrysotile field sample slides, the fiber counts between the two objectives were not significantly different. No statistically significant differences were observed in the visibility of blocks of the test slides between the two objectives. Also, the DM and standard objectives showed no pattern of differences in viewing the fine lines and/or dots of each species images on the eight-form diatom test plate. Among various potential factors that might affect the analysts' performance of fiber counts, this study supports the greater contrast caused by the different phase plate absorptions as the main cause of high counts for the AIHA PAT chrysotile slides using the DM objective. The comparison of fiber count ratios (DM/standard) between the AIHA PAT chrysotile samples and chrysotile field samples indicates that there is a fraction of fibers in the PAT samples approaching the theoretical limit of visibility of the phase-contrast microscope with 3-degree phase-shift. These fibers become more clearly visible through the greater contrast from the phase plate absorption of the DM objective. However, as such fibers are not present in field samples, no difference in counts between the two objectives was observed in this study. The DM objective, therefore, could be allowed for routine fiber counting as it will maintain continuity with risk assessments based on earlier phase-contrast microscopy fiber counts from field samples. Published standard methods would need to be modified to allow a higher aperture specification for the objective.
Topics: Air Pollutants; Asbestos, Amosite; Asbestos, Serpentine; Microscopy, Phase-Contrast; Mineral Fibers; National Institute for Occupational Safety and Health, U.S.; Observer Variation; Occupational Exposure; Reproducibility of Results; United States
PubMed: 25737333
DOI: 10.1093/annhyg/mev007 -
Journal of Biomedical Optics 2008Microscopic images of biological phase specimens of various optical thickness, acquired under off-axis illumination and apodized/conventional phase-contrast are... (Comparative Study)
Comparative Study
Microscopic images of biological phase specimens of various optical thickness, acquired under off-axis illumination and apodized/conventional phase-contrast are compared. The luminance profiles in appropriately filtered apodized phase-contrast images compare well with those in the original off-axis illumination images. The two unfiltered image types also yield similar results in terms of quasi-three-dimensional surface (pseudo-relief) rendering, and thus are comparable in terms of the information contents (optical thickness map). However, the overall visual impression is very different as the visual cues to depth structure are present in the off-axis illumination images only. The comparison demonstrated in the present paper was made possible owing to apodization, which substantially reduces the "halo"/shade-off artifacts in the phase-contrast images. The results imply the possibility of combining the off-axis illumination and apodized phase-contrast imaging to examine specimens of medium optical thickness, in which the phase visualization capability of the two imaging modes substantially overlaps (e.g., larger cells or cell clusters).
Topics: Algorithms; Image Enhancement; Image Interpretation, Computer-Assisted; Lighting; Microscopy, Phase-Contrast; Reproducibility of Results; Sensitivity and Specificity; Statistics as Topic
PubMed: 19021445
DOI: 10.1117/1.2966716 -
Physical Review Letters Jul 2009In biological samples the resonant coherent anti-Stokes Raman scattering signal of less abundant constituents can be overwhelmed by the nonresonant background,...
In biological samples the resonant coherent anti-Stokes Raman scattering signal of less abundant constituents can be overwhelmed by the nonresonant background, preventing detection of those molecules. We demonstrate a method to obtain the phase of the oscillators in the focal volume that allows discrimination of those hidden molecules. The phase is measured with respect to the local excitation fields using a cascaded phase-preserving chain. It is measured point-by-point and takes into account refractive index changes in the sample, phase curvature over the field-of-view, and interferometric instabilities. The detection of the phase of the vibrational motion can be regarded as a vibrational extension of the linear (refractive index) phase contrast microscopy introduced by Zernike around 1933.
Topics: HeLa Cells; Humans; Interferometry; Microscopy, Phase-Contrast; Spectrum Analysis, Raman
PubMed: 19659356
DOI: 10.1103/PhysRevLett.103.043905 -
Journal of Biomedical Optics Feb 2012Human retinal and choroidal vasculature was visualized by a differential phase-contrast (DPC) method using high-speed, swept-source optical coherence tomography (SS-OCT)...
Human retinal and choroidal vasculature was visualized by a differential phase-contrast (DPC) method using high-speed, swept-source optical coherence tomography (SS-OCT) at 1060 nm. The vasculature was recognized as regions of motion by creating differential phase-variance (DPV) tomograms: multiple B-scans of individual slices through the retina were collected and the variance of the phase differences was calculated. DPV captured the small vessels and the meshwork of capillaries associated with the inner retina in en-face images over 4 mm(2). The swept-source laser at 1060 nm offered the needed phase sensitivity to perform DPV and generated en-face images that capture motion in the inner choroidal layer exceeding the capabilities of previous spectrometer-based instruments. In comparison with the power Doppler phase-shift method, DPV provided better visualization of the foveal avascular zone in en-face images.
Topics: Angiography; Choroid; Equipment Design; Equipment Failure Analysis; Humans; Microscopy, Confocal; Microscopy, Phase-Contrast; Reproducibility of Results; Retinal Vessels; Retinoscopes; Sensitivity and Specificity; Tomography, Optical Coherence
PubMed: 22463043
DOI: 10.1117/1.JBO.17.2.026011 -
Blood Jan 1969
Topics: Abortion, Induced; Cesarean Section; Culture Techniques; Electrophoresis; Femur; Fetal Hemoglobin; Fetus; Gels; Hematopoiesis; Hemoglobinometry; Liver; Megakaryocytes; Microscopy, Phase-Contrast; Spleen; Starch
PubMed: 4884282
DOI: No ID Found -
Journal of Microscopy May 2018Automated cell segmentation plays a key role in characterisations of cell behaviours for both biology research and clinical practices. Currently, the segmentation of...
Automated cell segmentation plays a key role in characterisations of cell behaviours for both biology research and clinical practices. Currently, the segmentation of clustered cells still remains as a challenge and is the main reason for false segmentation. In this study, the emphasis was put on the segmentation of clustered cells in negative phase contrast images. A new method was proposed to combine both light intensity and cell shape information through the construction of grey-weighted distance transform (GWDT) within preliminarily segmented areas. With the constructed GWDT, the clustered cells can be detected and then separated with a modified region skeleton-based method. Moreover, a contour expansion operation was applied to get optimised detection of cell boundaries. In this paper, the working principle and detailed procedure of the proposed method are described, followed by the evaluation of the method on clustered cell segmentation. Results show that the proposed method achieves an improved performance in clustered cell segmentation compared with other methods, with 85.8% and 97.16% accuracy rate for clustered cells and all cells, respectively.
Topics: Automation, Laboratory; Cell Shape; Epithelial Cells; Humans; Image Processing, Computer-Assisted; Intravital Microscopy; Microscopy, Phase-Contrast
PubMed: 29280132
DOI: 10.1111/jmi.12673