Fig. 2

TIS window for two-photon microscopy. a1 A large-field and dual-channel two-photon microscopic image of bilateral hemispheric cortical blood vessels (in red, Texas red) and neurons (in green, EGFP) after the TIS window establishment (viewed with a 4 × objective). a2–a4 High magnification images of the corresponding color frames in (a1) near the sutura (viewed with a 20 × objective). a5-a10 High magnification (zoom in) images of the white frames (white arrow-indicated) in (a2–a4), respectively. (20 × objective, zoom 4). b Typical images of two-photon microscopy of neurons at various depths before and after the TIS window establishment, as well as after the removal of the skull. The axial step was set at 5 μm. c The SBRs of the images at various imaging depths under different conditions. The SBR was determined as follows: for each image of a certain imaging depth, firstly, a line across a typical structure (including the background around the structure) was drawn, and the intensity distribution along the line was obtained. Secondly, the obtained intensity values were normalized to 0–1. Thirdly, the ratio of the strongest value on the biological structure to the strongest value in the background was calculated as SBR. d Comparison between the TIS window and open-skull glass window in dendritic spine imaging. The excitation wavelength was 920 nm (80 MHz, 140 fs). Similar results were obtained in 4 mice