Fig. 3 ∣. Comparison of LN blood vessel imaging by 3PM and 2PM.

a, In vivo images of fluorescein⁺ blood vessels using 1,280 nm 3PE and 920 nm 2PE and Alexa-Fluor-647⁺ blood vessels using 1,280 nm 2PE and 1,680 nm 3PE at the same site of the same LN. For 920 nm 2PE, no image is shown at 600 μm depth because the maximum imaging depth achieved was about 300 μm. Scale bars, 50 μm. b, Fluorescence intensity profiles across the blood vessels along the yellow lines in a. c, SBRs measured at different depths. Each data point is the average and s.d. of SBRs measured in three blood vessels in one image. d, Normalized fluorescence signal intensity as a function of imaging depth measured in the same mouse as in a. The fluorescence signal strength at a particular depth is represented by the average value of the brightest 0.5% pixels in the xy image at that depth divided by the square (for 2PE) or cube (for 3PE) of the average power. The effective attenuation length (l_e) was the inverse of the slope divided by the order of the nonlinear process (two for 2PE and three for 3PE).