A low-cost FPGA-based approach for pile-up corrected high-speed in vivo FLIM imaging

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Abstract
Two-photon excited fluorescence microscopy (2PM) has revolutionized neuroscience, allowing the investigation of biological dynamics with subcellular and subsecond resolution in living animals.1,2 Fluorescent labeling with proteins and dyes has been used to elucidate the morphology and behavior of cells and blood vessels under physiological and pathological conditions, whereas functional readouts such as calcium imaging or blood flow measurements have shed light on dynamic phenomena such as neuronal activity and neurovascular coupling.3,4 However, despite its successes, intensity-based 2PM is heavily affected by confounding factors such as fluorophore expression, tissue scattering and absorption, and sample motion. Although these problems can be somewhat alleviated using ratiometric approaches, the wavelength dependence of scattering and absorption still prevents accurate quantitation. Thus, traditional 2PM is insufficient to investigate biological processes such as metabolism, which require the precise quantification of molecule concentrations.5,6
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Funding Information
  • Innosuisse Innovation Agency (Grant 52207.1)

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