Imaging phase-function contrast with masked aperture scattering oblique plane microscopy

Oblique plane microscopy (OPM) is a powerful tool for monitoring biological processes due to its capability for high-resolution, rapid, optically-sectioned imaging through a single objective lens. Recently, our group has demonstrated scattering-contrast OPM (sOPM) as a technique to image blood cells in situ and in vivo. In order to classify blood cells visualized with sOPM, scattering data could be further leveraged and better understood. We present here a visualization and analysis of the scattering signal by masking and imaging the final Fourier plane of the sOPM system. We demonstrate the angular distribution of the scattering signal and image with several aperture masks. Microsphere phantoms are imaged in the image plane and Fourier plane to demonstrate the change in scattering behavior for Mie scatterers with large (4 micron) diameters and small (190 nm), Rayleigh-like scatterers similar to subcellular features such as granules. Circular apertures were used to isolate the side scattering centered at 90 degrees compared to the angular extremes. A Michelson contrast of 0.20-0.25 was observed for 4 micron diameter spheres and 0.05-0.10 for 190 nm diameter spheres using a split aperture. Microsphere sizes are classified from images using split aperture contrast and confirmed by fluorescence. Leveraging differential scattering angle contrast will enable the visual classification of blood cells, particularly white blood cells where granules and other organelles present distinct side scattering signals. Finally, the quantitative nature of the differential scattering angle contrast may enable machine-learning based classification and cell counting.