Two-Wavelength Computational Holography for Aberration-Corrected Simultaneous Optogenetic Stimulation and Inhibition of In Vitro Biological Samples

Abstract

Optogenetics is a versatile toolset for the functional investigation of excitable cells such as neurons and cardiomyocytes in vivo and in vitro. While monochromatic illumination of these cells for either stimulation or inhibition already enables a wide range of studies, the combination of activation and silencing in one setup facilitates new experimental interrogation protocols. In this work, we present a setup for the simultaneous holographic stimulation and inhibition of multiple cells in vitro. The system is based on two fast ferroelectric liquid crystal spatial light modulators with frame rates of up to 1.7 kHz. Thereby, we are able to illuminate up to about 50 single spots with better than cellular resolution and without crosstalk, perfectly suited for refined network analysis schemes. System-inherent aberrations are corrected by applying an iterative optimization scheme based on Zernike polynomials. These are superposed on the same spatial light modulators that display the pattern-generating holograms, hence no further adaptive optical elements are needed for aberration correction. A near-diffraction-limited spatial resolution is achieved over the whole field of view, enabling subcellular optogenetic experiments by just choosing an appropriate microscope objective. The setup can pave the way for a multitude of optogenetic experiments, in particular with cardiomyocytes and neural networks.