Fig. 5: 2-photon shadow imaging in the mouse brain in vivo.

a Schematic of imaging technique based on a custom-built 2-photon microscope equipped with a spatial light modulator (SLM) and a high-NA objective with a correction collar to reduce optical aberrations. b Schematic of the in vivo labeling strategy of the ECS, where a highly concentrated dye solution (<5 μL of 50 mM of Alexa Fluor 488) was stereotaxically injected slowly (over 10 min) into the ipsilateral lateral ventricle. c Time course of average fluorescence labeling intensity of the ECS in somatosensory cortex after intraventricular dye injection. The fluorescence labeling appeared within 30 min and stayed elevated at workable levels for at least 4 h; the average intensity drop between 1 and 4 h was 47.5 ± 2.2% (mean ± SEM); n = 4. d Representative in vivo 2-photon shadow images acquired in the somatosensory cortex of an anesthetized mouse. Left: raw image, revealing cell bodies as large black ‘shadows’; Scale bar, 25 μm; right: same image after inversion of the look-up-table; dotted square: zoomed-in part showing cluster of cell bodies; examples of shadow images delineating perivascular spaces (green arrows) running along blood vessels and putative pericytes and/or astrocytic endfeet (red arrows) wrapping around blood vessels. Scale bars, 10 μm. The imaging was repeated independently with similar results in 15 animals. e Perivascular spaces and blood vessel diameters were geometrically measured by line profiles laid orthogonally across the blood vessels (left: capillary; right: arteriole/venule), returning their widths (FWHM), scale bar, 10 μm. f Quantitative measurements of the diameters of perivascular spaces and blood vessels (mean ± SEM; N_capillary = 105; N_PVS = 210; N_mice = 7). Source data are provided as a Source data file. g The widths of perivascular spaces and the diameters of the blood vessels they enclose are highly correlated (Pearson correlation coefficient r = 0.838; p < 0.0001; N = 79). Source data are provided as a Source data file.