Second harmonic generation (SHG) microscopy is expected to be a powerful tool for observing the cellular-level functionality and morphology information of thick tissue owe to its unique imaging properties. However, the near-infrared excitation is normally used in SHG microscopy, with the result that the maximum attainable spatial resolution is limited. In this paper, we report the use of pixel reassignment to improve the spatial resolution of SHG microscopy. The SHG signal is imaged onto a position-sensitive camera, instead of a point detector typically used in conventional SHG microscope. The data processing is performed through pixel reassignment and subsequent deblurring operation. We present the basic principle and a rigorous theoretical model for SHG microscopy using pixel reassignment (SHG-PR). And for the first time, the optimal reassignment factor for SHG-PR is derived based on the coherent characteristics and the dependence of wavelength in SHG microscopy. To evaluate the spatial resolution improvement, two nano-beads with different distances and simulated microtubule specimen is imaged. We gain about a 1.5-fold spatial resolution enhancement compared to conventional SHG microscopy. When a further deblurring operation is implemented, this method allows for a total spatial resolution enhancement of about 1.87. Additionally, we demonstrate the validity of SHG-PR for raw data with noise.
Keywords --- Microscopy , nonlinear optics, second harmonic generation, image processing, label free, resolution improvement, pixel reassignment