A brief description of the principles of confocal microscopy

The confocal microscope has its name from the arrangement of the light path. In a confocal microscope, the illumination and detection lightpaths share a common focal plane, which is achieved by 2 pinholes that are equidistant to the specimen (see figure). Commonly, Krypton/Argon and Helium/Neon mixed gas lasers are used that give you a range of different distinct wavelengths (see below). This light is sent through a pinhole and reflected by a beamsplitter to the objective and specimen. The beamsplitter is a dichroic filter that acts as a mirror for the excitation wavelengths and is transparent to all other wavelengths. Therefore, the emitted light from the specimen (which has a wavelength spectrum above the excitation wavelength) can go through the beamsplitter to the detection pinhole and the detector (actually the beamsplitter now has been replaced by an acousto-optical device, but for the sake of understanding the principle this doesn't matter here). As a consequence of the pinhole arrangement, light arriving at the detector comes predominantly from a narrow focal plane, which improves the z-resolution significantly compared to conventional microscopy. At the high end, it is possible to achieve axial resolution in the submicron range. In the following, I will try to go through the process of preparing and scanning a fluorescent specimen, explaining a little more about the technical features of the confocal microscope to the extent you need to know them in order to set the scanning parameters in a sensible way. For further, a little more detailed information about the principles of confocal microscopy, here are some websites that do a fairly good job:






...and if you're really keen, there is always "the bible":

Pawley JB, ed (1995) Handbook of Biological Confocal Microscopy, second Edition. New York, London: Plenum Press.

There are multiple copies of this book flying around at Brandeis. Ask Ed or me.