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

Confocal imaging has further improved the images obtained of specimens using multiple labeling." (Paddock, Fellers, and Davidson, 2009) The following illustration labeled Figure 3 shows a comparison of a conventional epifluorescence image with a confocal image of similar regions of a whole mount of a butterfly pupal wing epithelium stained with propidium iodide." (Paddock, Fellers, and Davidson, 2009)
Figure 3

Source: Paddock, Fellers, and Davidson (2009)

The most widely used confocal variation in biomedical research is the laser scanning confocal microscope. The confocal microscope was invented by Marvin Minsky in 1955 with the development of this approach stated to be driven by "the desire to image biological events as they occur in living tissue (in vivo)..." (Paddock, Fellers, and Davidson, 2009) The original configuration utilized by Minsky was one in which a "...pinhole was placed in front of a zirconium arc source as the point source of light. The point of light was focused by an objective lens at the desired focal plane in the specimen, and light that passed through it was focused by a second objective lens at a second pinhole having the same focus as the first pinhole (the two were confocal). Any light that passed the second pinhole struck a low-noise photomultiplier, which generated a signal that was related to the brightness of the light from the specimen. The second pinhole prevented light originating from above or below the plane of focus in the specimen from reaching the photomultiplier. The use of spatial filtering to eliminate out-of-focus light or flare, in specimens that are thicker than the plane of focus, is the key to the confocal approach." (Paddock, Fellers, and Davidson, 2009)

Paddock, Fellers, and Davidson (2009) report that the focused spot of light "must be scanned across the specimen" if one is to build an image through use of the confocal principle. Paddock, Fellers, and Davidson (2009) state that in the original instrument that Minsky built:

"...the beam was kept stationary and the specimen itself was moved on a vibrating stage. This arrangement has the advantage that the scanning beam is held stationary on the optical axis of the microscope, which can eliminate most lens defects that would affect the image. For biological specimens, however, movement of the specimen can cause wobble and distortion, resulting in a loss of resolution in the image. Furthermore, it is impossible to perform various manipulations on the specimen such as microinjection of fluorescently labeled probes when the stage and specimen are moving." (Paddock, Fellers, and Davidson, 2009)

In actuality, there was not the necessary technology available in 1955 for Minsky to fully develop and demonstrate "the potential of the confocal approach, especially for imaging biological structures." (Paddock, Fellers, and Davidson, 2009) Paddock, Fellers, and Davidson report that the information flow in the modern laser scanning confocal microscope is shown in the following figure which has been adapted from their work.

Figure 4

The information flow in a modern laser scanning confocal microscope

Source: Paddock, Fellers, and Davidson (2009)

The basic optics of the optical microscope are reported in the work of Paddock, Fellers, and Davidson to have "remained fundamentally unchanged for decades because the final resolution achieved by the instrument is governed by the wavelength of light, the objective lens, and the properties of the specimen itself. The dyes used to add contrast to specimens, and other technology associated with the methods of optical microscopy, have improved significantly over the past 20 years." (2009)

Modern technology has served to bring about both "growth and refinement" in the confocal approach largely due to optical microscopy renewal driven by modern technology advances. Confocal microscope classification of designs is one the "basis of the method by which the specimens are scanned." (Paddock, Fellers, and Davidson, 2009) Two basic means of scanning include:

(1) to scan the stage beam; or (2) to scan the illumination beam. (Paddock, Fellers, and Davidson, 2009)

Stated to be an alternative method for imaging biological systems and one that is more practical for use is "to scan the illumination beam across a stationary specimen. This approach is the basis of many of the systems that have evolved into the research microscopes that are in vogue today." (Paddock, Fellers, and Davidson, 2009)

The most popularly used method is single-beam scanning in which the scanning of the beam is generally accomplished through use of mirrors that are computer-controlled and "drive by galvanometers at a rate of one frame per second. To achieve faster scanning, at near video frame rates, some systems use an acousto-optical device or oscillating mirrors." (Paddock, Fellers, and Davidson,…