Imaging of cells by autofluorescence: a new tool in the probing of biopharmaceutical effects at the intracellular level

Abstract

The success of biopharmaceuticals relies on the ability to have reliable probes to interpret their mechanisms of action in situ at the intracellular level in terms of cell organelles and microcompartments. One of the most effective probes is the endogenous coenzyme NAD(P)H and its fluorescence transients obtained by the microinjection or perfusion of metabolic intermediates and modifiers, in the presence of drugs and inhibitors. The approach in fluorescence microtopography and microspectrofluorimetry is based on the premise that natural cell fluorescence (autofluorescence) holds a decisively greater potential in unravelling intracellular physiopathological processes than extrinsic fluorescence or artificial pseudocolouring. The mounting as a detector of a cooled charge‐coupled device camera or alternatively of a non‐cooled camera in conjunction with an image intensifier or an investigator (i.e. frame scan accumulator) to enhance sensitivity makes possible the detection of the low‐quantum‐yield NAD(P)H fluorescence at a level comparable to images previously obtained with high‐quantum‐yield fluorochromes. The modulation of mitochondrial autofluorescence by rotenone, carbonyl cyanide p‐ trifluoromethoxyphenylhydrazone and oligomycin, and of cytoplasmic and nuclear autofluorescence by glucose and iodacetamide in CV‐1 kidney epithelial cells, Ehrlich‐Lettre hypotetraploid CCL77 cells and Saccharomyces cerevisiae, provides examples of the usefulness of fluorescence imaging in the study of biopharmaceuticals. The method goes beyond NAD(P)H to the multiplicity of extrinsic and intrinsic probes already available or in development.