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

Wei-Ting Zhang; Tzu-Hao Harry Chao; Yue Yang; Tzu-Wen Wang; Sung-Ho Lee; Esteban A Oyarzabal; Jingheng Zhou; Randy Nonneman; Nicolas C Pegard; Hongtu Zhu; Guohong Cui; Yen-Yu Ian Shih

doi:10.1016/j.crmeth.2022.100243

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

Cell Rep Methods. 2022 Jun 29;2(7):100243. doi: 10.1016/j.crmeth.2022.100243. eCollection 2022 Jul 18.

Authors

Wei-Ting Zhang^{1

2

3}, Tzu-Hao Harry Chao^{1

2

3}, Yue Yang^{1

2

3

4}, Tzu-Wen Wang^{1

2}, Sung-Ho Lee^{1

2

3}, Esteban A Oyarzabal^{1

2

3}, Jingheng Zhou⁵, Randy Nonneman^{1

2

3}, Nicolas C Pegard^{6

7}, Hongtu Zhu^{2

4

8}, Guohong Cui⁵, Yen-Yu Ian Shih^{1

2

3

7}

Affiliations

¹ Center for Animal MRI, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
² Biomedical Research Imaging Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
³ Department of Neurology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁴ Department of Biostatistics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁵ In Vivo Neurobiology Group, Neurobiology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, NC 27709, USA.
⁶ Department of Applied Physical Sciences, UNC Neuroscience Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁷ Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁸ Department of Computer Science, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Abstract

Fiber photometry is an emerging technique for recording fluorescent sensor activity in the brain. However, significant hemoglobin absorption artifacts in fiber photometry data may be misinterpreted as sensor activity changes. Because hemoglobin exists widely in the brain, and its concentration varies temporally, such artifacts could impede the accuracy of photometry recordings. Here we present use of spectral photometry and computational methods to quantify photon absorption effects by using activity-independent fluorescence signals, which can be used to derive oxy- and deoxy-hemoglobin concentration changes. Although these changes are often temporally delayed compared with the fast-responding fluorescence spikes, we found that erroneous interpretation may occur when examining pharmacology-induced sustained changes and that sometimes hemoglobin absorption could flip the GCaMP signal polarity. We provide hemoglobin-based correction methods to restore fluorescence signals and compare our results with other commonly used approaches. We also demonstrated the utility of spectral fiber photometry for delineating regional differences in hemodynamic response functions.

Keywords: emission; excitation; genetically encoded calcium indicators; green fluorescent sensor; hemodynamic response function; hemoglobin absorption; isosbestic; photon traveling path lengths; red-shifted fluorescent sensor; spectral fiber photometry.

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

Authors

Affiliations

Abstract

Publication types

MeSH terms

Grants and funding