Chapter 58 Fluorescence Labeling of Flagellar Membranes

Lipophilic fluorescent dyes are used for examining cell structure and investigating the structure and function of specific organelles. One of these dyes, 3, 3′-dihexyloxacarbocyanide iodide [DiOC₆(3)] is used to investigate the properties of the endoplasmic reticulum in living and fixed cells. DiOC₆(3) diffuses through the plasma membrane and enters cell membranes, but in living cells the dye becomes concentrated within specific organelles. The concentration of DiOC₆(3) determines the organelles who appear stained. This selectivity of DiOC₆(3) staining reflects accumulation of the positively charged dye molecules in subcellular membranes that have negative membrane potential. The sensitivity of DiOC₆(3) accumulations to membrane potential is used to measure plasma membrane potential in mammalian cells. When Chlamydomonas reinhardtii cells are exposed to low concentrations of DiOC₆(3), flagella rather than mitochondria are specifically stained. Staining of mitochondria is observed only when the DiOC₆(3) concentrations is increased, and even then, the flagella continue to stain. At the highest DiOC₆(3) concentrations tested, other cellular membranes including the chloroplast membrane, also became labeled. If the staining characteristics of DiOC₆(3)reflect its membrane potential-sensitive partitioning into membranes, flagellar ion pumps are to maintain the higher negative membrane potential across the flagellar membranes than that of the rest of the plasma membrane, or even that of mitochondria. It is conceivable that this membrane potential may have functional roles in controlling flagellar length, autotomy, or aspects of flagellar beating. There is description of medium and culture conditions, preparation and storage of DiOC₆(3), staining of Chlamydomonas with DiOC₆(3), microscopy, staining patterns and prospects. Long-chain dyes, once incorporated into a membrane, the dye is retained within that membrane. DiOC₆(3) staining may be revealing physiologically relevant information concerning flagellar membrane potential. Use of an emission filter that blockslight from 555 to 1.2 nm eliminates chlorophyll autofluorescence, a necessity photography on a standard light microscope.