Two-Photon Microscopy in the Mammalian Brain

Two-photon fluorescence microscopy, first introduced in 1990 (Denk et al., 1990), dramatically improved the ability to see inside strongly scattering biological tissues. It has therefore emerged as a highly suitable modality for neural imaging and for noninvasive fluorescence microscopy in living animals (Zipfel et al., 2003). Development of membrane-permeable calcium sensitive fluorescent indicators and their in vivo demonstration for multi-cell loading in 2003 (Stosiek et al., 2003), combined with two-photon microscopy techniques, enabled the investigation of neural populations in vivo with the ability to monitor each neuron separately (Ohki et al., 2005; Garaschuk et al., 2006). The fluorescence of these indicators depends on Ca²⁺ concentration, and since the initiation of an action potential in neurons is associated with a fast inward Ca²⁺ flux, followed by slower return to baseline (Smetters et al., 1999), calcium concentration inside the neuron follows the action potentials pattern. In addition, temporal deconvolution and other spike inference methods can be applied to the data, allowing for the separation of temporally adjacent action potentials and therefore the improvement of temporal resolution (Yaksi and Friedrich 2006; Deneux et al., 2016; Pnevmatikakis et al., 2016).