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).

In recent decades, optogenetics has been transforming neuroscience research, enabling neuroscientists to drive and read neural circuits. The recent development in illumination approaches combined with two-photon (2P) excitation, either sequential or parallel, has opened the route for brain circuit manipulation with single-cell resolution and millisecond temporal precision. Yet, the high excitation power required for multi-target photostimulation, especially under 2P illumination, raises questions about the induced local heating inside samples. Here, we present and experimentally validate a theoretical model that makes it possible to simulate 3D light propagation and heat diffusion in optically scattering samples at high spatial and temporal resolution under the illumination configurations most commonly used to perform 2P optogenetics: single- and multi-spot holographic illumination and spiral laser scanning. By investigating the effects of photostimulation repetition rate, spot spacing, and illumination dependence of heat diffusion, we found conditions that make it possible to design a multi-target 2P optogenetics experiment with minimal sample heating.