Near-infrared (NIR) dyes are used as fluorescence markers in small animal imaging and in diffuse optical tomography. In these applications it is important to know whether the dyes bind to proteins or to other tissue constituents, and whether their fluorescence lifetimes depend on the targets they bind to. Unfortunately, neither the optical beam paths nor the detectors of commonly used in confocal and multiphoton laser scanning microscopes (LSMs) directly allow for excitation and detection of NIR fluorescence. This paper presents three ways of adapting existing LSMs with time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) systems for NIR FLIM: 1) confocal systems with wideband beamsplitters and diode laser excitation, 2) confocal systems with wideband beamsplitters and one-photon excitation by titanium-sapphire lasers, and 3) two-photon systems with optical parametric oscillator (OPO) excitation and non-descanned detection. A number of NIR dyes are tested in biological tissue. All of them show clear lifetime changes depending on the tissue structures they are bound to. We therefore believe that NIR FLIM can deliver supplementary information about the tissue composition and on local biochemical parameters.
Alteration of cellular energy metabolism is a principal feature of tumor and stem cells. Here we analyze the metabolic interactions between cancer cells and fibroblasts in a co-culture model and the metabolic heterogeneity of tumors and metabolic changes in mesenchymal stem cells during adipogenic differentiation based on the fluorescence of the metabolic cofactors NADH, NADPH, and FAD. We registered a metabolic switch from oxidative phosphorylation to glycolysis with slight acidification of the cytosol in cancer cells in a co-culture model. In the tumor tissue we detected metabolic heterogeneity with more glycolytic metabolism of cancer cells in the stroma-rich zones. The shift of cellular energy metabolism from glycolysis to oxidative phosphorylation and the activation of lipogenesiswere observed in adipocytes. Data aboutmetabolic alterations in cancer and stemcells are important formonitoring the progression of cancers, the development of anticancer drugs and stemcell therapy.