Flat-field super-resolution structured illumination microscopy with joint spatial-temporal light modulation

Abstract

AbstractSuper-resolution structured illumination microscope (SR-SIM) has been established as a powerful tool for visualizing subcellular dynamics and studying organelle interactions in live cells. However, the interfering Gaussian beams result in a limited and nonuniform field of view (FOV) which hinders its application for large whole-cell dynamics and pathological sample imaging. Here, we proposed a joint spatial-temporal light modulation (JSTLM) method to reshape the excitation light field into flat-field structured illumination without disturbing the interfering fringes. Our flat-field structured illumination microscopy (flat-field SIM) improves the uniformity across the whole FOV significantly, hence enabling SR image stitching. Skeleton dynamics and vesicle transportation in and between whole cells were visualized by flat-field SIM. With the stitching of multi-FOV flat-field SIM images, millimeter-sized SR images can be obtained which provides the possibility for cell heterogeneity studies and pathological diagnoses. The JSTLM method can be further incorporated with regions of interest to reduce unnecessary photodamage to live cells during multicolor imaging.ContributionsY.L. and X.H.C. conceived and designed the idea. Y.L., S.M.L., X.J., and G.W. built the SIM setup. Y.L. performed the data acquisitions. Y.L. and X.H.C. conducted the optical wave simulation. J.Z. prepared the cell samples. Y.T. and L.B.W. performed the image analyses. Y.L. prepared the illustrations. X.H.C. and J.G. supervised the project. Y.L. and H.L. wrote the manuscript.