Confocal diaphragm

Figure 9.29 Optical diagram of a Raman microscope. A pinhole spatial filter consists of a pinhole confocal diaphragm (Dj and D2). (Reproduced with permission from G. Turrell and J. Corset, Raman Microscopy, Developments and Applications, Academic Press, Harcourt Brace Company, London. 1996 Elsevier B.V.)...

The spatial resolving power is especially large, if the confocal technique is applied (Schrader et al., 1993, 1994). A focus of a laser beam is produced by a microscope objective of large numerical aperture at the spot of the sample to be investigated (Fig. 3.5-12). The radiation emerging from this spot is projected by the objective onto a diaphragm with a hole of the size of the projected laser focus. Only the radiation passing... 

Figure 3.5-12 Principle of a confocal microscope a LF fiber transporting the laser radiation, D dichroitic mirror, 0 objective, S sample, the Raman radiation produced in the illuminated spot of the sample is focused upon the diaphragm A, only the radiation from the spot is focused at the fiber SF, which transports the Raman radiation to the spectrometer b focal range in the illuminated sample, Ax spatial, Az depth resolution.

Hypothyroidism may result in motor unit type-specific adaptations in NMJ structure. Using confocal microscopy to analyze the effect of hypothyroidism on the three-dimensional structure of NMJs in the rat diaphragm muscle, Prakash et al. (1996a) found that hypothyroidism reduced the planar area of both axon terminals and motor end-plates at type 1 and Ila muscle fibers, and decreased the number of branches and mean branch length. In contrast, morphological adaptations were not evident for... 

Illumination and contra.st are central points in the discussion of imaging performance. Without sufficient contrast, neither the required magnification nor a maximal resolution can be obtained. A. Kohler developed optical illumination for microscopy in 1893 [8]. The main advantage of Kohler illumination is that in spite of the small filament of the light bulb, the entire area of the lamp field stop, as well as the illuminated part of the specimen, have a uniform luminance (Fig. 4). The twofold ray path of diaphragms and lenses suggests that adjustment of the condenser iris, being the illumination aperture, controls resolution, contrast, and resolution depth. This principle is also important for correct setup of the confocal microscope as well as electron microscopy. 

A slight enhancement of the lateral resolution. Theoretically, the confocal setup may decrease the spreading function, but it should be mentioned that this requires very small diaphragms to be used, which in turn may decrease the amount of detected intensity. 

Convolution should be applied to both confocal and wide-field data bnt requires very high positioning accuracy and a relatively constant, known PSF. It is thus important to remember that PSF varies with wavelength, objective s NA, field and apertnre diaphragm diameters, alignment of the microscope, and immersion medium. 

---