Fig. 4: 2-photon shadow imaging in acute brain slices.

a Schematic of imaging technique based on a commercial 2-photon microscope. b Acute brain slices were placed into the imaging chamber and a micro-manipulated patch pipette was used to inject the extracellular dye well below the surface of the slice (500 μM of Alexa488-Dextran). c 2-photon shadow images (representative of 15 independent experiments) of acute brain slices from different regions including hippocampus, corpus callosum and cerebellum, revealing their characteristic anatomical structure. Scale bars, 20 μm. d Shadow imaging of YFP-labeled tumor cells in acute brain slices that were implanted into the brains of mice four weeks before slicing, revealing their anatomical relationship with the cellular environment. Examples (representative of 5 independent experiments) from cortex and corpus callosum. Scale bars, 20 μm. Note instances of intimate ‘embrace’ of unlabeled structures/cells by tumor cells.