Two-photon optogenetics

Abstract

The use of optogenetics, the technology that combines genetic and optical methods to monitor and control the activity of specific cell populations, is now widely adopted in neuroscience. The development of optogenetic tools, such as natural photosensitive ion channels and pumps or calcium- and voltage-sensitive proteins, has been growing tremendously during the past 10 years, thanks to the improvement of their performances in terms of facilitating light stimulation. To this aim, efficient illumination methods are also needed. The most common way to photostimulate optogenetic tools has been, so far, widefield illumination with visible light. However, the necessity of addressing the complexity of brain architecture has recently imposed switching to the use of two-photon excitation, which provides a better spatial specificity and deeper penetration in scattering tissue. Two-photon excitation is still challenging, due to intrinsic characteristics of optogenetic tools (e.g., the low conductivity of light-sensitive channels), and efficient illumination methods are therefore essential for advancing in this domain. Here, we present a review on the existing two-photon optical approaches for photoactivation of optogenetic tools, and future perspectives for the widespread implementation of these techniques.

Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics

“…Many optogenetic applications require manipulating select cells within a population with high spatiotemporal precision. Two-photon digital holography (2P-DH) is an effective method to flexibly target light to many regions simultaneously, or to produce an extended image to excite large patches of membrane for summation of single-channel ionic currents (13). It was recently shown that 2P scanning excitation of MAG 2p could depolarize neurons, but scans of nearly 1 s were necessary (32).…”

“…Using digital holography (2P-DH) (13,34), we demonstrate that activation of L-MAG0 460 is compatible with functional imaging using the red genetically encoded calcium indicator R-GECO1.0 (35). This combination of optical tools facilitates fast, multicellular stimulation and recording, an important benchmark for the utility of PTLs for in vivo optogenetic studies.…”

“…In particular, the inherent spatial confinement of 2P absorption is critical for optical manipulation of individual cells (10) in the intact mammalian brain, where it is difficult to control the spatial extent of gene expression or confine soluble reagents. However, 2P-excited optogenetics is not yet as widespread in adoption as 2PM, being widely perceived to require sophisticated optical techniques (11)(12)(13) or reagent concentrations that compromise pharmacological specificity (2,14).…”

Voltage imaging to understand connections and functions of neuronal circuits

“…For instance, cellular resolution functional imaging will require novel 2p microscopes that are able to monitor activity patterns of a large number of neurons updated at approximately each millisecond. The current trend of 2p optical instrumentation advances toward solutions that involve spatial, temporal, and possibly spectral multiplexing strategies enabled by multifocal, temporal focusing and holographic technologies (Emiliani et al 2015;Oron et al 2012;Shtrahman et al 2015). Another possibility that should not be neglected is 1p approaches that resolve single cells in vivo such as GRIN lenses (Jung et al 2004;Tang et al 2015) or light sheet approaches (Bouchard et al 2015).…”

Superresolving dendritic spine morphology with STED microscopy under holographic photostimulation

“…[29][30][31] Both for optogenetics and uncaging, the light should ideally be delivered to the specimen in a way that allows targeting single excitable units, which can be specific parts of a tissue, single cells, or even subcellular compartments, such as neuronal dendrites or spines. [32][33][34] In the case of dendritic spine observation, this is all the more important since it has been shown that dendritic spines exhibit collective *Address all correspondence to: Valentina Emiliani, E-mail: valentina.emiliani@ parisdescartes.fr behaviors even when separated by several microns. 35,36 Also, nonlinear synaptic integration along dendrites has been found.…”