A Leica TCS SP5 II upright resonant scanning multi-photon microscope with a Leica 25X, 0.95NA water immersion objective was used throughout the entire experiment (Leica Microsystems, Germany). A Ti:sapphire laser (Spectra Physics, Irvine, California) was tuned to 720 nm for two-photon excitation in the UV range.
All in vivo vascular images were captured at 0.68 μm isotropic resolution with 8 line averages. For each image, 512 × 512 pixels were collected every 280 msec with non-descanned detectors and an 8 kHz resonant line scanning mirror. The vasculature dye ANEPPS channel consists of two dichroic mirrors with cutoff wavelengths at 510 nm and 560 nm (Semrock FF510-Di02 and FF560-Di01, respectively) and a 70 nm wide band-pass filter centered at 605 nm (Chroma Technology, ET605/70M). A short-pass filter with a cutoff wavelength at 680 nm is used to block the excitation light for all imaging experiments.
The 4.0 μm microsphere image stacks were captured at 0.4 × 0.4 × 0.4 μm and at 50 × 50 × 50 nm isotropic resolution with 2 line averages using two dichroic mirrors with cutoff wavelengths at 450 nm and 650 nm (cover full emission range of microspheres). The PSF images from the 0.2 μm microsphere were scanned at 52 × 52 × 142 nm resolution.
All in vivo vascular images were captured at 0.68 μm isotropic resolution with 8 line averages. For each image, 512 × 512 pixels were collected every 280 msec with non-descanned detectors and an 8 kHz resonant line scanning mirror. The vasculature dye ANEPPS channel consists of two dichroic mirrors with cutoff wavelengths at 510 nm and 560 nm (Semrock FF510-Di02 and FF560-Di01, respectively) and a 70 nm wide band-pass filter centered at 605 nm (Chroma Technology, ET605/70M). A short-pass filter with a cutoff wavelength at 680 nm is used to block the excitation light for all imaging experiments.
The 4.0 μm microsphere image stacks were captured at 0.4 × 0.4 × 0.4 μm and at 50 × 50 × 50 nm isotropic resolution with 2 line averages using two dichroic mirrors with cutoff wavelengths at 450 nm and 650 nm (cover full emission range of microspheres). The PSF images from the 0.2 μm microsphere were scanned at 52 × 52 × 142 nm resolution.