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

Methods Mol Biol. 2019:1929:15-26. doi: 10.1007/978-1-4939-9030-6_2.

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.

Keywords: Calcium imaging; Cortex; In vivo; Mouse; Neuronal activity; Two-photon microscopy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium / analysis*
  • Calcium Signaling
  • Fluorescent Dyes / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Microscopy, Fluorescence, Multiphoton / instrumentation*
  • Microscopy, Fluorescence, Multiphoton / methods
  • Neurons / metabolism*
  • Single-Cell Analysis / instrumentation
  • Single-Cell Analysis / methods
  • Visual Cortex / cytology*
  • Visual Cortex / metabolism

Substances

  • Fluorescent Dyes
  • Calcium