Part 1 Principles
Confocal Microscopy tutorial
Q: when do I need confocal microscope for my fluorescence specimen?
A: As discussed in confocal application: Fluorescence detecting section, confocal microscopy is not the first choice for ordinary fluorescence detecting nor for a simple co-localization of multi fluorescence labeling. Considering following situation:
You might not need confocal microscopy. Your tasks can be easily done by digital camera based conventional fluorescent microscopy without the bother of setting up more than 20 parameters, going through lengthy image optimization, and bleaching signal away before getting a satisfactory image: a frustrating process if the specimen does not have a high SNR required by confocal microscopy, as discussed in part 1: optical sectioning. The final results equal to or even worse than what you can easily get from a digital camera do not reward you for the efforts you have paid and the cost of consuming laser.
The folllowing four problematic situation in fluoresscence microscopy need confocal microscopy to deal with:
In digital camera based fluorescece microscopy, multi-labeling is handled sequentially by change filterset in turn and take separate shot then display them in different channel with or without an overlay. Time delay in several seconds between channels is inevitable. If the time delay is intolerable in your application, confocal is your only choice. In confocal system with maximum number of PMTs installed, up to six detecting channels can operate simultaneously without any time delay.
Background fluorescence is sometimes the headache
of fluorescence microscopy. It makes picture looking bad, buries
weak signal in background and reduce general contrast thus reduce obtainable
resolution. This problem is more severe in tissue section and thick specimen
than in cultured cell specimen. In digital camera based
system, the common way to overcome it is simply lessen the excitation intensity
or reduce the exposure time.
Cross-talking refers excitation spectra overlaps, emission spectra overlaps, excitation and emission spectra overlaps in multi-labeled specimen. It is a common problem for multi-labeled specimen. When a mixture of fluorophores exists and more than one excitation light apply to the mixture, there is a complex of interactions and it can be divided into three categories:
Recently, some new fluorophores appear in the market emitting at longer than 650 nm which is invisible to most of the eyes of human beings and many common CCD chips. These dyes, however, greatly expand the range of choice and possibility of three, or four fluorophores labeling. In this case, confocal can do a decent job for you to detect emission from 650 to 750 nm.
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