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

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1929; p. 15
• Main Authors: Birkner, Antje, Konnerth, Arthur
• Format: Journal Article
• Language: English
• Published: United States 2019
• Subjects: Calcium imaging; Two-photon microscopy; In vivo; Neuronal activity; Mouse; Cortex
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-9030-6_2

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.