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Abstract
.The excited state lifetime of a fluorophore together with its fluorescence emission spectrum provide information that can yield valuable insights into the nature of a fluorophore and its microenvironment. However, it is difficult to obtain both channels of information in a conventional scheme as detectors are typically configured either for spectral or lifetime detection. We present a fiber-based method to obtain spectral information from a multiphoton fluorescence lifetime imaging (FLIM) system. This is made possible using the time delay introduced in the fluorescence emission path by a dispersive optical fiber coupled to a detector operating in time-correlated single-photon counting mode. This add-on spectral implementation requires only a few simple modifications to any existing FLIM system and is considerably more cost-efficient compared to currently available spectral detectors.
Highlights
Fluorescence lifetime imaging (FLIM) microscopy is a microscopy technique that maps the fluorescence lifetime values at each voxel into image contrast
We propose a simple, cost-efficient method that, by the addition of an optical fiber to a conventional single detector multiphoton FLIM microscope, to obtain spectral information of emission signals which is otherwise unavailable without the fiber in addition to the lifetime information
A longer optical fiber will give a better spectral resolution, but it will suffer from larger signal attenuation
Summary
Fluorescence lifetime imaging (FLIM) microscopy is a microscopy technique that maps the fluorescence lifetime values at each voxel (the average time spent by the molecule in the excited state) into image contrast. FLIM can reveal spatial variations in the microenvironment of a sample by the virtue of the molecule’s available electronic states and the relaxation times from those levels to its ground state.[1] The technique of molecular probing using fluorescence lifetime has enabled the development of optical methods that reveal a wide range of properties, including molecular binding activity, and autofluorescence-based diagnostics.[2] Another fluorescence methodology that can provide information on the identity and microenvironment of a molecule is spectral or, when implemented across a broader sensing range, hyperspectral imaging (HSI).[3]. FLIM measures the fluorescence intensity as a function of time between excitation and fluorescence emission. Time-domain FLIM acquisition methods use high time-resolution electronics to measure the arrival time of the emission photon relative to the time of excitation photon pulse
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