Fig. 1

Microscope setup and imaging protocol. A Cranial window surgery allows optical access to the M2CA. Dashed line demarcates attachment of the temporal muscle to the cranium (a’). The arrowhead points towards the middle cerebral artery (MCA) (a’’). Position of the craniotomy is outlined (a’’’). The arrow points towards the M2CA and indicates the direction of blood flow (a’’’’). Hooks retracting the temporal muscle can be removed while the dental cement is still wet (arrows, a’’’’’). B Experimental outline of two-photon imaging of ischemic stroke in mice: Animals are implanted with cranial windows over the M2CA (arrow indicates direction of blood flow). Photothrombotic M2CAO is induced through the cranial window. Mice are immediately transferred to the microscope to image the acute phase of ischemic stroke. The cerebral vasculature can be followed from the acute, to the subacute and the chronic phase after ischemic stroke. Mouse schematic adapted from scidraw.io. C The M2CA before and directly after photothrombosis. Arrows indicate the direction of blood flow. D Quantification of TTC stainings of mouse brains 72 h after sham or stroke surgery. Sham-operated animals did not show any brain damage, while mice subjected to M2CAO had average infarct volumes of 12.7% of the ipsilateral hemisphere (n = 4 animals). E TTC stainings of mouse brains 72 h after sham or stroke surgery. Mice that underwent M2CAO surgery had visibly damaged tissue in the cortex and parts of the striatum (dashed line). F Schematic of the two-photon microscope used for dual-color imaging in the anesthetized living mouse. LP, long pass filter; BP, band pass filter; PMT, photomultiplier tube; HyD, hybrid detector. G Epifluorescent and two-photon images of wild-type C57BL/6 and endothelial cell reporter mice (Claudin5-GFP) intravenously injected with fluorescent tracers (FITC70, TRITC70). Scale bars (G) 100 µm; (g’-g’’) 25 µm. M-L, mediolateral axis; A-P, anterior–posterior axis