AbstractNear-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.
Optical Estimation of Absolute Membrane Potential Using One- and Two-Photon Fluorescence Lifetime Imaging Microscopy