Recent advances in patterned photostimulation for optogenetics

Abstract

An important technological revolution is underway in the field of neuroscience as we begin the 21st century. The combination of optical methods with genetically encoded photosensitive tools (optogenetics) offers the opportunity to quickly modulate and monitor a large number of neuronal events and the ability to recreate the physiological, spatial, and temporal patterns of brain activity. The use of light instead of electrical stimulation is less invasive, and permits superior spatial and temporal specificity and flexibility. This ongoing revolution has motivated the development of new optical methods for light stimulation. They can be grouped in two main categories: scanning and parallel photostimulation techniques, each with its advantages and limitations. In scanning approaches, a small light spot is displaced in targeted regions of interest (ROIs), using galvanometric mirrors or acousto-optic deflectors, whereas in parallel approaches, the light beam can be spatially shaped to simultaneously cover all ROIs by modulating either the light intensity or the phase of the illumination beam. With amplitude modulation, light patterns are created by selectively blocking light rays that illuminate regions of no interest, while with phase modulation, the wavefront of the light beam is locally modified so that light rays are directed onto the target, thus allowing for higher intensity efficiency. In this review, we will describe the principle of each of these photostimulation techniques and review the use of these approaches in optogenetics experiments by presenting their advantages and drawbacks. Finally, we will review the challenges that need to be faced when photostimulation methods are combined with two-photon imaging approaches to reach an all-optical brain control through optogenetics and functional reporters (Ca2+ and voltage indicators).

Wide-area all-optical neurophysiology in acute brain slices

“…Point-scanning based mesoscopes have achieved pixel rates of ~2×10 7 /s over 0.6 × 0.6 mm FOVs but the requirement to translate the beam long distances limits pixel rates over large FOVs (4.4 × 4.2 mm) to 5.6×10 6 /s. Acousto-optical steering allows fast 2P random-access imaging 38 For precisely targeted single-cell stimulation, 2P optics are essential, but for wide-area optogenetic stimulation, 1P optics are preferable, as follows: 2P optogenetic stimulation requires timeaverage optical powers of 20 -80 mW/cell, [2][3][4] . Maximal safe steady-state 2P optical power into intact brain tissue is ~200 mW 39 , limiting simultaneous 2P stimulation to at most a few tens of neurons at a time.…”

“…TTX (1 µM) silenced activity in both the voltage (pink) and Ca 2+ (grey) channels, confirming that signals arose from neural activity and not optical crosstalk. Voltage imaging was performed at 500 Hz with 0.7 W/cm 2 640 nm light and calcium imaging was performed at 20 Hz with 1.1 W/cm 2 Fig. 3d.…”

“…All-optical neurophysiology (AON)-simultaneous optical stimulation and optical readout of neural activity-provides a promising approach to mapping neural excitability and synaptic strength across wide regions of brain tissue 1,2 . Recent advances in two-photon (2P) calcium imaging AON in vivo have enabled input-output measurements on circuits containing up to ~100 near-surface neurons in small cortical regions.…”

“…Recent advances in two-photon (2P) calcium imaging AON in vivo have enabled input-output measurements on circuits containing up to ~100 near-surface neurons in small cortical regions. [2][3][4] However, most of the intact rodent brain remains inaccessible to optical microscopy, and one would ideally like to perform AON simultaneously on many thousands of neurons across multiple brain regions to map spatial variations in function. Acute brain slices in principle enable wide-area optical mapping across any brain region.…”

Multiplexed temporally focused light shaping for high-resolution multi-cell targeting

“…This can be seen as follows: the temporal resolution for scanning 20 illumination, TR,scan, roughly equals the illumination time per spot (tdwell) multiplied by the number of scanned positions and by the number of targets, while that for parallel approaches, TR,paral, is only given by tdwell. As a consequence, for volumetric multi-cell targeting, TR,scan can strongly exceed the value of TR,paral and 3D parallel illumination remains the only option to achieve millisecond temporal resolution 39 .…”