Sp5 x mp multiphoton microscope

Immunostained tissues were imaged using an inverted Leica SP5-X MP multiphoton Leica microscope connected to a Ti-Sapphire laser (Spectra Physics MaiTai HP DeepSee Laser), Spectral Physics (tunable wavelength: 690 –1040 nm). The objective used was a HCX PL APO lambda blue 63 × 1.20 NA WATER UV. Tissue sections containing GFP-expressing cells were stained for expression of SCRG1 (Alexa 555) and Nidogen-2 (Alexa 647). Fibrillar collagen was imaged by means of second harmonic generation (SHG) using two-photon excitation at 892 nm and emission between 426 and 446 nm was detected using a hybrid detector (HyD SP, Leica). GFP-expressing cells were simultaneously excited using the 892 nm two-photon excitation and emitted GFP-light was collected between 505 and 550 nm using a PMT detector. SCRG1 (Alexa 555) and Nidogen-2 (Alexa 647) was excited with a supercontinuum white light laser (WLL) and emitted light (Alexa 555: 567–612 nm and Alexa 647: 650–710 nm) were detected using the hybrid detector (HyD SP, Leica). All images are from back-scattered light and captured with a resolution of 1024 × 1024 pixels, at 200 Hz.