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Biotechnology and Applied Biochemistry (1998) 28, (25–32) (Printed in Great Britain)

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

Marc Dellinger*, Marc Geze†, René Santus†, Elli Kohen‡¹, Cahide Kohen‡, Joseph G. Hirschberg§ and Marco Monti§

*Laboratoires de Photobiologie et de Biophysique, INSERM U201,CNRS URA 481, 43 rue Cuvier 75231, Paris cedex 05, France, †Laboratoire de Photobiologie, INSERMU312, Museum National d'Histoire Naturelle, 43 rue Cuvier 75231, Paris cedex 05, France, ‡Department of Biology, University of Miami, POB 2249118, Coral Gables, FL 33124, U.S.A. and §Department of Physics, University of Miami, PO Box 249118, Coral Gables, FL 33124, U.S.A

¹ To whom correspondence should be addressed.

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.

Received 16 October 1997/26 February 1998; accepted 26 February 1998

Portland Press Ltd © 1998