Fig. 1: MD-FSS enables rapid aberration measurement for AO correction through highly turbid medium.

a Schematics of the MD-FSS system. The inset illustrates the measurement of aberrated PSF electric field by MD-FSS: Multiple phase-modulated weak focal points are spatially shifted by equal distances to simultaneously probe the electric field. \({E}_{{stat}}\), focal field of strong stationary beam positioned on the guide star; \({E}_{{scan}}\), focal field of the multiple weak scanning beams; \({E}_{{psf}}^{i}\), measured complex-valued PSF of \({i}_{{th}}\) multiplexed beam at down-sampled scanning scheme; \({f}_{n}\), modulation frequency of each weak scanning beam. b Comparison of the measured PSFs using SD-FSS and MD-FSS. Top: Diagram illustrating the focal field and scanning scheme of SD-FSS (left), accompanied by the amplitude (mid) and phase (right) of the full-sampled PSF measured by SD-FSS; Middle: Diagram illustrating the scanning scheme of one down-sampled scanning beam in MD-FSS (left), along with the amplitude (mid) and phase (right) of one down-sampled PSF; Bottom: Diagram illustrating the merging of multiple down-sampled PSFs from all scanning beams (left), along with the amplitude (mid) and phase (right) of the merged full-sampled PSF measured by MD-FSS; ds: down-sampled scanning, full: full-sampled scanning. c, d 2PF image of \(1\,\mu m\) fluorescence beads at a depth of \(400\,\mu m\) through a \(50\,\mu m\) thinned skull, shown without AO correction (left), with SD-FSS AO correction (middle) and with MD-FSS correction (right); Signal intensity with AO correction was enhanced by 50-fold. e Line profile and full width at half maximum (FWHM) measured along the dashed white line in (c) (left) and (d) (right). f Comparison of AO correction result between SD-FSS and MD-FSS on awake behaving Thy1-YFP mouse through a thinned-skull window; Images of neuron dendrites without AO correction (left), with SD-FSS AO correction (middle), and with MD-FSS AO correction (right). g Line profile along the dashed white line in (f). h Measured amplitude and phase of aberrated PSF using SD-FSS (top) and MD-FSS (bottom) in (f) on awake behaving mouse; (i) AO correction phase pattern calculated from PSF in (h), using SD-FSS (top) and MD-FSS (bottom). The results (f–i) demonstrate that MD-FSS’s fast measurement speed enables the PSF sensing to be less affected by motion artifacts. Scale bars: \(1\,\mu m\) in (c, d); \(20\,\mu m\) in (f). Figures (a) brain and objective model created using BioRender (https://biorender.com/).