Fig. 2: AO-enhanced 3PM and AI-based denoising allows near-diffraction limited resolution at large image depths and high SNR imaging.

a 3D renderings showing 2PM and 3PM imaging down to approximately 1000 µm. Gamma values were adjusted for 3D visualization. b SNR of 2PM and 3PM along depths, normalized to the SNR at the brain surface. The dashed red line in (a, b) indicates the imaging depth at which biological structures cannot be clearly discerned anymore in 2PM in contrast to 3PM (approximately 450 µm). c Scheme of 3PM and adaptive optics setup. Modified from Fig. 1a in Ref. 22. d Exemplary GBMC imaged without (top left) and with (bottom left) AO optimization and the corresponding images on the THG channel (top and bottom right). The line indicates the line segment averaged over to produce the line profiles in (e), (n = 6 experiments with similar results) showing the effect of uncorrected optical aberration on the visibility of fine cellular structures. The inset on each panel shows the frequency ___domain power spectrum of the image, with the ring indicating 1 µm length scale. e Line profile comparisons for both mGFP and THG channels showing intensity enhancement. f The averaged radial profile of the frequency maps is shown, allowing easier estimation of the respective frequency cut-offs. g Exemplary 3PM images with and without AO optimization on the mGFP channel at different depths in deep cortex and CC (left and middle) and on the THG channel within the CC (right). Zoom-ins are shown on the right. Arrowheads show TMs (left), a shadow of a cell nucleus that is caused by the membrane-bound GFP labeling (middle), and a blood vessel (right) on AO on and off images. TMs, the cell nucleus, and the blood vessel are not clearly visible without AO (n = 6 experiments with similar results). Source data are provided as a Source Data file.