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Improved deep two-photon calcium imaging in vivo

Cell Calcium. 2016 Dec 21. pii: S0143-4160(16)30215-9. doi: 10.1016/j.ceca.2016.12.005. [Epub ahead of print]

Authors/Editors: Birkner A, Tischbirek CH, Konnerth A.
Publication Date: 2016



Two-photon laser scanning calcium imaging has emerged as a useful method for the exploration of neuralfunction and structure at the cellular and subcellular level in vivo. The applications range from imagingof subcellular compartments such as dendrites, spines and axonal boutons up to the functional analysisof large neuronal or glial populations. However, the depth penetration is often limited to a few hundredmicrometers, corresponding, for example, to the upper cortical layers of the mouse brain. Light scatteringand aberrations originating from refractive index inhomogeneties of the tissue are the reasons for theselimitations. The depth penetration of two-photon imaging can be enhanced through various approaches,such as the implementation of adaptive optics, the use of three-photon excitation and/or labeling cellswith red-shifted genetically encoded fluorescent sensors. However, most of the approaches used so farrequire the implementation of new instrumentation and/or time consuming staining protocols. Here wepresent a simple approach that can be readily implemented in combination with standard two-photonmicroscopes. The method involves an optimized protocol for depth-restricted labeling with the red-shifted fluorescent calcium indicator Cal-590 and benefits from the use of ultra-short laser pulses. Theapproach allows in vivo functional imaging of neuronal populations with single cell resolution in allsix layers of the mouse cortex. We demonstrate that stable recordings in deep cortical layers are notrestricted to anesthetized animals but are well feasible in awake, behaving mice. We anticipate that theimproved depth penetration will be beneficial for two-photon functional imaging in larger species, such as non-human primates.

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