Excitation and detection in the wavelength range above 800nm is a convenient and relatively inexpensive way to increase the penetration depth in optical microscopy. Moreover, detection at long wavelength avoids the problem that tissue autofluorescence contaminates the signals from endogenous fluorescence probes. FLIM at NIR wavelength may therefore be complementary to multiphoton microscopy, especially if the lifetimes of NIR fluorophores report biological parameters of the tissue structures they are bound to. Unfortunately, neither the excitation sources nor the detectors of standard confocal and multiphoton laser scanning systems are directly suitable for excitation and detection of NIR fluorescence. Most of these problems can be solved, however, by using ps diode lasers or Ti:Sapphire lasers at their fundamental wavelength, and NIR-sensitive detectors. With NIR-sensitive PMTs the detection wavelength range can be extended up to 900 nm, with InGaAs SPAD detectors up to 1700 nm. Here, we demonstrate the use of a combination of laser scanning, multi-dimensional TCSPC, and advanced excitation sources and detectors for FLIM at up to 1700 nm. The performance was tested at tissue samples incubated with NIR dyes. The fluorescence lifetimes generally get shorter with increasing absorption and emission wavelengths of the dyes. For the cyanine dye IR1061, absorbing around 1060 nm, the lifetime was found to be as short as 70 ps. Nevertheless the fluorescence decay could still be clearly detected. Almost all dyes showed clear lifetime changes depending on the binding to different tissue constituents.
Wolfgang Becker and Vladislav Shcheslavsky, "TCSPC FLIM in the wavelength range from 800 nm to 1700 nm
(Conference Presentation)," Proc. SPIE 9723, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications VIII, 97230K (Presented at SPIE BiOS: February 16, 2016; Published: 27 April 2016); https://doi.org/10.1117/12.2217500.4848770180001.
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