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

Abstract

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.