Department of Pathology: Confocal Facility

Why Use Confocal?:

Standard epifluorescent, widefield microscopy has been used to visualize fluorescently labeled molecules in samples. Using this technology, the entire sample is bathed by an illumination source (in our facility this is a mercury arc lamp). The excited fluor is then viewed using appropriate filters for the emitted light of the fluor. Due to the 3-dimensional nature of most samples, parts of the sample of sometimes out of the focal plane for viewing. However, fluorescence from these out of focus regions can interfere with capture and interpretation of the fluorescently labeled molecule. To eliminate this problem, Confocal microscopy utilizes imaging principles developed by Marvin Minsky (patented in 1957).

At a basic level, confocal combines focused illumination of a sample with one or more beams of light and collection of emitted light by a photomultiplier tube. Because only a portion of the sample is illuminated during a given scan of the sample, high resolution images can be collected from a specific layer of the sample. This is particularly useful when analyzing thick sections of tissue or when the specific subcellular localization of the illuminated molecule is of interest. Because confocal sections are very thin (our confocal microscope can capture a section 0.2 uM in thickness), simultaneous labeling of 2 or more proteins can be analyzed for their proximity in the cell. This co-localization of molecules can be determined to within .2 uM. More complicated methods such as fluorescence resonance energy transfer (FRET) can be used to determine if molecules are within 50 angstroms of eachother.

Confocal can also be used to capture sections of a thicker sample which can be re-assembled afterward to demonstrate a high-resolution image of 3-dimensional structure without out of focus fluorescence. Other uses of confocal microscopy include fluorescent recovery after photobleaching (FRAP), quantification of fluorescent changes between samples, and overlaying 2 or more fluorescently labled proteins.