Spectral fiber-photometry derives hemoglobin concentration changes for accurate measurement of fluorescent sensor activity

Abstract

Fiber-photometry is an emerging technique for recording fluorescent sensor activity in the brain. Here we demonstrate significant hemoglobin-absorption artifacts that may be misinterpreted as sensor activity changes. Because hemoglobin exists in nearly every location in the brain and its concentration varies over time, such artifacts could significantly impede the accuracy of photometry recording results. We present a novel use of spectral photometry technique and propose a computational method to quantify photon absorption effect from an activity-independent fluorescent protein, which can be used to derive oxy- and deoxy-hemoglobin concentration changes and correct target sensor activities across spectra. We further demonstrate the utility of this method for delineating brain regional differences in neurovascular transfer functions and shed light on the interpretation of hemodynamic-based neuroimaging data.

A flexible and versatile system for multicolor fiber photometry and optogenetic manipulation

“…To confirm these results and to exclude artefacts that could be caused by in vivo preparations (such as breathing, locomotion, or hemodynamics)(Lerner et al, 2015; Marshall et al, 2016; Zhang et al, 2021), we performed analog experiments in organotypic slice cultures. Brain slices were transduced with an identical combination of ChrimsonR and NIR-GECO2.…”

Simultaneous recording of neuronal and vascular activity in the rodent brain using fiber-photometry

“…The proposed three techniques utilize a spectrometer-based fiber-photometry system ( Meng et al., 2018 ). For labs using non-spectrometer-based photodetectors ( Gunaydin et al., 2014 ; Kim et al., 2016 ; Lazarjan et al., 2021 ), this protocol could be adopted by using multiple photodetectors with different bandpass filters ( Zhang et al., 2022 ). The protocol described below focuses on the measurement of neuronal and vascular activity changes simultaneously.…”

“…On the right are the high-resolution images of the brain area with fluorescence signals showing GCaMP alone (top), tdTomato alone (middle), and merged images (bottom). Image is reprinted with permission from ( Zhang et al., 2022 ). CRITICAL: After the injection, the micro syringe needle should stay unmoved for 10 min for virus diffusion.…”

“…This technique is applicable to different fluorescent sensors and provides a more accurate measurement of hemodynamic response function in any location of the rodent brain. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022) .…”

“…For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022) .…”