Material for “ Two - photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion ”

Modified two-photon laser scanning microscope. We use a locally-designed TPLSM for the studies described here that has been modified to allow delivery of green laser light for photothrombotic clotting (Supplementary Fig. 1). Tightlyfocused 100-fs, 800-nm pulses from the femtosecond laser oscillator (Mira 900-F, Coherent, Inc.) are scanned throughout the brain of the rat using galvanometric scan mirrors and axial translation of the objective. The energy per pulse of the imaging beam was adjusted by passing the beam through a rotating half-wavelength (λ/2) plate and keeping only one polarization with a polarizing beam splitter. Two-photon excited fluorescence was separated by a dichroic mirror (600-nm long pass; dichroic 2 in Supplementary Fig. 1), spectrally filtered with BG-39 colored glass to block residual 800-nm light, and detected on a photomultiplier tube (PMT) (no. R6357; Hamamatsu). The PMT signal was amplified (10 transimpedance gain), low-pass filtered (4 pole Bessel at fc = 300 kHz), and digitized. LabViewTM-based software of local design controlled the scan mirrors and microscope objective stepper motor and recorded the PMT signal as a function of the scan mirror and objective position. Using the metal head frame that is affixed to the rat’s skull, the rat is bolted onto a two-dimensional translation stage underneath the microscope objective, allowing precise positioning of the rat relative to the TPLSM field of view. Individual planar images that spanned the area surrounding the target vessel, or sets of images at 1-μm axial spacing, were obtained both before and after photodisruption. Planar images were also used to monitor the production of the vascular disruptions in real-time.