For fluorescence imaging, confocal microscopy has a number of advantages compared to conventional epi- fluorescence microscopy. The two most important ones are the ability to eliminate out-of-focus noise (better resolution and contrast) and increased detection sensitivity.20 In addition to light detectors of high sensitivity, the confocal system has the advantage of being able to accumulate (or average) images over time. Also, because of the controllable laser source, there is a reduced risk of specimen bleaching.20
1.1.3. Fluorescence microscopy
By using appropriate fluorochromes, it is possible to identify cells and sub-cellular components with a high degree of specificity amidst non-fluorescing entities or those with different fluorescence properties; and the availability of hundreds of fluorescent labels with known excitation and emission curves and well-understood biological structure targets has accelerated the application of fluorescence microscopy in both clinical laboratories and research.
The basic task of a fluorescence microscope is to provide excitation light to irradiate the specimen, and then separate the much weaker ( 103X) fluorescent light from the excitation light, so that only the emission is observed or detected. Conventional widefield microscopy and confocal microscopy are also the two key techniques for fluorescence imaging.26, 27 Fluorescence signal emanating from above and below the focal plane creates an out-