Reliable targeting method for in vivo photo-stimulation of neurons*

Recent advances in optical neuroscience have provided powerful tools to explore neuronal circuitry of the brain in novel ways. In vivo calcium imaging allows for monitoring the activity of tens to hundreds of neurons in real time. Simultaneous photo-stimulation of target neurons can be achieved using an integrated holographic stimulation beam path. However, the persistent jitter in awake behaving animals hinders our ability to accurately read out neural activity or to reliably target neurons for photo-stimulation. Real time capable tracking-based motion correction (Aghayee et al, 2017) corrects for the jitter and therefore allows for a valid read out of neural activity essential for identification of target neurons. We show that motion compensation can be used to estimate the real-time position of neurons more accurately as opposed to using the average position. In addition, we address the optical accessibility and off-target illumination problem in holographic microscopy by introducing a simple optical configuration that reduces speckles and yields more uniform intensity across target points.