Fig. 2. AO enables high-resolution 3P deep brain imaging.
Indirect modal-based AO correction and ECG-gated 3PM at 1,300 nm excitation. a,c,e, Representative images showing dendrites and spines in (a) layer VI and (c,e) hippocampus of the in vivo mouse brain (EGFP–Thy1(M)) through a cranial window. Images were recorded with two different conditions: AO system (wavefront correction of system aberrations) and AO full (wavefront correction of system and brain tissue aberrations). Maximum intensity projection images with system and full aberration correction at layer VI in cortex (a) as well as hippocampus (c,e) (scale bar, 20 μm; on zoomed regions, 2 µm). White boxes in a indicate magnified views of spines. The white cross in a,c,e shows orthogonal view along dendrite. Respective wavefront maps for aberration correction are displayed in the top and bottom corners. Aberration measurement location for e was identical to c. b, Intensity profile along spines (right; white dotted line) and across dendrite (left; white cross), respectively, in cortical layer VI (a). Norm., normalized. d, Intensity profile along neuron somata (white dotted line in c) and across dendrite (white dotted line in e). f, Lateral resolution analysis of AO correction improvement. Spectral power map as a function of spatial frequency (wavenumber) at (left) layer VI of cortex (a) and (right) hippocampus (e). Middle, average radial profile of spectral power maps for maximum intensity images. g, Axial resolution analysis. Effective PSF of microscope inferred from beads in solution and compared to in vivo measurements performed on dendrites, with system and full AO at a depth of 653 μm (cortex), 822 μm (cortex), and 1,000 and 1,327 μm (hippocampus). For AO at 1,327 µm, the AO measurement was performed at 1,000 µm depth. The effective illumination NA was reduced at a greater depth to increase objective transmission. h, Spatial analysis of lateral (green) and axial (blue) intensity enhancement (AO on/AO off) with respect to distance from location of AO measurement in cortex (left) and hippocampus (right). Median values are shown as dots and top 75th and bottom 25th percentile as shadows. Dotted lines show exponential fits. The red box indicates the area where the intensity enhancement (AO on/AO off) <1. Same data as Extended Data Figs. 6 and 7 for Z35. i, Analysis of wavefront distortion (peak-to-valley (PV) and root-mean-square error (r.m.s.e.)) displayed on DM for aberration correction in cortex (C) and hippocampus (HPC) and pooled from Extended Data Fig. 8. Only datasets with <100 μm lateral off-set between cortex and hippocampus in the same animal were included in the analysis. Representative datasets obtained from several imaging sessions in n = 7 mice.