Existing super-resolution fluorescence microscopes compromise acquisition speed to provide subdiffractive sample information. We report an analog implementation of structured illumination microscopy that enables 3D super-resolution imaging with 145 nm lateral and 350 nm axial resolution, at acquisition speeds up to 100 Hz. By performing image processing operations optically instead of digitally, we removed the need to capture, store, and combine multiple camera exposures, increasing data acquisition rates 10–100x over other super-resolution microscopes and acquiring and displaying super-resolution images in real-time. Low excitation intensities allow imaging over hundreds of 2D sections, and combined physical and computational sectioning allow similar depth penetration to confocal microscopy. We demonstrate the capability of our system by imaging fine, rapidly moving structures including motor-driven organelles in human lung fibroblasts and the cytoskeleton of flowing blood cells within developing zebrafish embryos.
We demonstrate 3D super-resolution in live multicellular organisms using structured illumination microscopy (SIM). Sparse multifocal illumination patterns generated by a digital micromirror device (DMD) let us physically reject out-of-focus light, enabling 3D subdiffractive imaging in samples 8-fold thicker than previously demonstrated with SIM. We imaged a variety of samples at one 2D image per second, at resolutions down to 145 nm laterally and 400 nm axially. In addition to dual-labeled, whole fixed cells, we imaged GFP-labeled microtubules in live transgenic zebrafish embryos at depths greater than 45 μm. We also captured dynamic changes in the zebrafish lateral line primordium and observed the interactions between myosin IIA and F-actin in cells encapsulated within collagen gels, obtaining two-color 4D super-resolution datasets spanning tens of time points and minutes without apparent phototoxicity. Our method uses commercially available parts and open-source software and is simpler than existing SIM implementations, allowing easy integration with widefield microscopes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.