Supplementary Figure 8: Axial resolution of 2P holographic spots

Top (a) and lateral (b) view of the fluorescence emission generated by a 10 µm diameter holographic spot on a thin fluorescent layer. The fluorescence is collected by a second bottom objective and relayed to a CCD camera; the z-stack is acquired by scanning the vertical position of the upper objective. Scale bar, 10 µm. Measured performed on setup 1 (λ = 1030 nm). (c) Normalized axial intensity profile from the z-stack of panel b. (d) Simulated axial dependence of the photocurrent’s peak at different excitation powers using the excitation spot of panels a,b. The black curve represents a Gaussian fit of the spot’s axial profile shown in panel c (FWHM = 17.5 µm). Colored curves represent the simulated axial dependence of the photocurrent for different excitation powers, taking into account the experimental photocurrent dependence on the excitation power shown in the inset (see Methods for more details on the model used) Inset: Black dots represent measured normalized photocurrent from a CoChR expressing CHO cell (λ = 1030 nm, setup 1), fitted by an exponential (red curve). (e) Experimental (violet dots; n = 2 CoChR expressing cells) and simulated (red line) photocurrent’s peak axial profile for the 10 µm diameter holographic spot of panels a,b using an excitation power of 80 µW/µm². The blue curve is a Gaussian fit of the experimental data (FWHM = 43 µm). The black curve represents the Gaussian fit of the spot’s axial profile shown in panel c. (f) Lateral (x,z) view of the fluorescence emission generated by a 10 µm diameter holographic temporally focused spot on a thin fluorescent layer. Scale bar, 10 µm. Measurement performed on setup 2, modified by adding a diffraction grating for temporal focusing. (g) Experimental (violet dots) axial dependence of the photocurrent’s peak and Gaussian fit (blue line) generated by using the temporally focused spot shown in (f) (n = 1 CoChR expressing cell; n = 1 soCoChR expressing cell). The black curve represents the optical axial resolution of the temporally focused spot of panel f.