Part 1 Principles
Confocal Microscopy tutorial
Part 2 application of confocal microscopy
6. fluorescence detecting in confocal microscopy
Before digital camera is widely available to many labs, confocal microscopy is the only convenience way to get co-localization data from multi fluorescence labeled specimen. Simple co-localization study was the major application of confocal microscopy in the middle and late 1990s.
Since high resolution and high sensitivity digital camera become affordable for many labs, it is not necessary to use confocal for simple co-localization study any more. A digital camera can do equally good job or even better job in case of weak signal. As discussed in part 1, section 6 of this tutorial, the superior suppression of out-focal-plane signal in confocal microscopy is achieved by discard lot of emission signal from specimen. If you have a generally weak signal that can not afford this luxury discard, or if you have a low SNR that can not meet the high SNR demanding from confocal microscopy, confocal will frustrate you. The efforts for "getting a good picture" by confocal from a weak and noisy staining will certainly fail.
If your task is a simple co-localization study, spatial distribution of the labeling or tomography of the cell structure has no impact on your study, if you do not need a z-series for 3-D reconstruction, if you do not have a thick specimen that is difficult to be resolved on conventional microscope, you really don't need confocal microscopy and be bothered by lot of parameter settings and the lengthy image optimization process, bleach your valuable fluorescent signal away before you get a satisfactory image from it. 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 situation in simple fluoresscence detecting need confocal microscopy:
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:
They will be discussed in following sections.
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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