The upright laser scanning microscope (BX61WI, Olympus, Tokyo, Japan) attached to a Ti:sapphire pulsed laser system (80 MHz repetition rate, <100 fs pulse width, Spectra Physics, Santa Clara, CA, USA) and software (Fluoview1000, Olympus, Japan) was used for two-photon fluorescence imaging. 20× water immersion (NA, 1.00; WD, 2 mm, Olympus), and 40× water-immersion objectives (NA 0.80, WD; 3.3 mm, Olympus) were selectively chosen for fluorescence imaging in vivo. 830-nm irradiation wavelength was used to excite calcein and dextran, and emission light was differentiated and detected with 525/50 and 615/50 filters, respectively. The average laser power for imaging was <30 mW.
Based on vessel diameter and blood flow direction, arterioles were discriminated and chosen as photo-thrombosis targets. System administration of Rose Bengal (RB, Sigma-Aldrich, USA; 100 µL, 10 mg/mL) was used to induce photo-thrombosis on specific arterioles under 563 nm laser beam illumination (intensity, 0.8–1.6 mW and stimulation duration, 80–100 s). All the procedure finished in 15 min after RB injection.
Based on vessel diameter and blood flow direction, arterioles were discriminated and chosen as photo-thrombosis targets. System administration of Rose Bengal (RB, Sigma-Aldrich, USA; 100 µL, 10 mg/mL) was used to induce photo-thrombosis on specific arterioles under 563 nm laser beam illumination (intensity, 0.8–1.6 mW and stimulation duration, 80–100 s). All the procedure finished in 15 min after RB injection.