Digital imaging fluorescence microscopy

This chapter discusses the unique features of digital imaging microscopy and suggests several research areas in lipoprotein metabolism where the technology can be applied to great advantage. The relationship between the plasma lipoproteins and atherosclerosis is elusive because of the complexity of interactions between lipoproteins and the different cell types in the normal blood vessel wall. Action of the lysosomal enzymes on low-density lipoprotein (LDL) is necessary for subsequent regulatory processes. As the protein moiety must be degraded, regulation involves the lipid components. Although lipid-protein interactions can be studied directly in the test tube using spectroscopic techniques, similar studies of the regulatory role of lipids in situ are technically impossible using conventional biochemical methods. One strategy is to combine the use of fluorescent lipid analogs with fluorescence microscopy. The best approach is to apply existing hardware and software to digitize images of living cells exposed to a particular fluorescent lipid. The ability to make quantitative fluorescence measurements depends on the resolution of three major experimental problems: (1) elimination of photobleaching, (2) calibration with fluorescent standards, and (3) elimination of out-of-plane contributions to the fluorescence intensities.