Reported physiological point spread functions (PPSFs) using two-photon optogenetics vary. Here’s a table from the paper attempting to summarize values from the past years of work, which I hope will be useful to all. If your paper is not cited, or cited incorrectly, pls tell me!
We sought to understand how one specific factor, optical resolution, affects the resolution of photostimulation under specific conditions: spiral scanning C1V1-Kv2.1 expressing neurons with a low repetition rate (2 MHz) laser.
We held all variables constant in the experiment except for the optical resolution of the system, which we varied using a spatial light modulator to underfill the back aperture of the objective. We measured the optical PSF of our system to confirm the change in resolution.
Under these conditions, the spatial precision of photostimulation (measured by the width of the GCaMP6s response versus axial position at half-max) does NOT change even with more than a two-fold change in optical resolution!
Your mileage WILL vary with different opsins, different expression strategies, different lasers, different measurements, and different optical systems.
You may note specifically the legend to the table (posted above) in the paper states: “The lowest axial FWHM values tend to come from papers using perisomatic restriction and/or temporal focusing. Note the best consistent results from zebrafish neurons at 10 and 15 μm.”
A key take-home message is that cell size and shape determine the spatial precision under these conditions. This is confirmed by a simple model convolving the optical PSF with a cell and showing how the size of the cell dictates the physiological PSF above a certain size.
This project has been led by @robmlees, whose attention to detail, rigour, and strong desire to “get it right” have resulted in these wonderfully well-controlled and clear results. With crucial contributions from the always thoughtful and inspiring @brunopichler!
We thank our funding sources @ERC_Research and @wellcometrust. Shout outs to @OxNeuro, @DPAGAthenaSwan, and @UniofOxford for supporting this work and where this research took place!
Would love to hear what you all think and especially so for those whose papers we were inspired by! @EmilianiLab @UNC_Optics @ianOldenburg @HayleyBounds @FellinLab @OferYizhar @selmaanchettih @jonvgill @HistedLab @ShohamLab @JimMarshel @KarlDeisseroth @CarrilloReid @yusterafa ...