Phase-contrast microscope

F. Zernike (Groningen) demonstration of the phase contrast method and invention of the phase contrast microscope. 

Techniques for differentiating between amorphous and crystalline are (i) sharp melting point, (ii) sharp peaks in the solid state infrared fingerprint region, (iii) optical birefringence observed when solid is viewed in a phase contrast microscope and (iv) sharp peaks in the powder X-ray diffraction pattern. 

Fig. 7 Phase contrast microscope images of fibroblast (L929) cells on TCPS and in the PMBV/...

Fig. 13 Phase contrast microscope images of mouse embryonic stem cells in the PMBV/PVA hydrogel left) and on TCPS (right)...

Figure 14.2 Phase contrast microscopic images of conditionally immortalized cells forming the inner blood-retinal barrier (A) and time-course of [3H] adenosine uptake by TR-iBRB cells (B). A Conditionally immortalized rat retinal capillary endothelial cell line TR-iBRB, retinal pericyte cell line TR-rPCT and Muller cell line TR-MUL. B The [ H]adenosine (14 nM) uptake was performed at 37°C in the presence (closed circle) or absence (open circle) of Na+.

Figure 22. Human embryonic kidney cells (A), rat vascular smooth muscle cells (B, C) and human osteoblast-like MG 63 cells (D) in cultures on micropattemed surfaces. A, B PTFE irradiated with UV light produced by a Xe2 -excimer lamp for 30 min in an ammonia atmosphere through a mask with holes 100 pm in diameter and center-to-center distance 300 pm C PE irradiated with Ar ions (energy 150 keV, ion dose lO ions/cm ) through a mask with holes 100 pm in diameter and center-to-center distance 200 pm fullerenes Qo deposited through a mask with rectangular holes with an average size of 128 3 pm per 98 8 pm on glass coverslips. Day 7 after seeding. A native cells in an inverted phase-contrast microscope B, C cells stained with hematoxylin and eosin, Olympus microscope IX 50 D cells stained with fluorescence-based LIVE/DEAD viability/cytotoxicity kit (Invitrogen), Olympus microscope IX 50. Bars 300 pm (A), 200 pm (B, D), Imm (C) [10,11].

In a phase contrast microscope, an incident wavefront present in an illuminating beam of light becomes divided into two components upon passing through a phase specimen. The primary component is an undeviated (or undiffracted) planar wavefront, commonly referred to as the surround (S) wave. It passes through and around the specimen but does not interact with it. In addition, a deviated or diffracted spherical wavefront (D-wave) is also produced. It becomes scattered in many directions. After leaving the specimen plane, sur-... 

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. 

The spatial structure of the channel has been investigated for a long time. In the beginning, light microscopists described intercalated disks which appeared as bands transverse to the longitudinal axis of the cardiac muscle fiber [Eberth, 1866]. With modem phase contrast microscopes they can easily be seen as shown in figure 2. 

Young anther sacs were dissected and meiocytes squashed according to the technique of Beeks (1955). These were viewed with a Zeiss phase-contrast microscope. 

Using the large vesicles, the fusion process can be monitored in a phase contrast microscope. In different fusion techniques 80 82) very small, submicroscopic vesicles (<100 nm) were used and fusion could only be followed by indirect methods. 

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+. 

Fig. 9 Inverted phase contrast microscope equipped with a CCD camera and a laser. Galvano mirrors allow for scanning of the laser focus across the sample...

Fig. 19 Phase-contrast microscope image of human dermal fibroblast cells attached to and spread on the surface of the waveguide chip...

Changes in clinical semm and urinary parameters, as well as microscopic examination of tissues, generally indicated severe liver and kidney damage. Acute renal and/or hepatic failure was the probable cause of death. Severe hepatic, renal, and capillary damage was also indicated by light and phase-contrast microscope (Ben-Hur et al. 1972 Ben Hur and Appelbaum 1973). 

Fig. 1. Optical pathway of (a) a compound microscope and (b) a phase-contrast microscope.

Cells, scraped from the growing surface or sedimented from a suspension culture are washed in PBS to remove serum and other unwanted medium components and then homogenised for example, using 5 strokes of a Potter-Elvehjem homogeniser or, for small volumes, by pipetting. Cell densities can be as low as 106 cells per ml. Disruption is checked using a phase contrast microscope. The nuclei are readily sedimented at 800 g for 10 min. 

Zeiss and many other phase contrast microscopes produce the equivalent of a central stop dispersion stain by using a phase 2 16X phase contrast objective with the phase 3 condenser ring in place. Leitz manufactures a phase contrast microscope which produces central stop dispersion colors at 400X by using the number 5 condenser ring position. If the microscopes in use at other labs do not produce a central stop dispersion stain in this way, a "dispersion stainer" is available (36). The color resolution (not the particle resolution) of a McCrone dispersion stainer is a little better than that obtained from mismatching phase rings. 

Such experiments with E. coli were subsequently refined through the introduction into the medium of sucrose for osmotic protection of the spheroplasts formed, and the sequence of events during the action of penicillin was photographed under the phase contrast microscope (Fig. 6)S1). 

Fig. 7a-c. Phase contrast microscope pictures of a blend of ethylene-vinyl acetate copolymer (40% vinyl acetate) with chlorinated polyethylene (43 % chlorine) before and after phase separation. Since both polymers are elastomers the mobility is quite high. The original pictures are coloured red and green. These black and white pictures have enhanced contrast to make the phase separation clear... 

Leave for 10 min at room temperature. Examine a sample of the cells with a phase contrast microscope (40-lOOx objectives) to check for good conversion of E. cob. rods to round spheroplasts. Compare to the original cells (5eeNote 2). If the spheroplast suspension is good, only one-half (5 mL) is required for the fusion. 

Living or stained specimens often yield poor images when viewed in bright-field illumination. To help with this, scientists developed a phase-contrast microscope... 

Differential Phase-Contrast Microscope with a Split Detector... 

FIG. 16.5 Optical system for readout of 3D memory. For nondestructive readout, a near-IR, differential phase-contrast microscope is used. 

As described in Section 16.4.5, the detection of refractive-index change between two isomers in a photochromic material is the most promising technique for nondestructive readout.It is necessary to develop a readout system that is sensitive to refractive-index distribution. For this purpose, several methods, such as phase-contrast microscope, differential phase-contrast microscope, and reflection confocal microscope configurations have been proposed. 

FIG. 16.21 (a) Readout data by confocai phase-contrast microscope wid (b) its cross section,... 

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