Real-time 3D movement correction for two-photon imaging in behaving animals

Two-photon microscopy is widely used to investigate brain function across multiple spatial scales. However, measurements of neural activity are compromised by brain movement in behaving animals. Brain motion-induced artifacts are typically corrected using post hoc processing of two-dimensional image...

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Published inNature methods Vol. 17; no. 7; pp. 741 - 748
Main Authors Griffiths, Victoria A., Valera, Antoine M., Lau, Joanna YN, Roš, Hana, Younts, Thomas J., Marin, Bóris, Baragli, Chiara, Coyle, Diccon, Evans, Geoffrey J., Konstantinou, George, Koimtzis, Theo, Nadella, K. M. Naga Srinivas, Punde, Sameer A., Kirkby, Paul A., Bianco, Isaac H., Silver, R. Angus
Format Journal Article
LanguageEnglish
Published New York Nature Publishing Group US 01.07.2020
Nature Publishing Group
Subjects
631/1647/328
631/1647/328/2057
631/1647/334/1874/345
631/378
Acousto-optics
Animal models
Animals
Balances (scales)
Bioinformatics
Biological Microscopy
Biological Techniques
Biomedical and Life Sciences
Biomedical Engineering/Biotechnology
Brain
Dendrites
Digital integrated circuits
Female
Field of view
Field programmable gate arrays
Fluorescence
Imaging
Imaging, Three-Dimensional - methods
Life Sciences
Male
Mice
Mice, Inbred C57BL
Microscopy
Microscopy, Fluorescence, Multiphoton - methods
Movement
Neuroimaging
Neurons
Photons
Proteomics
Real time
Synapses
Three dimensional motion
Zebrafish
United Kingdom
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Summary:Two-photon microscopy is widely used to investigate brain function across multiple spatial scales. However, measurements of neural activity are compromised by brain movement in behaving animals. Brain motion-induced artifacts are typically corrected using post hoc processing of two-dimensional images, but this approach is slow and does not correct for axial movements. Moreover, the deleterious effects of brain movement on high-speed imaging of small regions of interest and photostimulation cannot be corrected post hoc. To address this problem, we combined random-access three-dimensional (3D) laser scanning using an acousto-optic lens and rapid closed-loop field programmable gate array processing to track 3D brain movement and correct motion artifacts in real time at up to 1 kHz. Our recordings from synapses, dendrites and large neuronal populations in behaving mice and zebrafish demonstrate real-time movement-corrected 3D two-photon imaging with submicrometer precision. Real-time 3D movement correction by tracking a fluorescent bead in the field of view enables functional imaging with 3D two-photon random-access microscopy in behaving mice and zebrafish.
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VG, and RAS conceived the project and RAS supervised it. VG, GK, K.M.N.S.N., P.A.K. and R.A.S designed the RT-3DMC system. VG and GK developed the closed loop FPGA code. A.V., B.M. and G.E. developed the MATLAB imaging software; P.A.K., K.M.N.S.N., G.K. and R.A.S. designed and built the AOL microscope. K.M.N.S.N., T.K. and S.A.P. developed and tested the LabVIEW imaging software. V.G., A.V., and R.A.S. designed the experiments on mice. D.C., T.Y., H.R. and C.B. performed viral transduction and surgeries. A.V, H.R., TY and C.B. performed the mouse experiments. J.Y.N.L., A.V., I.H.B. and R.A.S. designed the experiments on fish. J.Y.N.L. and A.V. performed the fish experiments. A.V. and V.G. analysed the data and AV made most of the figures. V.G., A.V. and R.A.S. wrote the manuscript with contributions from all authors.
Equal contribution Victoria A. Griffiths and Antoine M. Valera
Author contributions
ISSN:1548-7091
1548-7105
DOI:10.1038/s41592-020-0851-7