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 maximum attainable resolution obtainable by SHG microscopy is limited by the use of long-wavelength, near-infrared excitation. 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, images of nano-beads separated by different distances and of a microtubule array have been simulated. 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.
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