Confocal advantage

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. 

Note that G as derived here relates the FRET-induced sensitized emission in the S channel to the loss of donor emission in the D channel and that it is identical to the correction factor y/ [2] or G [6, 14]. Note however, that if the correction factors or 5 change, G and

correction factor C [3] is a constant that depends only on fluorophore properties and filter settings, and therefore it does not change with excitation intensity or detector gain. This is a clear advantage for confocal filterFRET. C (Eq. (7.14)) and G (Eq. (7.19)) are related as ... 

In this chapter, it was shown that filterFRET is an easy, intuitive and quantitative alternative to record sensitized emission and FRET efficiency. The major advantages of filterFRET over donor-based FRET detection methods (FLIM) are that it can be carried out with standard wide-held or confocal fluorescence microscopes that are available in most laboratories, and that it yields additional data on the acceptor population. FilterFRET is also fast, requiring just two confocal scans (if need be on a line-by-line basis) which minimizes the risk of artifacts due to, for example, organelle movement in living cells, and acquisition can be optimized for each channel independently. However, quantitative... 

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 laser scanning fluorescence microscopy was used to study the exposure of the avidin-specific binding sites in the Av-GEB platform by the immobilization of a small and flexible biotinylated fluorescein molecule as a fluorescence marker. Fluorescence microscopy thus confirms that Av-GEB platform exposes active binding sites for biotin, acting as affinity matrix (Fig. 21.2B). After use, the electrode surface can be renewed by a simple polishing procedure for further uses, highlighting a clear advantage of this new material with respect to surface-modified approaches such as classical biosensors and other common... 

Infrared microscopic imaging provides the significant advantages of direct spatially resolved concentration and molecular structure information for sample constituents. Raman microscopy (not further discussed in this chapter) possesses the additional benefit of confocal acquisition of this information and a 10-fold increase in spatial resolution at the expense of reduced signal-to-noise ratios compared with IR. The interested reader is urged to check the seminal studies of the Puppels group in Rotterdam,38 0 as well as our own initial efforts in this direction.41 The current section describes the initial applications of IR microspectroscopic imaging to monitor the permeation and tissue distribution of the dermal penetration enhancer, DMSO, in porcine skin as well as to track the extent of permeation of phospholipid vesicles. 

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