Fig. 6: 2-photon shadow imaging to estimate ECS volume fraction in vivo and reveal anatomical context of invading tumor cells.

a Z-stack of 2-photon shadow images acquired over >100 μm in depth (see also SI Movie 11). Scale bar, 25 μm; N = 4 mice; n = 18. b Regions of interest in neuropil analyzed for ECS volume fraction, based on normalized fluorescence intensity. Scale bars, 10 μm (left), 5 μm (zoom ins). c Group data of ECS volume fraction (VF) of cortical neuropil, based on normalized fluorescence intensity values, indicating that VF is substantial in mouse cortical neuropil in vivo, consistent with biophysical measurements in the literature. VF = 23 ± 0.7% (mean ± SEM); N = 4 mice; n = 36 regions of interest. Source data are provided as a Source data file. d In vivo 2-photon shadow images in motor cortex of mice implanted with YFP-labeled cells from a human GBM tumor cell line. The Alexa Fluor 488 (ECS) and YFP (tumor cells) fluorescence signals were spectrally detected. The images shown here were merged and pseudo-colored based on signal intensity (see also SI Movie 12). Scale bar, 20 μm. The imaging was repeated independently with similar results in 6 animals.