Flat-top beam illumination for polarization-sensitive second-harmonic generation microscopy

Wide-field second-harmonic generation (SHG) was used to obtain the second-harmonic signal from the entire image area for rapid imaging, despite the fact that conventional Gaussian beam illumination has low energy utilization efficiency, which makes it easy to overexpose the intensity of the image center area. However, flat-top beam illumination has uniform spatial distribution, thereby improving the photon excitation efficiency in the entire image region and reducing laser damage and thermal effect. By combining flat-top beam illumination and wide-field SHG polarization measurement, we can calculate more myosin fibril symmetrical axis orientations through polarization analysis of 16 images at a fast imaging speed while expanding the field of view. More importantly, the application of a flat-top beam can further improve the capability of polarization measurement in SHG microscopy.