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Numerous highly fluorescent macrophages (MU), designated ''smoker cells,'' exist in the lungs of smokers and subjects who have quit smoking within 5 years. The brightly fluorescent MU, however, are not present in the lungs of never smokers. Some investigators have speculated that the intense fluorescence of the MU is due to smoke-induced changes in the autofluorescence of naturally occurring (i.e., endogenous) compounds (e.g., NADP). In contrast, other researchers have theorized that the fluorescence is due to the uptake of tobacco smoke particulates (i.e., ''tar''). Studies reported herein were undertaken to test the hypothesis that the origin of the MU fluorescence could be profiled with the novel technologies afforded by spectral confocal laser scanning microscopy (sCLSM) and multispectral cytometry (MSC). To this end, spectral emissions were obtained by sCLSM of optical sections of live MU isolated from fresh surgically excised human lung tissue and in air-dried lung tissue imprints. Confirmation of spectral profiles of these single cell observations was obtained in population studies with the use of high-throughput MSC in which multispectral analyses were performed with three different lasers. Proof of concept experiments demonstrated that relatively nonfluorescent MU from the lungs of nonsmokers became fluorescent upon short-term ex vivo exposure to tobacco smoke tar. Summarily, the studies reported herein document that the fluorescence of human lung MU is due to tobacco tar. Microsc. Res. Tech. 67:79-89, 2005. V V C 2005 Wiley-Liss, Inc.

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