Super-resolution microscopy gives researchers invaluable opportunities and continues to make great strides in terms of performance and applicability. Clever developments in stimulated emission depletion (STED) microscopy have pushed the doors open wider for many applications. Here, we discuss three examples: first, how time-resolved detection unlocks new information; then, live-cell imaging enabled by intelligent illumination schemes; and finally, deep tissue imaging with dynamic aberration correction. As an outlook, we examine MINFLUX as an approach for molecular resolution with fluorescence.

Three-dimensional fluorescence microscopy is a key technology for inspecting biological samples, ranging from single cells to entire organisms. We recently proposed a novel approach called spatially modulated Selective Volume Illumination Microscopy (smSVIM) to suppress illumination artifacts and to reduce the required number of measurements using an LED source. Here, we discuss a new strategy based on smSVIM for imaging large transparent specimens or voluminous chemically cleared tissues. The strategy permits steady mounting of the sample, achieving uniform resolution over a large field of view thanks to the synchronized motion of the illumination lens and the camera rolling shutter. Aided by a tailored deconvolution method for image reconstruction, we demonstrate significant improvement of the resolution at different magnification using samples of varying sizes and spatial features.