Part 1 Principles
Confocal Microscopy tutorial
Part 2 application of confocal microscopy
In practical, almost all specimen for confocal microscope are fluorescent specimen, but don't assume confocal microscopy as a "high quality image fluorescence microscopy". Confocal microscope is not always the best tool for fluorescence detecting.
Confocal microscope is capable of detecting multiple fluorescence channel simultaneously or sequentially, but do not take confocal microscope simply as a tool for co-localization study.
These are two common misconception about confocal microscopy.
As discussed in the first part of this tutorial, the true power of confocal microscopy lies on its strong suppression of out-of-focal-plane signals, namely, the depth discrimination or optical sectioning. The major applications of confocal microscopy rooted from this feature including:
The feature of multiple channel simultaneous detecting without any time delay benefits applications including:
The effect of suppressing stray light improves confocal image quality, but this effect is not always apparent on every case, and can be compromised by its vulnerability to SNR in case of weak, noisy signal.
Other advantages of confocal come from the use of laser for its mono-chromatism, coherent and high intensity, use of PMT for its high sensitivity, high dynamic range and wide adjustable gain and offset control, use of powerful digital image processor and post-acquisition manipulation of raw image data. All these have nothing to do with confocal effects, but they are indispensable parts of confocal system and add extra power to confocal microscopy.
Simply taking, confocal microscopy is not the first choice for ordinary fluorescence detecting nor for simple co-localization of multi fluorescence labeling. These tasks can be easily done by digital camera based conventional fluorescent microscopy without the bother of setting up loads of parameters and go through lengthy image optimization, and bleach 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 frequently-heard notion "the fluorescence in my specimen is weak and noisy, I want to get a good picture from confocal microscope" is a common misconception. You might get a even worse picture in this case. For details, please refer the following section: fluorescence detecting in confocal system.
To help users understanding how these features are in practical use, examples are given below in different categories. As you can see, most of the specimen are stained by fluorescence dyes, but keep in mind, the purpose here is not simply for detecting fluorescence.
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