Title High-throughput feedback-enabled optogenetic stimulation and spectroscopy in microwell plates Authors: Saachi Datta 1,✝ , William Benman 1,✝ , David Gonzalez-Martinez 1 , Gloria Lee 1 , Juliette Hooper 1 , Grace Qian 1 , Gabrielle Leavitt 1 , Lana Salloum 1 , Gabrielle Ho 1 , Sharvari Mhatre 1 , Michael S. Magaraci 1 , Michael Patterson 1 , Sevile G. Mannickarottu 1 , Saurabh Malani 2 , Jose L. Avalos 2,3,4 , Brian Y. Chow 1 , Lukasz J. Bugaj 1,5,6 * Affiliations: 1 Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, 19104, USA 2 Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544 3 The Andlinger Center for Energy and the Environment, Princeton, NJ 08544 4 High Meadows Environmental Institute, Princeton, NJ 08544 5 Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, 19104, USA 6 Institute of Regenerative Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA ✝ equal contribution *corresponding author Correspondence: bugaj@seas.upenn.edu ABSTRACT The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent programming of light patterns in single wells of microwell plates. However, there is currently a lack of instrumentation to monitor such experiments in real time, necessitating repeated transfers of the samples to stand-alone instruments and limiting the types of experiments that could be performed. Here we address this gap with the development of the optoPlateReader (oPR), an open-source, solid-state, compact device that allows automated optogenetic stimulation and spectroscopy in each well of a 96-well plate. The oPR integrates an optoPlate optical stimulation module with a module called the optoReader, an array of 96 photodiodes and LEDs that allows 96 parallel light measurements. The oPR was optimized for stimulation with blue light and for measurements of optical density and fluorescence. After calibration of all device components, we used the oPR to measure growth and to induce and measure fluorescent protein expression in E. coli. We further demonstrated how the optical read/write capabilities of the oPR permit computer-in-the-loop feedback control, where the current state of the sample can be used to adjust the optical stimulation parameters of the sample according to pre-defined feedback algorithms. The oPR will thus help realize an untapped potential for optogenetic experiments by enabling automated reading, writing, and feedback in microwell plates through open-source hardware that is accessible, customizable, and inexpensive. . CC-BY-NC 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 13, 2022. ; https://doi.org/10.1101/2022.07.13.499906 doi: bioRxiv preprint