Super-resolution Radial Fluctuations Enables Polarization-resolved Nonlinear Optical Nanoscopy

Abstract

Second harmonic generation microscopy (SHG) is a powerful imaging modality which has found applications in investigating both biological and synthetic nanostructures. Like all optical microscopy techniques, the resolution of SHG is limited to approximately half the wavelength of the excitation light. Because of this several groups have proposed techniques to enable super-resolution SHG imaging. However, these techniques often involve quite complicated optical setups compared to standard SHG microscopes, a major impediment towards more widespread utilization. Here we apply super-resolution radial fluctuations (SRRF), a commonly used technique for super-resolution fluorescence imaging, to enable super-resolution SHG microscopy. By imaging individual nanostructures, we demonstrate that SRRF can provide resolution enhancement of up to 3x compared to a laser scanning SHG microscope, which is comparable to the best resolution enhancement reported in the literature. Additionally, we show that SRRF maintains the polarization dependence of SHG, therefore enabling super-resolution polarization SHG imaging. Finally, we perform SRRF processing on third harmonic generation images to demonstrate the significant potential of SRRF for other super-resolution nonlinear optical microscopy. Importantly, since SRRF can achieve super-resolution purely through image processing, the technique demonstrated here could be used to enhance the resolution of images obtained using a wide variety of nonlinear optical microscopy setups including both laser scanning and widefield configurations.