Optimization of wide-field second-harmonic generation microscopy for fast imaging of large sample areas in biological tissues

Second-harmonic generation (SHG) microscopy is a label-free imaging method that can be used to visualize the detailed arrangement of collagen structures in biological tissues. Here, we sought to optimize the speed of microscopic SHG image acquisition of macroscopic fixed tissue sample areas by employing the wide-field imaging with a high power and medium, 1 MHz pulse repetition frequency laser in combination with a mechanical sample scanning. Unlike in the conventional laser-scanning microscopy, the optimum of the wide-field acquisition entails an interplay between the size of the illuminated area and the intensity of the generated signal. We delineate quantitative procedures to set the image parameters for the maximum speed of the tiled image acquisition, and also describe the possible optimization of the laser parameters for further enhancement of the speed of acquisition.