Microscope/microscopy phase contrast

Light Microscopic Method. Phase contrast microscopy (PCM) accurately assesses fiber exposure levels for fibers 5 pm in length and >0.25 pm in diameter. Furthermore, PCM cannot differentiate between asbestos and nonasbestos fibers. Currently, the standard method for the determination of airborne asbestos particles in the workplace is NIOSH Method 7400, Asbestos by Phase Contrast Microscopy (NIOSH 1994a). OSHA considers that sampling and analytical procedures contained in OSHA Method ID-160 and NIOSH Method 7400 are essential for obtaining adequate employee exposure monitoring. Therefore, all employers who are required to conduct monitoring are required to use these or equivalent methods to collect and analyze samples (OSHA 1994). In NIOSH Method 7400, asbestos is collected on a 25 mm cellulose ester filter (cassette-equipped with a 50 mm electrically-conductive cowl). The filter is treated to make it... 

Optical microscopy Phase contrast microscopy Polarized light microscopy Scanning electron microscopy Scanning ion conductance microscope Scanning probe microscopy Scanning thermal profiler... 

Mann, S. Meyer, J. Dietzel, I., Integration of a scanning ion conductance microscope into phase contrast optics and its application to the quantification of morphological parameters of selected cells. Journal of Microscopy 2006, 224, 152-157. 

The vesicles were observed on an inverted microscope in phase-contrast and confocal fluorescence modes, as follows. The white cloud of lipid was gently dispersed in the tube and introduced into an observation chamber. The chamber was filled with the same solution as the internal solution except that 0.1 M sucrose was replaced with 0.1 M glucose (external solution). Because of the difference in the reffactivity of the internal and external solutions, the contrast of the vesicle s images was enhanced in the phase-contrast mode. For the confocal fluorescence microscopy, a confocal scanner unit (CSU 10, Yokogawa, Japan) was used, and the lipophilic fluorescent dye Nile Red (Molecular Probes, Inc., OR, USA) was added to the starting lipid solution at 0.3 wt% of the lipid. 

Microscopy provides detailed information about miscibility and about phase morphology, i.e. the actual geometry of the phases. Optical microscopy resolves structures down to about 1 im. The samples may need staining prior to examination. In other cases, where the refractive index mismatch is sufficiently large, direct examination can be made in the microscope using phase-contrast or interference-contrast optical microscopy. 

Light microscopy allows, in comparison to other microscopic methods, quick, contact-free and non-destmctive access to the stmctures of materials, their surfaces and to dimensions and details of objects in the lateral size range down to about 0.2 pm. A variety of microscopes with different imaging and illumination systems has been constmcted and is conunercially available in order to satisfy special requirements. These include stereo, darkfield, polarization, phase contrast and fluorescence microscopes. 

Visual imaging of cell population in vitro) using low signal-to-noise ratio phase contrast microscopy can enable systematic monitoring measurements of cell quality, development and apoptosis. In the present study, microscopic evaluations as seen in Fig. 11 did not reveal any significant alteration in cellular morphology upto 1000 ng/ml. 

Barer, R. Some applications of phase-contrast microscopy. Quart. J. microscop. Sci- 88, 491—500 (1947)-... 

Figure 9.29 Membrane formation by meteoritic amphiphilic compounds (courtesy of David Deamer). A sample of the Murchison meteorite was extracted with the chloroform-methanol-water solvent described by Deamer and Pashley, 1989. Amphiphilic compounds were isolated chromatographically on thin-layer chromatography plates (fraction 1), and a small aliquot ( 1 p,g) was dried on a glass microscope slide. Alkaline carbonate buffer (15 p,l, 10 mM, pH 9.0) was added to the dried sample, followed by a cover slip, and the interaction of the aqueous phase with the sample was followed by phase-contrast and fluorescence microscopy, (a) The sample-buffer interface was 1 min. The aqueous phase penetrated the viscous sample, causing spherical structures to appear at the interface and fall away into the medium, (b) After 30 min, large numbers of vesicular structures are produced as the buffer further penetrates the sample, (c) The vesicular nature of the structures in (b) is clearly demonstrated by fluorescence microscopy. Original magnification in (a) is x 160 in (b) and (c) x 400.

Later, differential interference microscopy was developed, enabling the detection of difference in levels as sensitively as phase contrast microscopy, and, because this technique was easier to use, it came to be used in preference to the former techniques [6]. Differential interference microscopy is superior to phase contrast microscopy in the observation of vicinal or curved surfaces, which are impossible to observe under a phase contrast microscope because the contrast is too high. 

Powerful methods that have been developed more recently, and are currently used to observe surface micro topographs of crystal faces, include scanning tunnel microscopy (STM), atomic force microscopy (AFM), and phase shifting microscopy (PSM). Both STM and AFM use microscopes that (i) are able to detect and measure the differences in levels of nanometer order (ii) can increase two-dimensional magnification, and (iii) will increase the detection of the horizontal limit beyond that achievable with phase contrast or differential interference contrast microscopy. The presence of two-dimensional nuclei on terraced surfaces between steps, which were not observable under optical microscopes, has been successfully detected by these methods [8], [9]. In situ observation of the movement of steps of nanometer order in height is also made possible by these techniques. However, it is possible to observe step movement in situ, and to measure the surface driving force using optical microscopy. The latter measurement is not possible by STM and AFM. 

Jhe theoretical lower limit of resolution of the light microscope is about 0.2 micron—i.e., 2000 A. This figure can be reduced in favorable circumstances by using phase-contrast or interference microscopy. Ultraviolet microscopy of coal was attempted at the Division of Coal Research, but without success owing to the opacity of coal to ultraviolet radiation. Vigorous attempts are being made to develop x-ray microscopy, and its limit of resolution is already an order better than that of the light microscope. 

Microscopy. This is a powerful tool for studying visually the distribution of the two phases in the polyblend. One can tell not only the domain size of the dispersed phase but also which polymer forms the dispersed phase from refractive index. A phase contrast light microscope can detect heterogeneity at the 0.2-10 /x level. If the sample can be stained preferentially and sectioned with microtome, then under favorable conditions electron microscopy can show heterogeneity to a very fine scale. In a study of PVC-poly(butadiene-co-acrylonitrile) blend,... 

Routine observation of cultured is usually carried out by phase contrast microscopy, utilizing the inverted phase contrast microscope. More recently, more detailed observations have become possible utilizing fluorescent tags and inverted fluorescent microscopes. Fluorescent tags currently in use permit the assessment of oxidant status and mitochondrial function as well as the intracellular concentration of sulfhydryl groups, Ca2+,H+,Na+, andK+. 

In phase-contrast microscopy, the light microscope is adapted to alter the phase of the light waves to produce an image in which the degree of brightness of a region of the specimen depends on its refractive index. 

In electron microscopy as in any field of optics the overall contrast is due to differential absorption of photons or particles (amplitude contrast) or diffraction phenomena (phase contrast). The method provides identification of phases and structural information on crystals, direct images of surfaces and elemental composition and distribution (see Section H below). Routine applications, however, may be hampered by complexities of image interpretation and by constraints on the type and preparation of specimens and on the environment within the microscope. 

Figure 17-2. Microscopic examination of cultured I-cell skin fibroblasts. (A) Phase contrast microscopy. (B) Electron microscopy showing the inclusions. Photographs courtesy of Dr. Robert DeMars.

Analogous results were also obtained microscopically. Interactions occurred spontaneously and large aggregates were formed, which were visible with phase contrast optical microscopy. These particles interact further giving rise to even larger aggregates, which in certain cases encapsulate smaller aggregates. 

Preliminary examination of the latex involved centrifugation and optical microscopy. Only a marginal tendency to fractionate was noticed after 2 hr of centrifuging several 10-ml samples. The approximate diameter of particles separable by normal centrifuging was near 0.5 fi. An optical microscope was equipped with an oil immersion lens (1000 X) and a phase contrast stage. The polymer particles were noticeable but only marginally visible. Their diameters were near the threshold of reso-... 

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