Ndd pmt detectors

A multiphoton inverted stand Leica SP5 microscope (Leica Microsystems Gmbh, Wetzlar, Germany) was used for tissue imaging. A Ti:Sapphire Chameleon Ultra (Coherent, Saclay, France) with a center wavelength at 810 nm was used as the laser source for second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) signals. The laser beam was circularly polarized to ensure isotropic excitation of the sample regardless of the orientation of fibrillar collagen. A Leica Microsystems HCX IRAPO 25×/0.95 W objective was used to excite and collect SHG and TPEF. Signals were detected in epi-collection through a 405/15-nm and a 525/50 bandpass filter, respectively, by NDD PMT detectors (Leica Microsystems) with a constant voltage supply, at constant laser excitation power, allowing direct comparison of SHG intensity values. Two fixed thresholds were chosen to distinguish biological material from the background signal (TPEF images) and specific collagen fibers (SHG images). SHG score was established by comparing the area occupied by the collagen relative to the sample surface. Image processing and analysis (thresholding and SHG scoring) were performed using ImageJ homemade routines (https://imagej.nih.gov/ij/) as previously described [8 (link)]. Results were normalized to control C57/BL6 mice.

A multiphoton inverted stand Leica SP5 microscope (Leica Microsystems Gmbh, Wetzlar, Germany) was used for tissue imaging as previously described [13 (link)]. A Ti:Sapphire Chameleon Ultra (Coherent, Saclay, France) with a centre wavelength at 810 nm was used as the laser source for generating second harmonic generation (SHG) and two-photon excited fluorescence signals (TPEF). The laser beam was circularly polarized and we used a Leica Microsystems HCX IRAPO 25×/0.95 W objective. SHG and TPEF signals were detected in epi-collection through a 405/15-nm and a 525/50 bandpass filter, respectively, by NDD PMT detectors (Leica Microsystems) with a constant voltage supply, at constant laser excitation power, allowing direct comparison of SHG intensity values. LAS software (Leica, Germany) was used for laser scanning control and image acquisition.

The images were obtained using an inverted stand Leica SP5 microscope (Leica Microsystems GmbH, Wetzlar, Germany) coupled with a femtosecond Ti:sapphire laser (Chameleon, Coherent, Saclay, France) tuned at a wavelength of 810 or 850 nm for all experiments. The laser beam was circularly polarized and a Leica Microsystems HCX IRAPO 25×/0.95 W objective was used. SHG (collagen structure) signal was detected in epi-collection through a 405/15 nm bandpass filters, respectively, by NDD PMT detectors (Leica Microsystems) with a constant voltage supply, at constant laser excitation power, allowing direct comparison of SHG intensity values. All images were then analyzed using CT-FIRE software to obtain the width and the length of the collagen fibers. To calculate the curvature ratio, line regions were drawn along the length of the fibers (A) and along the linear distance between the start and the end of the fibers (B), the curvature ratio was calculated CR = A/B. For each image at least, 15 fibers were analyzed. Fiber alignment was determined using the directionality plugin in Image J. The fiber alignment was defined by the CV of the angle for all fibers per image. The smaller the CV is, the more aligned the fibers are.