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Authors & Affiliations
Emir Karamehmedovic
Abstract
Intensity fiber photometry has contributed to significant advances in neuroscience over the past ten years. Recording the calcium-dependent change in fluorescence intensity enables the correlation of the mouse brain activity with different behaviors and external excitations. However, intensity, even normalized using the isosbestic excitation point of the calcium sensor, is not a reliable parameter when comparing the different brain regions or animals, as conformational changes and oscillations in the pH value modulate the signal. Also, for some compounds, there are no intensiometric sensors. In some cases, measuring fluorescence lifetime enables better quantification and process monitoring, as fluorescence lifetime is an absolute parameter independent of the signal strength. We introduce a novel Fluorescence Lifetime Fiber Photometry (FLiP) method that relies on waveform sampling rather than TCSPC. The FLiP system demonstrates resilience to background illumination and fiber autofluorescence. Measurements on solutions that are typically used as fluorescent labels such as Coumarine 6, fluorescein, and Lucifer yellow in different solvents, with fluorescence lifetimes in the range of 2ns to 10ns, show excellent agreement with the results reported in the literature. We show that the technique allows data acquisition of up to 10Hz in samples relevant to neuroscience. The instrument's precision depends on the integration time, fluorophore concentration, excitation peak power, and fiber type, and is typically around 10ps for a single dye solution. Measurements are done with fluorophore concentrations from 10nM to 400nM. The new waveform sampling FLiP system offers simultaneous lifetime measurements at three excitation wavelengths and detection in two detection windows.