Deep Two-Photon Imaging In Vivo with a Red-Shifted Calcium Indicator

Part of the Methods in Molecular Biology book series (MIMB, volume 1929)


Two-photon calcium imaging became in recent years a very popular method for the functional analysis of neural cell populations on a single-cell level in anesthetized or awake behaving animals. Scientific insights about single-cell processing of sensory information but also analyses of higher cognitive functions in healthy or diseased states became thereby feasible. However, two-photon imaging is generally limited to depths of a few hundred micrometers when recording from densely labeled cell populations. Therefore, such recordings are often restricted to the superficial layers 1–3 of the mouse cortex, whereas the deep cell layers 4–6 are hardly accessible with standard two-photon imaging. Here, we provide a protocol for deep two-photon calcium imaging, which allows imaging of neuronal circuits with single-cell resolution in all cortical layers of the mouse primary cortex. This technique can be readily applied to other species. The method includes a reduction of excitation light scattering by the use of a red-shifted calcium indicator and the minimization of background fluorescence by visually guided local application of the fluorescent dye. The technique is similar to previously published protocols for in vivo two-photon calcium imaging with synthetic calcium dyes (Stosiek et al. Proc Natl Acad Sci U S A 100:7319–7324, 2003). Hence, only minor changes of a generic two-photon setup and some adaptations of the experimental procedures are required.

Key words

Calcium imaging Two-photon microscopy Neuronal activity Mouse Cortex In vivo 



This work was supported by the Deutsche Forschungsgemeinschaft (IRTG 1373 and SFB 870) and a European Research Council Advanced Grant, all to A.K.


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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Neuroscience, Technical University of MunichMunichGermany
  2. 2.Munich Cluster for Systems Neurology (SyNergy) and Center for Integrated Protein Sciences (CIPSM)MunichGermany

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