Studying the responses of retinal ganglion cell (RGC) populations is of major importance to the fields of neuroscience and vision research. Multiphoton excitation of optogenetic probes has recently become the leading imaging approach for recording fluorescence signals in neural populations and has  specific advantages  for  imaging  retinal  activity  during  visual  stimulation,  because  it  leads  to  reduced  direct excitation of the photoreceptors. However, multiphoton imaging of retinal activity is not straightforward: point-by-point scanning leads to repeated neural excitation and is inherently slow due to the challenge of collecting enough photons from every scanned location. In this study, we present an enabling optical design that facilitates multi-photon imaging of responses to visual stimuli in mouse retinas.

First, we introduce a rapid functional imaging  technique  based  on  Scanning  Line  Temporal  Focusing (SLITE)  for capturing neural activity in the isolated retina expressing the genetically encoded calcium indicator GCaMP6-type, with a temporally focused line rather than a point, increasing the scan speed and reducing the impact of repeated excitation, while maintaining high optical sectioning.

Next, we demonstrate significant neural responses to various stimuli and present our signal processing approach for advanced neural activity analysis.  We present our signal analysis work scheme, which includes automated video processing and signal extraction, artifact removal, fluorescence signal smoothing, and spike inference. Finally, we summarize and visualize spatial and temporal features of RGCs following analysis of the interaction between the projected stimuli and the neural response.

In sum, the new optical design and signal analysis methods overcome a number of outstanding obstacles, allowing the study of rapid calcium signals, thereby bringing us a step closer toward distributed monitoring of retinal neural activity during vision.