Wednesday, September 14, 2011

Fluorescence and Autofluorescence

Epi fluorescence is one of the most common techniques used in scientific research today. Samples are "tagged" with certain compounds that attach to a specific part of the tissue or cell and then exposed to a discrete wavelength of light to "excite" the compounds. The wavelength of light used is matched to specific compounds, or fluorochromes, so that the resulting image only shows the structure that has been labeled. For example, a very common fluorochrome is DAPI, which is used to attach to the nucleus of a cell. The wavelength of light used to excite DAPI is 325-375 nanometers (nm), which is in the ultraviolet range. When the DAPI is excited by this wavelength of light, it releases energy at a longer wavelength, in this case 435-485nm, which is blue light. So the sample is illuminated in the ultraviolet, but the color that is reflected back to the eyepieces is in the blue. When you see images of cells in literature, they are almost always blue.

Other fluorochromes all have a distinctive color associated with them. FITC is excited by blue light, but reflects back green so FITC images will be green. TRITC is excited by green light, but reflects back orange-red, so those images will be in the orange-red range.

Some material, especially plant material has the inherent ability to fluoresce, or react to specific wavelengths of light, without having any dyes or fluorochromes attached to the material. This is called "autofluorescence" and can interfere with using this research technique effectively. It is however, helpful for other tasks, such as using a plant sample when you are working to align the fluorescence illumination. This autofluorescence does not fade or quench as do biological samples so you are not damaging your cells while performing the alignment. It also allows practice in setting up photography without damaging the sample. And the bonus is that it results in beautiful images!