Second-harmonic generation microscopy with synthetic aperture and computational adaptive optics

Abstract

Second-harmonic generation (SHG) microscopy is a powerful label-free imaging tool widely used to visualize collagen and muscle in biological tissues. However, traditional laser-scanning SHG microscopy requiring voxel scanning is time-intensive. Wide-field SHG microscopy was designed to bypass this restriction, but its application to deep tissue imaging is limited due to vulnerability to scattering and sample-induced aberrations.We introduce synthetic aperture SHG (SA-SHG) microscopy to attenuate the effect of multiple scattering noises. Our SA-SHG method coherently integrates amplitude and phase maps of wide-fieldSHGfields taken for different illumination angles, thereby enhancing the signal-to-noise ratio.We also develop computational adaptive optics SHG (CAO-SHG) microscopy to computationally correct the sample-induced aberrations. Our algorithm optimizes SHG fields' aperture synthesis to identify aberration maps, enabling the restoration of diffraction-limited imaging. We successfully apply this approach to real biological samples, demonstrating its potential for high-resolution imaging in complex biological environments. © 2024 Optica Publishing Group.