Super-resolved structured illumination microscopy (SR-SIM) is an important tool for fluorescence microscopy. SR-SIM microscopes perform multiple image acquisitions with varying illumination patterns, and reconstruct them to a super-resolved image. In its most frequent, linear implementation, SR-SIM doubles the spatial resolution. The reconstruction is performed numerically on the acquired wide-field image data, and thus relies on a software implementation of specific SR-SIM image reconstruction algorithms. We present fairSIM, an easy-to-use plugin that provides SR-SIM reconstructions for a wide range of SR-SIM platforms directly within ImageJ. For research groups developing their own implementations of super-resolution structured illumination microscopy, fairSIM takes away the hurdle of generating yet another implementation of the reconstruction algorithm. For users of commercial microscopes, it offers an additional, in-depth analysis option for their data independent of specific operating systems. As a modular, open-source solution, fairSIM can easily be adapted, automated and extended as the field of SR-SIM progresses.

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.

Liver sinusoidal endothelial cells (LSECs) act as a filter between blood and the hepatocytes. LSECs are highly fenestrated cells; they contain transcellular pores with diameters between 50 to 200 nm. The small sizes of the fenestrae have so far prohibited any functional analysis with standard and advanced light microscopy techniques. Only the advent of super-resolution optical fluorescence microscopy now permits the recording of such small cellular structures. Here, we demonstrate the complementary use of two different super-resolution optical microscopy modalities, 3D structured illumination microscopy (3D-SIM) and single molecule localization microscopy in a common optical platform to obtain new insights into the association between the cytoskeleton and the plasma membrane that supports the formation of fenestrations. We applied 3D-SIM to multi-color stained LSECs to acquire highly resolved overviews of large sample areas. We then further increased the spatial resolution for imaging fenestrations by single molecule localization microscopy applied to select small locations of interest in the same sample on the same microscope setup. We optimized the use of fluorescent membrane stains for these imaging conditions. The combination of these techniques offers a unique opportunity to significantly improve studies of subcellular ultrastructures such as LSEC fenestrations.

Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of significantly longer tracks in sptPALM with minimum effort. Main textFluorescent proteins (FPs) and in particular green-to-red photoconvertible fluorescent proteins (PCFPs) have become indispensable tools for advanced imaging such as singlemolecule localization microscopy (SMLM) or single-particle tracking photoactivated localization microscopy (sptPALM). Both techniques are however limited by blinking, a process in which the fluorophores stochastically enter reversible dark states. PCFPs display blinking on multiple timescales, arising from different underlying photochemical processes. 1 Fluorescence intermittencies shorter than the typical exposure times used in these imaging methodologies (~tens of milliseconds), such as caused by intersystem crossing to the triplet state, reduce the apparent brightness of the label. Intermittencies longer than the exposure time, in contrast, can cause severe complications such as multiple counting of target molecules in quantitative SMLM and interruptions of single-molecule tracks in sptPALM. 2 However, the mechanistic origin of long-lived dark states in PCFPs has remained unclear and there is presently no strategy to eliminate these. We set out to investigate the nature of long-lived fluorescence intermittencies in photoconverted (red) mEos4b, 3 one of the latest probes in a series of highly popular greento-red PCFPs. Individual molecules of mEos4b were immobilized in a polyacrylamide (PAA) matrix, converted to the red emissive state using 405-nm illumination, and the fluorescence emission visualized in time using a sensitive widefield microscope. The single-molecule fluorescence traces (Fig. 1A) displayed reversible and long-lived intermittencies, which we identified as the blinking giving rise to interpretation difficulties in SMLM and sptPALM. Histograms of the intermittency duration (Supplementary Fig. 1) revealed the presence of at least two dark states, as previously reported in mEos2 or Dendra2 4,5 (Supplementary Note 1). While the shorter-lived dark state was insensitive to the intensity of the employed 561-nm illumination (Supplementary Figure 2), the rate at which the longer-lived dark state returned to the emissive state increased with the illumination intensity, reaching a saturation regime at ~0.8 s -1 above a power density of ~ 1.5 kW/cm² (Fig. 1B), indicating a sensitivity to light.