Out-of-focus image

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 focal depth of the conventional optical microscopes is usually deep in comparison to its lateral resolution. This leads to the superposition of the out-of-focus images, and hinders the 3D interpretation of the images. Con-focal microscopy has overcome this disadvantage by introducing a pinhole just in front of the detector as shown in Fig. 2. The solid line shows the opti-... 

There are also laser-scanning confocal microscopes rapidly overtaking the TSM as a means of confocal microscopy. There is also a computer software program that produces a "confocal" image by recogni2ing the shapes of out-of-focus detail, ie, halos, and subtracting these from the in-focus image. 

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.

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,... 

For samples thicker than the depth of field, the images are blurred by out-of-focus fluorescence. Corrections using a computer are possible, but other techniques are generally preferred such as confocal microscopy and two-photon excitation microscopy. It is possible to overcome the optical diffraction limit in near-field scanning optical microscopy (NSOM). 

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 ... 

When fluorescently labeled biological specimens are viewed with a conventional wide-field microscope, a haze of out-of-focus fluorescence is usually created hy the overlapping structures within the sample. As we focus through the specimen, our hrains have a remarkable ability to discern substantial structural detail. However, the resolution of the images we record on film is degraded hy the out-of-focus fluorescence. The confocal microscope can reject out-of-focus information and enhance the contrast of an image because the illumination and the detection are confined to an identical (small) region of the specimen. An overview of the basic principles of a confocal microscope is presented in Fig. 1 and outlined helow. 

Optical microscopes have one serious drawback, their resolution, resulting from the fundamental physics of lenses. Lord Rayleigh, over 100 years ago, defined the currently accepted maximum optical lens resolution to be one-half of the wavelength of the imaging radiation. In truth, conventional optical microscopy did not achieve this level of definition mainly because of out-of-focus light. This prevented the observation of atoms and single molecules. 

One of the major problems in the photography of a volume of air containing droplets is the lack of an adequate depth of focus. Only those particles contained within a shallow depth present true images. It is difficult to make measurements when the majority of droplets are out of focus. La Mer and Lee (SF) have developed a method which places all the drops within a very shallow band. An aerosol stream is forced to pass through an extremely fine slot, so that stream thicknesses of only 0.02 mm. can be formed. 

The pinhole value sets how much out-of-focus light is allowed to reach the detector. For dark samples it is sometimes not possible to close the pinhole. In this case, use position 2 and give more light to the image with the gain bottom of the microscope. 

Land (1983) reports that the viewed image can be surprisingly far out of focus without an impact on color perception. However, in this case a sharp color edge turns into a smooth color gradient. It therefore seems unlikely that human color perception uses some type of threshold. Also, Marr (1982) notes that one is able to see a rainbow. If the retinex algorithm were used by the visual system, one would assume that the rainbow were thresholded out because a rainbow is a smooth transition of all colors of the spectrum. 

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