Super-Resolution Fluorescence Microscopy Methods for Assessing Mouse Biology.

Super-resolution (diffraction unlimited) microscopy was developed 15 years ago; the developers were awarded the Nobel Prize in Chemistry in recognition of their work in 2014. Super-resolution microscopy is increasingly being applied to diverse scientific fields, from single molecules to cell organelles, viruses, bacteria, plants, and animals, especially the mammalian model organism Mus musculus. In this review, we explain how super-resolution microscopy, along with fluorescence microscopy from which it grew, has aided the renaissance of the light microscope. We cover experiment planning and specimen preparation and explain structured illumination microscopy, super-resolution radial fluctuations, stimulated emission depletion microscopy, single-molecule localization microscopy, and super-resolution imaging by pixel reassignment. The final section of this review discusses the strengths and weaknesses of each super-resolution technique and how to choose the best approach for your research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.

Second harmonic generation microscopy using pixel reassignment.

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.

Post-processing strategies in image scanning microscopy.

Image scanning microscopy (ISM) coupled with pixel reassignment offers a resolution improvement of √2 over standard widefield imaging. By scanning point-wise across the specimen and capturing an image of the fluorescent signal generated at each scan position, additional information about specimen structure is recorded and the highest accessible spatial frequency is doubled. Pixel reassignment can be achieved optically in real time or computationally a posteriori and is frequently combined with the use of a physical or digital pinhole to reject out of focus light. Here, we simulate an ISM dataset using a test image and apply standard and non-standard processing methods to address problems typically encountered in computational pixel reassignment and pinholing. We demonstrate that the predicted improvement in resolution is achieved by applying standard pixel reassignment to a simulated dataset and explore the effect of realistic displacements between the reference and true excitation positions. By identifying the position of the detected fluorescence maximum using localisation software and centring the digital pinhole on this co-ordinate before scaling around translated excitation positions, we can recover signal that would otherwise be degraded by the use of a pinhole aligned to an inaccurate excitation reference. This strategy is demonstrated using experimental data from a multiphoton ISM instrument. Finally we investigate the effect that imaging through tissue has on the positions of excitation foci at depth and observe a global scaling with respect to the applied reference grid. Using simulated and experimental data we explore the impact of a globally scaled reference on the ISM image and, by pinholing around the detected maxima, recover the signal across the whole field of view.