Two-Photon Optogenetics by Computer-Generated Holography

Part of the Neuromethods book series (NM, volume 133)


Light patterning through spatial light modulators, whether they modulate amplitude or phase, is gaining an important place within optical methods used in neuroscience, especially for manipulating neuronal activity with optogenetics. The ability to selectively direct light in specific neurons expressing an optogenetic actuator, rather than in a large neuronal population within the microscope field of view, is now becoming attractive for studies that require high spatiotemporal precision for perturbing neuronal activity in a microcircuit. Computer-generated holography is a phase-modulation light patterning method providing significant advantages in terms of spatial and temporal resolution of photostimulation. It provides flexible three-dimensional light illumination schemes, easily reconfigurable, able to address a significant excitation field simultaneously, and applicable to both visible or infrared light excitation. Its implementation complexity depends on the level of accuracy that a certain application demands: Computer-generated holography can stand alone or be combined with temporal focusing in two-photon excitation schemes, producing depth-resolved excitation patterns robust to scattering. In this chapter, we present an overview of computer-generated holography properties regarding spatiotemporal resolution and penetration depth, and particularly focusing on its applications in optogenetics.

Key words

Light patterning Phase modulation Spatial light modulator Temporal focusing Optogenetics Opsin kinetics 



We thank Marco Pascucci and Benoît C. Forget for helpful discussions on the fitting procedure to extract opsins kinetics parameters; Valeria Zampini for recording data on CoChR-expressing neurons (Fig. 9); Dimitrii Tanese and Nicolò Accanto for help with preparation of Figs. 3, 4 and 6. We thank the National Institutes of Health (NIH 1-U01-NS090501-01), the “Agence Nationale de la Recherche” (grants ANR-10-INBS-04-01; France-BioImaging Infrastructure network, ANR-14-CE13-0016; HOLOHUB, ANR-15-CE19-0001-01; 3DHoloPAc) and the Human Frontiers Science Program (Grant RGP0015/2016) for financial support.


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© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Wavefront-Engineering Microscopy group, Neurophotonics Laboratory, CNRS UMR8250Paris Descartes UniversityParis Cedex 06France
  2. 2.Institut national de la santé et de la recherche médicale–InsermParisFrance

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