Video microscopic image processing facilitates the evaluation of light microscopic autoradiography at high magnification
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SUMMARY Light microscopic autoradiographs of H‐thymidine labelled unstained semithin sections of Xenopus laevis embryonic nuclei were examined with conventional Nomarski differential interference contrast, phase‐contrast and video microscopy. Whereas at low magnification it was possible to obtain a photograph of the nuclear structure and the silver grains in one focal plain, at high magnification, with small depths of focus, a satisfactory image was not attainable. Therefore, we stored the images of the two different focus levels with a digital image processing system and combined both images by an arithmetic operation. This video microscopic technique allows the use of high magnification light microscopy with oil immersion objectives and the application of additional electronic contrast enhancing methods for an adequate and rapid analysis of light microscopic autoradiographs.Keywords:
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Biomedical applications aimed toward understanding livecell dynamics have rekindled an interest in optical microscope modes that may provide additional information beyond the widely used ¦uorescence modalities that include confocal, structured illumination microscopy, multiphoton, and STED imaging. ™e three microscope modes discussed in this chapter-dark-›eld, phase contrast, and dižerential interference contrast (DIC) microscopy-are techniques that readily address the challenges of live-cell imaging. ™eir ability to image cell structure dynamics without having to introduce any chemical probes or dyes can be of great advantage in ensuring that cell function is not being altered. In addition, the ability to observe living cells over time, without introducing the potentially damaging ežects of high-intensity light sources, ožered by these techniques is o²en desirable for many biological investigations.
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Like phase contrast, Nomarski interference contrast microscopy can be used to examine unstained specimens in biology and medicine. The properties of both contrast enhancement techniques are illustrated by various examples. The phase contrast method is especially suited for thin specimens with small differences in refractive index, whereas the interference contrast method supplies good results even of thick specimens. Interference contrast is a valuable supplement to the phase contrast method, and expands the application of microscopy in biology
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The use of phase contrast and differential interference contrast (DIC) microscopy enhances the observation of structural detail within a cell. Phase-contrast microscopy is more widely available to veterinarians than DIC microscopy due to the higher cost of the DIC equipment. Wet mount preparation, as used in phase-contrast and DIC microscopy, causes less physical damage to spermatozoa. This chapter discusses the processes involved in the preparation of a wet mount. It also provides a description of DIC microscopy and phase-contrast microscopy. Sperm morphology should be evaluated under 400× magnification or higher. If the sample is too concentrated, spermatozoa will overlie each other, making evaluation of morphology difficult. The sample should be appropriately diluted to allow examination of individual spermatozoa.
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Frozen bull semen was analyzed after fixation with glutaraldehyde (0.2% sol. in PBS) by clear field microscopy (after staining with Rose Bengal and Victoria blue B), phase contrast microscopy and differential-interference-contrast microscopy performed by two observers who analysed sets of 100 and 200 spermatozoa. The results obtained with phase-contrast microscopy did not differ significantly from those obtained using interference contrast microscopy, performed by two observers on sets on 100 and 200 spermatozoa, concerning the following sperm abnormalities: abnormal detached heads, acrosome ruptures, tail abnormalities and total abnormalities. In view of the importance of extending the evaluation of sperm cytomorphology among artificial insemination centres, and the fact the phase-contrast microscopy system is less expensive than a differential-interference-contrast system and easier to operate, the authors recommend the use of phase-contrast microscopy for the routine study of sperm cytomorphology.
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In the field of biology and medicine, observation object of the microscope has been changing from the thin specimen
to the thick living tissue. Furthermore, observation of the internal structure of a living tissue is also desired by low
invasion. However, the real structure of a phase object with three-dimensional distribution such as a living tissue is
difficult to observe, because of the influence of the phase distribution before and behind of observation position. We
enabled observation of the internal structure of living tissue without stain, by adding a new function to reduce the
influence of phase distribution to our Retardation-Modulated differential interference contrast (RM-DIC) microscope
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Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the phase halos typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.
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Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the phase halos typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.
Interference microscopy
Bright-field microscopy
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