Brain Function: Novel Technologies Driving Novel Understanding - Bioinspired Approaches for Human-Centric Technologies - page 297

imaging is useful for investigating the activity of nonneuronal cells, in particular
astrocytes, which are a subtype of glial cells that play important modulatory roles in
the brain (Haydon 2001 ; Volterra and Meldolesi 2005 ; Fellin 2009 ). Thus, by using
a combination of two-photon microscopy and calcium imaging, it is now possible to
monitor the excitability of both neurons and glia in the intact central nervous
system.
The value of this optical approach in studying the functional properties of
cellular networks is easy to appreciate. Because the interactions between different
cells generate the complex ensemble dynamics that must form the basis of brain
function, preserving the structure and function of the network circuitry is critical.
Because light penetrates the tissue without causing mechanical disturbances, fluo-
rescence calcium imaging allows the investigation of the function of brain cells and
their interactions with the external world with minimal invasiveness. Furthermore,
in vivo fluorescence microscopy allows simultaneous visualization of the function
and structure of hundreds of cells with single-cell resolution (Stosiek et al. 2003 ;
Gobel et al. 2007 ), which is not possible with current electrophysiological
approaches.
From an optical point of view, however, recording fluorescent signals generated
deep within the brain is not a trivial task. The presence of many molecules and
compartments with different optical properties renders the brain optically
nonhomogeneous, with large variations in its refractive index (Helmchen and
Denk 2005 ). These differences in optical homogeneity cause the deflection of
light rays from their original path, a phenomenon termed scattering. Light scatter-
ing plays a fundamental role in the progressive degradation of fluorescence imaging
at increasing depths below the brain surface, which renders the signal generally
impossible to detect in regions deeper than 1 mm (Helmchen and Denk 2005 ). Most
importantly, light scattering is inversely related to the wavelength of the light that is
used; thus, blue-shifted light (of a shorter wavelength) is highly scattered, whereas
red-shifted light (of a longer wavelength) is scattered to a lesser extent. The success
of two-photon microscopy for in vivo fluorescence imaging relies heavily on using
infrared-shifted light to significantly decrease light scattering compared to imaging
using the visible wavelength range (Denk et al. 1990 ; Denk and Svoboda 1997 ;
Zipfel et al. 2003 ; Svoboda and Yasuda 2006 ). This approach permits the detection
of fluorescent signals from deeper (up to 900-1,000 mm) regions of the brain
(Theer et al. 2003 ) compared to imaging using single-photon excitation (up to
50-100 mm) while providing sufficient spatial resolution to monitor cellular and
subcellular structures. Two-photon microscopy is increasingly combined with the
use of genetically encoded calcium indicators (Looger and Griesbeck 2012 ). Com-
pared to synthetic calcium dyes, the genetically encoded indicators have the
advantage that they can be targeted to either specific cells in the brain or specific
subcellular compartments, thus facilitating the identification of the cellular source
of the signal. Moreover, since their expression is stable, functional imaging of
calcium signals over extended periods of time (from weeks to months) is possible.