Double-labeling immunofluorescence is stated to detect "localization of a protein of interest as well as the distribution of the protein relative to another marker such as a neurochemical or organelle marker." (Volpicelli-Daley and Levey, 2003 Fluorescence imaging labeled tissue through use of confocal makes provision of "high-resolution analysis of the extent of colocalization, with a theoretical limit of resolution of 0.1 to 0.2 um." (Volpicelli-Daley and Levey, 2003) Immunofluorescence techniques are stated to "in general...utilize secondary antibodies conjugated to a flurosphore." (Volpicelli-Daley and Levey, 2003) It is important according to Volpicelli-Daley and Levey to choose flurosphores with "minimal background staining and a minimum overlap of excitation/emission spectra...when performing double labeling experiments." (2003)
IV. FLUORESCENCE MICROSCOPY
The work of Coling and Kachar (1997) entitled: "Theory and Application of Fluorescence Microscopy" states that fluorescence is the luminescent emission that results form absorption of photons. Fluorescence is distinguished form its counterpart, a longer-lasting afterglow call phosphorescence, by the magnitude of the decay time." Coling and Kachar report that there is an abrupt ceasing of fluorescent emission at the time the "exciting energy is shut off." (Coling and Kachar, 1997)
Fluorescent imaging is used in various spectroscopy techniques and is stated to have particular usefulness in fluorescence microscopy." (Coling and Kachar, 1997) The primary use of fluorescent microscopy is the examination of specimens that have been treated with special fluorescent reagents which have the ability to absorb a certain wavelength of light and emit light "...of a certain wavelength slightly shifted toward the red end of the spectrum from the absorbed light." (Coling and Kachar, 1997) Selective examination of a specific component of a complex bimolecular assembly is enabled by fluorescence microscopy." (Coling and Kachar, 1997)
Coling and Kachar report that the importance in biology and in neurobiology of florescence microscopy is because of:
(1) the extraordinary development of new fluorescent molecular probes; and (2) the development of improved low light level imaging systems and confocal microscopy techniques." (1997)
V. CONFOCAL MICROSCOPY
Confocal microscopy is reported to produce "sharp images of structures within relatively thick specimens" or those up to several hundred microns. (Paddock, Fellers, and Davidson, 2009) Confocal microscopy is stated to be especially useful in the examination of specimens that are fluorescent. When viewing thick fluorescent specimens from a conventional widefield fluorescent microscope they appear fuzzy and lacking in contrast since fluorosphores within the specimens' entire depth are "illuminated and fluorescence signals are collected not only from the plane of focus but also from areas above and below." (Paddock, Fellers, and Davidson, 2009)
Advantages of confocal microscopy overconventional optical microscopy include those of:
(1) shallow depth of field;
(2) elimination of out-of-focus glare; and (3) the ability to collect serial optical sections from thick specimens. (Paddock,
Fellers, and Davidson, 2009)
In the biomedical sciences a major application of confocal microscopy is stated to involve "imaging either fixed or living cells and tissues that have usually been labeled with one of more fluorescent probes." (Paddock, Fellers, and Davidson, 2009) The following illustration shows the principal light pathways in confocal microscopy.
Figure 1
Source: Paddock, Fellers, and Davidson (2009)
The majority of confocal microscopes are reported to be "...relatively easy to operate" and it is stated that these have "...become part of the basic instrumentation of many multi-user imaging facilities." (Paddock, Fellers, and Davidson, 2009)
The laser scanning confocal microscope (LSCM) is stated to be superior to that in the conventional widefield optical microscope however, it is still "...considerably less than that of the transmission electron microscope, it has in some ways bridged the gap between the two more commonly used techniques." (Paddock, Fellers, and Davidson, 2009)
While in a conventional widefield microscope "...the entire specimen is bathed in light from a mercury or xenon source, and the image can be viewed directly by eye or projected directly onto an image capture device or photographic film. In contrast, the method of image formation in a confocal microscope is fundamentally different. The illumination is achieved by scanning one or more focused beams of light, usually from a laser, across the specimen. The images produced by scanning the specimen in this way are called optical sections. This terminology refers to the noninvasive method by which the instrument collects images, using focused light rather than physical means to section the specimen." (Paddock, Fellers, and Davidson, 2009) This is shown in the following illustration labeled Figure 2.
Figure 2
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