<title>Toward noninvasive microspectrofluorometry of skin lesions for diagnostic and prognostic evaluation of cell metabolism and organelle interactions</title>

Abstract

Photoinduced modifications of NAD(P)H attributed autofluorescence of CHO cells in asingle-beam gradient force optical trap (optical tweezers) were studied. Fluorescence spectra of single cells in the optical trap were measured using a modified microscope with an JR microbeam at 1064 and 760 nm for trapping, UVA radiation at 365 nm for fluorescence excitation, and an optical multichannel analyzer for spectral recording. No strong effect of the 1064 nm trapping beam on fluorescence intensity and spectral characteristics was found, even for power densities up to 70 MW/cm2. In contrast, 760 nm microirradiation resulted in a significant fluorescence increase, probably indicating cell damage due to absorption by heme-containing molecules. UVA exposure (1 W/cm2) of the trapped cells generated within seconds an initial fluorescence decrease, followed by a significant increase up to 5X of the value prior to irradiation. The UVA-induced modifications reflect NAD(P)H autooxidation and irreversible cell damage due to oxidative stress.

Microspectrofluorometry and fluorescence imaging in the study of human cytopathology

“…With a panel of endogenous metabolic, exogenous organelle-, or enzyme-selective fluorescent probes, used in combination with conventional biochemistry, microspectrofluorometry (Hirschberg et al, 1994(Hirschberg et al, , 1995Hirschberg and Kohen, 1996;Kohen et al, 1986Kohen et al, , 1990Kohen et al, , 1991aKohen et al, ,b, 1992aKohen et al, -d, 1993Kohen and Hirschberg, 1998); computer-controlled confocal fluorescence imaging (Lansing Taylor et al, 1989;Salmon and Cassimeris 1989), fluorescence micrography (Kohen et al, 1986(Kohen et al, , 1990(Kohen et al, , 1991a(Kohen et al, ,b. 1992a(Kohen et al, -d, 1993, morphometry and ultrastructural analysis (Hirschberg et al, 1995), we have established optical methods for studying the cytopathology and cytopharmacology of single or small groups of living cells.…”

“…We also had considerable published information for evaluating our biochemical and morphometry studies (Hirschberg et al, 1994(Hirschberg et al, , 1995Kohen et al, 1991Kohen et al, , 1992c. Mitochondria-toxic dicarboxylic fatty acids (azelaic, sebacic, decanoic, tetramethylazelaic) were applied to melanoma cells because of previous background information on the metabolic and ultrastructural action of these agents, plus knowledge of their effectiveness in the topical treatment of melanoma (Hirschberg et al, 1994(Hirschberg et al, , 1995Kohen et al, 1991Kohen et al, , 1992c. All 8,255 melanoma cells and Harding-Passey melanoma (HPM73) cells were grown in Eagle's medium with 10% fetal calf serum (FCS).…”

“…Microinjection of metabolic intermediates such as glucose-6-phosphate (G6P), 6-phosphogluconate (6-PG), and malate was carried out by microelectrophoresis (Hirschberg et al, 1994(Hirschberg et al, , 1995Kohen et al, 1991Kohen et al, , 1992c.…”

“…For microspectrofluorometry-microinjection and fluorescence micrography, cells were transferred to a microincubation chamber used in conjunction with an inverted fluorescence microscope (Hirschberg et al, 1994(Hirschberg et al, , 1995Kohen et al, 1991Kohen et al, , 1992c. Microinjection of metabolic intermediates such as glucose-6-phosphate (G6P), 6-phosphogluconate (6-PG), and malate was carried out by microelectrophoresis (Hirschberg et al, 1994(Hirschberg et al, , 1995Kohen et al, 1991Kohen et al, , 1992c.…”

“…The current paper describes the application of rapid fluorescence spectral imaging to melanoma cells, spontaneously transformed cultured cells, long-term malignant cells, and gene-defective cells lacking a particular enzyme vs. cells with a normal complement of enzymes (Dellinger et al, 1998;Hirschberg et al, 1994Hirschberg et al, , 1995Kohen et al, 1983Kohen et al, , 1990Kohen et al, , 1991aKohen et al, ,b, 1992aKohen et al, -c, 1993. Drug effects on these cell systems using rapid fluorescence spectral imaging were also investigated (Dellinger et al, 1998).…”