Chameleon vision s

Mice were imaged as previously described^{31 (link)}. In short, mice were sedated and placed face-up in a custom-designed imaging box. Time-lapse images of the entire tumor volume were acquired for a maximum of 13 hours. The minimal time interval between serial images was set to 45 minutes. For tile-scans, images of the complete z-stack of the tumor were acquired to a depth of 300 μm, with a step size of 3 µm (typically 70–100 images in each z-stack). In a group of 4 mice, blood vessels were imaged by intravenous injection of 70 kDa Dextran–Texas Red (Invitrogen Life Technologies) or 2.000.000 kDa Dextran-Rhodamine (Thermofisher scientific).
Imaging was performed on an inverted Leica SP8 multiphoton microscope with a chameleon Vision-S (Coherent Inc., Santa Clare, CA, www.coherent.com), equipped with a 25 x water objective (HCX IRAPO NA0.95 WD 2.5 mm) with four HyD detectors: HyD1 (560–650 nm), HyD2 (500–550 nm), HyD3 (455–490 nm), and HyD4 (<455 nm). Images were acquired at a wavelength of 960 nm, where H2B Dendra2 as well as both Dextran reagents showed sufficient signal intensity. Collagen (second harmonic generation) was detected using HyD4, H2B-Dendra2 was detected with HyD2 and Dextran reagents were detected with HyD1. Scanning was performed in a bidirectional mode at 400 Hz and 12 bit, with a zoom of 1 × , 512 × 512 pixels.

We applied our previously developed MNLO imaging method [20 (link),21 (link),22 (link)]. Briefly, we used two separate laser systems: a picosecond-pulsed laser system (pico-EMERALD; HighQ Laser, APE, Berlin, Germany) for CARS imaging, and a femtosecond-pulsed laser system (Chameleon Vision-S; Coherent Inc., Santa Clara, CA, USA) for TPEF imaging. To obtain CARS signals corresponding to Raman peaks 1 and 2 of PM_2.5 and SRM 2786, we set the center wavelengths of the pump beam to 827 nm and 817 nm, respectively, and the center wavelength of the Stokes beam to 1064 nm. To obtain TPEF and SHG signals, we set the wavelength of the fs-pulsed laser to 810 nm. All images were obtained using FluoView software (FV10-ASW; Olympus Corp., Tokyo, Japan).
For en face 3D MNLO imaging, human skin biopsy samples were mounted with the stratum corneum (SC) region facing down on a coverglass-bottom chamber and then covered with a coverslip. z-depth images were obtained to measure depths of 0–150 μm. For side-view imaging, samples were cross-sectioned into 50 μm thick slices.