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. LAY DESCRIPTION: Second harmonic generation (SHG) microscopy has emerged as a powerful imaging technique in clinical diagnostics and biological research. SHG microscopy is label-free and provides intrinsic optical sectioning for three-dimensional (3D) imaging. However, a near-infrared excitation wavelength results a restriction in the maximum attainable spatial resolution of SHG microscopy. In this paper, we present a simple resolution-enhanced SHG imaging method, SHG microscopy using pixel reassignment (SHG-PR). We demonstrate a rigorous theoretical model for SHG-PR and derive the optimal reassignment factor. The simulation result shows the clear improvement of the image resolution and contrast in the SHG-PR after deblurring operation. The FWHM value of single microtubule shows that SHG-PR enables a spatial resolution enhancement by a factor of 1.5, compared to conventional SHG microscopy. After a proper deblurring operation, this method allows for a total spatial resolution enhancement of about 1.87. The improvements of spatial resolution and contrast are still valid for raw data with noise. It is expected that this method can contribute towards new insights in unstained tissue morphology, interaction of cells, and diseases diagnosis.
Keywords: Image processing; microscopy; nonlinear optics; pixel reassignment; resolution improvement; second harmonic generation.
© 2020 Royal Microscopical Society.