Super-resolution microscopy opened diverse novel research directions by overcoming the classical resolution limit. Revealing structures beyond the diffraction limit was made possible by exploiting the fluorescent emission of individual fluorophores. Involving sample properties to apply these techniques entails a redefinition of the resolution parameter. Here, we propose a new method for assessing the resolution of individual super-resolved images based on image partial phase auto-correlation. The novel algorithm is model-free and does not require any user-defined parameters. We demonstrate its performance on a wide variety of imaging modalities, including diffraction-limited techniques. Finally, we show how our method can be used to optimize image acquisition and post-processing in superresolution microscopy.

Super-resolution fluorescence microscopy provides unprecedented insight into cellular and subcellular structures. However, going `beyond the diffraction barrier' comes at a price since most far-field superresolution imaging techniques trade temporal for spatial super-resolution. We propose the combination of a novel label-free white light quantitative phase tomography with fluorescence imaging to provide high-speed imaging and spatial super-resolution. The non-iterative phase reconstruction relies on the acquisition of single images at each z-location and thus enables straightforward 3D phase imaging using a classical microscope. We realized multi-plane imaging using a customized prism for the simultaneous acquisition of 8 planes. This allowed us to not only image live cells in 3D at up to 200 Hz, but also to integrate fluorescence super-resolution optical fluctuation imaging within the same optical instrument.This 4D microscope platform unifies the sensitivity and high temporal resolution of phase tomography with the specificity and high spatial resolution of fluorescence imaging.

Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labeling via bio-orthogonal tetrazine chemistry combines minimal label size with rapid labeling kinetics. At the same time, fluorogenic tetrazine–dye conjugates exhibit efficient quenching of dyes prior to target binding. However, live-cell compatible long-wavelength fluorophores with strong fluorogenicity have been difficult to realize. Here, we report close proximity tetrazine–dye conjugates with minimal distance between tetrazine and the fluorophore. Two synthetic routes give access to a series of cell-permeable and -impermeable dyes including highly fluorogenic far-red emitting derivatives with electron exchange as the dominant excited-state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multicolor and super-resolution STED, and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size.

We thank the Deutsche Forschungsgemeinschaft (DFG) for their financial support (EXC81, SFB623). We also acknowledge Stephen Hashmi (Heidelberg University) for fruitful discussions. Volker Huch is gratefully acknowledged for X-ray crystallography. Michael Schwering and Dominik Brox have continuously supported the project with their expertise in microscopy.Supporting information for this article, including details of reagents used, instruments, and analytical data, including spectroscopic characterization, is available on the WWW under http://dx.