Confocal plane

Bolton If it happens in another confocal plane then it is diffusing from there. 

Somljo It is not a question of diffusion, it is because the confocal microscope does not have a 1 A confocal plane. At best, given a x 40 objective with a numerical aperture of 1.3, it will have a -axis resolution of about 0.7 [im. 

Bolton This problem has been investigated by us and others. My understanding is that if you have a spark which is of the same size, duration and spatial spread as the spark in your confocal plane, and it is in another confocal plane then, if you see it, it will have a slower time course because diffusion will have occurred. If it is not in your confocal plane you will not see it at the beginning, so it has to spread to be visible. As it diffuses it will take longer, so the timecourse is very critical for deciding which confocal plane it is in (Gordienko et al 1998). 

The optical principles of confocal microscopy can be understood by examining the CLSM optical path that has reflected illumination as illustrated in Figure 1.45. The laser beam is focused as an intense spot on a certain focal plane of the specimen by a condenser lens, which is also serves as an objective lens to collect the reflected beam. A pinhole aperture is placed at a confocal plane in front of the light detector. The reflected beam from the focal plane in a specimen becomes a focused point at the confocal plane. The pinhole aperture blocks the reflected light from the out-of-focal plane from entering the detector. Only the light signals... 

In a confocal microscope a small aperture is placed in the plane where rays coming from a particular plane in the object come to a point (see Fig. 6.1, the plane is called the confocal plane). A light detector is placed behind the aperture. The aperture selects one point (x, y) in the plane, and also cuts out most of the light coming from other planes in the specimen. If the illumination is focused onto the selected point in the object, then... 

Another confocal scanning method uses a rapidly rotating disk containing many holes in the confocal plane (a Nipkow disk). These holes act as confocal apertures and mechani-... 

The extension of the voxel in a radial direction gives infomiation on the lateral resolution. Since the lateral resolution has so far not been discussed in temis of the point spread function for the conventional microscope, it will be dealt with here for both conventional and confocal arrangements [13]. The radial intensity distribution in the focal plane (perpendicular to the optical axis) in the case of a conventional microscope is given by... 

Figure 1 Principle of confocal Raman microscopy A laser spot in the focal plane passes through the pinhole P. A laser spot at a distance z from the focal plane is projected in the image plane with size P, and is largely blocked by the pinhole P. [L — lens M = beam splitter fi, f2 = focal length of lens L, and L2, respectively b2 — image distance of out-of-focus laser spot). Reproduced from Tabaksblat et al. [14], with permission of the Society for Applied Spectroscopy. 2000.

Drummond, D. R., Carter, N. and Cross, R. A. (2002). Multiphoton versus confocal high resolution z-sectioning of enhanced green fluorescent microtubules Increased multiphoton photobleaching within the focal plane can be compensated using a Pockels cell and dual widefield detectors. J. Microsc. 206, 161-9. 

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,... 

Fig. 12 CNTs act as a vector for drug delivery into living cells. After incubation of HeLa cells with AlexaFluor594-labeled SWNTs for 12 h at 37 °C, living cells were observed under confocal fluorescence microscope for a CNT uptake study, (a) Images show dual confocal detection of AlexaFluor594-SWNT (red) internalized into cells with the membrane stained by AlexaFluor488 (green), (b) Series of images of different z-focal scanning planes down through cells. (Adapted from [61])...

Figure 4b depicts for the case bAk -0.3 (b is the confocal parameter of the gaussian and Ak is the linear plane-wave, wave-vector mismatch, Ak=3 (n -no )/c). One can see that bAk>0 results in... 

FIG. 2. Confocal image of an isolated smooth muscle cell from guinea-pig ileum which has been permeabilized with staphylococcal os-toxin and incubated with 150 /iM Fluo-3 acid which stains the SR. The SR is predominantly localized to the periphery of this type of smooth muscle as seen on the left hand side where the image plane is through the centre of the cell, whereas an extensive network is seen where the image plane is adjacent to the plasma membrane as seen in the right hand portion of the cell. 

In a confocal microscope, invented in the mid-1950s, a focused spot of light scans the specimen. The fluorescence emitted by the specimen is separated from the incident beam by a dichroic mirror and is focused by the objective lens through a pinhole aperture to a photomultiplier. Fluorescence from out-of-focus planes above and below the specimen strikes the wall of the aperture and cannot pass through the pinhole (Figure 11.3). 

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 ... 

Hexagonal mesophases can be recognized by their typical fan-shape texture (Fig. 7a). Lamellar mesophases typically show oily streaks with inserted maltese crosses (Fig. 7b). The latter are due to defects, called confocal domains, that arise from a concentric rearrangement of plane layers. In some lamellar mesophases these defects prevail. Hence no oily streaks occur but maltese crosses are the dominant texture (Fig. 7c). 

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