Second Harmonic Generation (SHG) and imaging

Both native tissue and decellularized liver scaffolds were cryoprotected and sections were then set in a mold and covered in OCT cold embedding media and frozen. The OCT blocks containing the liver tissue/scaffolds were cryosectioned to a thickness of 20 μm and mounted on glass slides. Prior to measurements, samples were thawed for 15 min in PBS.

All SHG images were obtained using a custom built multiphoton microscope incorporating an upright confocal microscope (SP5, Leica) and a mode-locked Ti: Sapphire Laser (Mai Tai, Newport Spectra-Physics). Images of the SHG signal from collagen I were collected using an 820 nm excitation with SHG signal obtained with a 414/46 nm bandpass filter and multiphoton autofluorescence signal obtained with a 525/40 nm bandpass filter. A 25X, 0.95 NA water-immersion objective (Leica) was used to deliver the excitation signal and to collect the SHG emission signal from the sample. Images with a 600 μm × 600 μm field of view were obtained with 2048 pixel resolution and a line rate of 10 Hz giving a pixel resolution of ~0.3 μm with 3X averaging on each acquisition to reduce the effect of noise.