Extended Data Fig. 3. Three-photon adaptive optics microscopy at 1300 nm excitation of intact Thy1-GFP mouse brain down to the edge of the CA1 stratum lacunosum at ~1.45 mm depth.

a, 3D reconstruction of three-photon image stack of THG signal (cyan) and GFP-labelled neurons (green). Image stack was acquired with AO correction based on AO measurements at three different depths (system 0 µm, cortex 600 µm, hippocampus 1000 µm), and utilized AO system correction for 0-500μm depth, AO Z35 (cortex) for 500-900μm depth, and AO Z35 (hippocampus) for 900-1450um depth. Lateral FOV 126×126 μm. b-i, Maximum intensity projection images of GFP (green) and THS (cyan) at various depths. b,f, Layer V/VI cortex where pyramidal cell bodies are imaged. c,g, At ~876-972μm depth the fibres of the corpus callosum (white matter) are visible. d, The CA1 pyramidal cell somata are visualized at ~1048-1128μm depth in the stratum pyramidale. e,h,i, At depth >1332μm the edge of the CA1 stratum lacunosum moleculare is reached, and the THS signal indicates denser tissue which suggests reaching the edge of the outer molecular layer or envelope of the hippocampal sulcus (h). Depth values correspond to axial motor position. Taking the refractive index difference between the coverslip/immersion media and various brain tissue into account, the actual imaging depth is ~5-10% larger⁴. Representative result of (n=3) experiments.