For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells.

To enhance the resolution of a confocal laser scanning microscope the additional information of a pinhole plane image taken at every excitation scan position can be used (Sheppard 1988). This photon reassignment principle is based on the fact that the most probable position of an emitter is at half way between the nominal focus of the excitation laser and the position corresponding to the (off centre) detection position. Therefore, by reassigning the detected photons to this place, an image with enhanced detection efficiency and resolution is obtained. Here we present optical photon reassignment microscopy (OPRA) which realizes this concept in an all-optical way obviating the need for image-processing. With the help of an additional intermediate optical beam expansion between descanning and a further rescanning of the detected light, an image with the advantages of photon reassignment can be acquired. However, just as in computational photon reassignment, a loss in confocal sectioning performance is caused by working with relatively open pinholes. The OPRA system shares properties such as flexibility and ease of use with a confocal laser scanning microscope, and is therefore expected to be of use for future biomedical routine research.

Super-resolved structured illumination microscopy (SR-SIM) is among the fastest fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-build instruments are able to deliver two-fold resolution enhancement with high acquisition speed. SR-SIM is usually a two-step process, with raw-data acquisition and subsequent, time-consuming post-processing for image reconstruction. In contrast, wide-field and (multi-spot) confocal techniques produce high-resolution images instantly. Such immediacy is also possible with SR-SIM, by tight integration of a video-rate capable SIM with fast reconstruction software. Here we present instant SR-SIM by VIGOR (Video-rate Immediate GPU-accelerated Open-Source Reconstruction). We demonstrate multi-color SR-SIM at video frame-rates, with less than 250 ms delay between measurement and reconstructed image display. This is achieved by modifying and extending high-speed SR-SIM image acquisition with a new, GPU-enhanced, network-enabled image-reconstruction software. We demonstrate high-speed surveying of biological samples in multiple colors and live imaging of moving mitochondria as an example of intracellular dynamics.

Super-resolution microscopy (SRM) is a fast-developing field that encompasses fluorescence imaging techniques with the capability to resolve objects below the classical diffraction limit of optical resolution. Acknowledged with the Nobel prize in 2014, numerous SRM methods have meanwhile evolved and are being widely applied in biomedical research, all with specific strengths and shortcomings. While some techniques are capable of nanometre-scale molecular resolution, others are geared towards volumetric three-dimensional multi-colour or fast live-cell imaging. In this editorial review, we pick on the latest trends in the field. We start with a brief historical overview of both conceptual and commercial developments. Next, we highlight important parameters for imaging successfully with a particular super-resolution modality. Finally, we discuss the importance of reproducibility and quality control and the significance of open-source tools in microscopy. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.