Second harmonic generation and third harmonic generation (SHG and THG) microscopy are nonlinear optical imaging techniques which have found a diverse range of applications in the investigation of both biological and synthetic nanostructures. Like all optical imaging techniques, the spatial resolution achievable using SHG and THG is limited by diffraction to around half of the excitation wavelength, a major impediment towards applications in nano imaging. Because of this, several groups have developed methods for super-resolution SHG and THG imaging, however these approaches have all involved the use of nonstandard microscope components which presents a technical and financial barrier to entry for those interested in applying these techniques. Here we investigate the application of several computational super-resolution techniques for SHG and THG imaging, with a focus on enabling super-resolution polarization-resolved SHG microscopy. We find that even though computational super-resolution was originally developed for incoherent imaging modalities such as fluorescence, it is able to provide a lateral resolution enhancement of up to 3.4× when imaging isolated nanostructures compared to a standard laser scanning harmonic generation microscope. While currently available CSR techniques are not able, in general, to correct for the inherent ambiguity between density of emitters and signal intensity within coherent imaging processes, we show that CSR can still provide improved localization in harmonic generation microscopy. We additionally show that two computational super-resolution techniques, super-resolution radial fluctuations and deblurring by pixel reassignment, preserve the polarization dependence of SHG signal, thereby allowing super-resolution polarization-resolved SHG measurements. These results are obtained with no modification to our microscope system and little change to the experimental workflow, and therefore present exciting opportunities for future applications of super-resolution SHG and THG microscopy.
Harvey et al. (Tue,) studied this question.