Blake Russell

Reduced stimulation responses are not limited to targeted cells and are amplified across the field of view. This can be mitigated by decreasing the imaging dose and increasing the inter-stimulus interval, crucial for anyone doing all-optical.

Thanks to Rob Lees, @laklab.bsky.social and Adam Packer

Out today in Neurophotonics, we show that cross-talk between the imaging laser and opsin in all-optical experiments can affect response dynamics beyond what has been accounted for. Imaging conditions subthreshold for evoking spikes can accelerate opsin desensitization:
doi.org/10.1117/1.NP...

Desensitization of opsin responses during all-optical interrogation depends on imaging parameters https://www.biorxiv.org/content/10.1101/2025.10.02.680058v1

Overall, this study shows that cross-talk may be impacting all-optical studies beyond what is typically accounted for.

Thankfully, this can be mitigated with methodological optimisations that will help reduce a source of trial-by-trial variability in future all-optical experiments! (5/5)

This can have a significant effect on how injected activity is propagated to the local network.

We find that a combination of longer inter-stimulus intervals and shorter imaging doses can ameleorate the observed response desensitization across the field of view (4/5).

This is a longstanding problem in the field, and has been addressed in previous studies by reducing the dwell time per cell and power on sample of the imaging laser.

However, we find that even imaging conditions that are subthreshold for evoking spikes can accelerate opsin desensitization (3/5).

All-optical experiments combine simultaneous two-photon imaging of a fluorescent reporter of neuronal activity with two-photon optogenetic stimulation. However, the reporter and actuator often have some spectral overlap. This can result in cross-talk where the imaging laser evokes spiking. (2/5)

Desensitization of opsin responses during all-optical interrogation depends on imaging parameters Significance The combination of two-photon calcium imaging and two-photon optogenetic stimulation, termed all-optical interrogation, provides spatial and temporal precision when recording and manipula...

🚨New pre-print!🚨

Can we truly independently read and write neuronal activity at the same time using all-optical techniques? Here we show that even two-photon imaging over large fields of view with low dwell time per cell can bias neural dynamics!

Read on for some potential fixes: (1/5)

Desensitization of opsin responses during all-optical interrogation depends on imaging parameters Significance The combination of two-photon calcium imaging and two-photon optogenetic stimulation, termed all-optical interrogation, provides spatial and temporal precision when recording and manipula...

All-optical experiments are great but @neurorussell.bsky.social found that opsin desensitization due to cross-talk between the imaging laser and the opsin causes major issues. These can be ameleorated with long inter-stimulus intervals & short imaging doses. All here: www.biorxiv.org/content/10.1...