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Flow Cytometry at San Jose State University

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Fluorescence Microscopy

Fluorescence is the luminescence of a substance excited by high-energy radiation such as ultraviolet light. Fluorescence microscopy achieves magnification of a cell that has been stained with fluorescent dyes or fluorochromes. The result is an image that enables the investigator to visualize both the microbial cell and the specific component of the cell stained by a fluorochrome. Several substances within a microbial cell fluoresce naturally, without dyes. Materials such as chlorophyll, other pigments, or storage granules emit non-specific autofluorescence when excited with high energy light in the ultraviolet range. Fluorochromes are used to stain specific features of interest on or inside a cell.

Many different dyes can be used as fluorochromes. The widely used fluorochrome, fluorescein isothiocyanate (FITC), will absorb light  at about 490 nm and emit at about 555-570 nm. Cellular components stained with FITC will therefore appear green under the fluorescent microscope (Fig. 3).

 Fig. 3. Wet mount of Tetrahymena sp. that has been fed on yeast cells (Saccharomyes cerevisieae var. ellisoides) that had been previously stained with the fluorescein isothiocyanate (FITC). The picture on the left is a magnified image of the large, granlular Tetrahymena sp. cells viewed with dim, white light illuminating the specimen. The picture on the right is the same field (note the position of the cells) where the whit light has been further dimmed, and the excitation short wavelength light has excited the FITC in the yeast cells which appear to glow green both inside and outside the larger Tetrahymena sp. cells.

The FITC-labeled yeast in Figure 3 are visible as bright green fluorescent objects because they have been excited by high energy light that has been directed down through the objective lens by a dichroic mirror (Fig 4). Longer wavelength light passes through the mirror, the ocular lens and finally into the eye of the observer. Note that the dichroic mirror diverts any high energy light that might damage the observer’s eyes. The information from fluorescence microscopy is visual; the location, morphology, and orientation of the cells and fluorescent components can be determined. However, the quantitation of florescence intensity and evaluation of large populations of cells is quite difficult using fluorescence microscopy.

Fig. 4. Schematic representation of fluorescence microscope.