Ti sapphire laser

TPEF images were acquired on a Leica TCS SP2 confocal microscope (Wetzlar, Germany) equipped with a Ti:sapphire laser (Spectra Physics, Mountain View, CA). Samples were placed on glass coverslips, excited with 755, 800 and 860 nm light and imaged using a 63×, 1.2 NA water immersion objective. The microscope was equipped with a microincubation system that kept samples in a humid chamber maintained at 37°C and 5% CO₂ (Okolabs, Ottaviano, Italy). TPEF images were acquired using emitter bandpass filters centered at 460 and 525 nm. Image stacks from one to four regions from 43 independent EETE tissues (n_{C_HFK} = 10, n_HKc/DR = 4, n_{16E6_HFK} = 10, n_{16E7_HFK} = 6 and n_{16E6E7_HFK} = 13) were acquired. TPEF 3D images were rendered in ImageJ (v10.2) and OsiriX (v3.0.2).

An Olympus BX51WI upright microscope and a water-immersion LUMPlan FL/IR 20×/0.50 W objective were used. Excitation (740 nm) was provided by a Prairie View Ultima multiphoton laser scan unit powered by a Millennia Prime 10 W diode laser source pumping a Tsunami Ti: sapphire laser (Spectra-Physics, Mountain View, CA, USA). Blood plasma was labeled by i.v. injection of tetramethylrhodamine isothiocyanate dextran (155 kDa) in physiological saline (5 % wt/ vol). All microvessels in an imaging volume (500 × 500 × 300 μm) were scanned at each study point, measuring the diameter and blood flow velocity in each vessel (3–20 μm Ø). Tetramethylrhodamine fluorescence was band pass filtered at 560–600 nm and NADH autofluorescence at 425–475 nm. Imaging data processing and analysis were carried out using the NIH ImageJ processing package.

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.