Confocal microscopy advantages

Another significant advantage of confocal microscopy is the increase in resolution achieved by the absence of the out-of-focus images. A microscope yielding a resolution of 220 nm, if confocal, yields a resolution of 160 nm. 

The major advantage of TIRF is that fluorophores outside the evanescent wave (typically more than 200 nm away from the surface) are not excited. Hence, TIRF has an intrinsic sectioning capability. Of interest is that the section capability (z-resolution) is far better than for confocal microscopy systems, which typically have a z-resolution of about 1 /mi. In addition and in contrast to confocal microscopy, TIRF does not cause out-of-focus bleaching because only the molecules at the surface will sense the evanescent wave. However, in comparison with confocal microscopy, a clear limitation of TIRF is that only one z-plane can be imaged the molecules immediately adjacent to the surface. As a consequence,... 

Two-photon excitation provides intrinsic 3-D resolution in laser scanning fluorescence microscopy. The 3-D sectioning effect is comparable to that of confocal microscopy, but it offers two advantages with respect to the latter because the illumination is concentrated in both time and space, there is no out-of-focus photo-bleaching, and the excitation beam is not attenuated by out-of-focus absorption, which results in increased penetration depth of the excitation light. 

Confocal microscopy (CM) is another microscope technique for apparent optical sectioning, achieved by exclusion of out-of-focus emitted light with a set of image plane pinholes. CM has the clear advantage in versatility its method of optical sectioning works at any plane of the sample, not just at an interface between substances having dissimilar refractive indices. However, other differences exist which, in some special applications, can favor the use of TIRF ... 

Confocal and scanning electrochemical microscopy of fluorescent and electroactive species, respeetively, have provided complementary information to that from the other microscopy techniques. Both these newer methods have the advantage of using the skin without fixation, thus avoiding possible artifacts associated with its chemical preservation. Confocal microscopy has... 

Although STED and confocal microscopy are easily combined to each others advantage in the same setup, STED is not an extension of confocal microscopy, because it does not require the imaging of the fluorescence onto a pinhole. In principle, one could detect the fluorescence signal right at the sample. Therefore, parallelized camera-based STED microscopy will also be possible with arrays of doughnuts or lines (Fig. 19.Id) [88]. 

Fluorescent labelling of the particles for confocal microscopy may be achieved in three ways. The first involves the use of polymerisable dyes. These dyes have been chemically modified to include a reactive group that can be chemically attached to the particle as they are produced. The advantage of this procedure is that the dye will not leave the particle once it is incorporated. For sterically-stabilised PMMA particles this involves adding a methacrylate group to the dye, and several such procedures [23-27] have been described in the literature. The most commonly used dye is 7-nitrobenzo-2-oxa-l,3-diazole-methyl methacrylate (NBD-MMA) [26,27], which is excited at 488 nm and emits at 525 nm, while the red end of the spectrum is well served by (rhodamine isothiocyanate)-aminostyrene (RAS) [27]. 

Many of these dyes and their applications have been reviewed in detail elsewhere (50). Of note, probes are available for both UV and 488 nm excitation and many of these calcium probes (Fura-l, Indo-1, Calcium Green-2) have the advantage of utilizing a ratiometric fluorescence read-out. These dyes have been successfully used in multiparametric analyses, using both flow cytometry and confocal microscopy, which has led to an enhanced understanding of the role of [Ca2+]i in apoptosis. Burchiel et al. (51) have recently published a review of multiparametric flow cytometric Ca2+ analysis. 

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