Confocal systems

Fig. 3. Comparisons of wide-field (A) and confocal fluorescence images (B, mesoglea level C, apical) of rhodamine phalloidin-stained F-actin in a whole-mount hydra tentacle. The hydra was fixed and stained as described in Chapter 18. The bar represents 25 pm. All images were collected with a Nikon (New York) Microphot FX microscope (x40 objective lens). Confocal images were collected with the microscope connected to a Bio-Rad (Hercules, CA) MRC600 laser-scanning confocal system.

Fig. 5. Optical sectioning of rhodamine phalloidin-stained F-actin in a neutrophil migrating through a 5-pm pore of a polycarbonate membrane. The neutrophil migration is stimulated in response to 10 M Af-formytmethionyl-leucy 1-phenylalanine. (A), (B), and (C) correspond to O.S-pm optical sections indicated as sections A, B, and C, respectively, in Fig. 4. The bar represents 10 pm. The images were collected with a Nikon Microphot FX microscope (x60 Plan-apochromat lens, numerical aperture, 1.6) connected to a Bio-Rad MRC600 laser-scanning confocal system.

Imaging is performed using a sensitive, high-resolution confocal system like the Opera instrument (Perkin Elmer). Table 1 summarizes the relevant imaging parameters used (see Note 5). 

Abstract Thin and flexible probes made with hollow-optical fibers may be useful for remote spectroscopy. Experimental results showed that these probes are useful for endoscopic measurements of infrared and Raman spectroscopy. A hollow-fiber probe has been used for remote FT-IR spectroscopy in the form of endoscopic measurement of infrared reflectometry spectra inside the body. This measurement was made possible by the hollow-fiber probe s flexibility, durability, nontoxicity, and low transmission loss. A hoUow-fiber probe with a ball lens at the end works as a confocal system for Raman spectroscopy. It can thus detect the molecular structure of biotissues with a high signal-to-noise ratio. Owing to their small diameter, the probes are useful for in vivo, noninvasive analysis using a flexible endoscope. 

The calculation of Atuik is complex and should be considered very carefully to obtain the correct value. Because confocal Raman microscopy is becoming more popular, the calculation of SEF based on a confocal system is given here [61]. 

The thickness of the water layer over the electrode surface can also affect the vertical spatial resolution of the confocal system. For example, with smaller pinhole size (300 pm), the spatial resolution is about 17 pm and 19 pm, without and with the solution layer, while when the size is about 600 pm, the spatial resolution amounts to 21 and 53 pm for the surface without... 

Unlike the confocal system, sample introduction and focusing is rather trivial as the image from the surface can be observed on the CCD. Once the microscope is focused (by making small adjustments to the objective position using its closed-loop piezoelectric stage) the sample can then be translated to a nearby area for measurement (as the area initially used for focusing will be bleached). Electronic shutters or attenuators should be incorporated into the optical path to limit photobleaching of the sample. 

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