Resolution assessment of super-resolution microscopy imaging: structural and technical dependencies for cell biology.

Towards validation and map quality assessment in electron cryo-microscopy

Super-resolution fluorescence microscopy techniques have enabled dramatic development in modern biology due to their capability to discern features smaller than the diffraction limit of light. Recently, super-resolution radial fluctuations (SRRF), an analytical approach that is capable of generating super-resolution images easily without the need for specialized hardware or photoswitchable fluorophores, has been presented. While SRRF has only been demonstrated on fluorescence microscopes, in principle, this method can be used to generate super-resolution images on any imaging platforms with intrinsic radial symmetric point spread functions. In this work, we show that SRRF can be utilized to enhance the resolution and quality of transmission electron microscopy (TEM) images. By including an image registration algorithm to correct for sample drift, the SRRF-TEM approach substantially enhances the resolution of TEM images of three different samples acquired with a commercial TEM system. We quantify the resolution improvement in SRRF-TEM and evaluate how SRRF parameters affect the resolution and quality of SRRF-TEM results.

Super-resolution radial fluctuation (SRRF) microscopy is a novel computational imaging technique that bypasses the optical diffraction limit (lateral resolutions of 200-300nm), achieving lateral resolutions of approximately 50-100nm while being compatible with live-cell imaging. Unlike traditional super-resolution methods such as stimulated emission depletion (STED) and single molecule localization microscopy (SMLM), SRRF minimizes phototoxicity and hardware complexity by analyzing fluorescence intensity fluctuations in standard wide-field microscopy data. This is achieved by calculating local gradient convergence ("radiality") across time-series images, enabling the reconstruction of sub-diffraction structures without specialized fluorophores or high-intensity illumination. Implemented through the open-source NanoJ-SRRF platform, SRRF optimizes parameters like ring radius and radiality magnification to enhance resolution, suppress noise, and maintain computational efficiency. Its advantages include low phototoxicity, compatibility with conventional dyes, and integration with various imaging modalities, allowing dynamic visualization of subcellular processes (e.g., mitochondrial fission, microtubule dynamics). Despite its limitations in axial resolution and potential artifacts in high-density structures, recent advancements like enhanced SRRF (eSRRF) and variance reweighted radial fluctuations and enhanced SRRF (VeSRRF) address these challenges, facilitating real-time, multicolor imaging. Applications range from ultrastructural studies to clinical pathology, with future developments in AI processing and multimodal integration promising further enhancements in imaging capabilities. SRRF stands to significantly impact the understanding of dynamic subcellular processes and biomedical research.

The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE). Plenty of nuclear pore complexes (NPCs) embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D) movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT) techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms.

Recent Developments in Correlative Super-Resolution Fluorescence Microscopy and Electron Microscopy

Super-resolution fluorescence microscopy is a widely used technique in cell biology. Stimulated emission depletion (STED) microscopy enables the recording of multiple-color images with subdiffraction resolution. The enhanced resolution leads to new challenges regarding colocalization analysis of macromolecule distributions. We demonstrate that well-established methods for the analysis of colocalization in diffraction-limited datasets and for coordinate-stochastic nanoscopy are not equally well suited for the analysis of high-resolution STED images. We propose optimal transport colocalization, which measures the minimal transporting cost below a given spatial scale to match two protein intensity distributions. Its validity on simulated data as well as on dual-color STED recordings of yeast and mammalian cells is demonstrated. We also extend the optimal transport colocalization methodology to coordinate-stochastic nanoscopy.

Strategies to maximize performance in STimulated Emission Depletion (STED) nanoscopy of biological specimens.

Recent advances in fluorescence super-resolution microscopy have allowed sub-cellular features and synthetic nanostructures down to ~15 nm in size to be imaged. However, direct optical observation of individual molecular targets (~5 nm) in a densely packed biomolecular cluster remains a challenge. Here, we show that such discrete molecular imaging is possible using DNA-PAINT (points accumulation for imaging in nanoscale topography) - a super-resolution fluorescence microscopy technique that exploits programmable transient oligonucleotide hybridisation - on synthetic DNA nanostructures. We examined the effects of high photon count, high blinking statistics, and appropriate blinking duty cycle on imaging quality, and developed a software-based drift correction method that achieves <1 nm residual drift (r.m.s.) over hours. This allowed us to image a densely packed triangular lattice pattern with ~5 nm point-to-point distance, and analyse DNA origami structural offset with angstrom-level precision (2 Å) from single-molecule studies. By combining the approach with multiplexed Exchange-PAINT imaging, we further demonstrated an optical nano-display with 5×5 nm pixel size and three distinct colours, and with <1 nm cross-channel registration accuracy. This method opens up possibilities for direct and quantitative optical observation of individual biomolecular features in crowded environments.

Decision letter: Deciphering a hexameric protein complex with Angstrom optical resolution

We present LAFTER, an algorithm for de-noising single particle reconstructions from cryo-EM.Single particle analysis entails the reconstruction of high-resolution volumes from tens of thousands of particle images with low individual signal-to-noise. Imperfections in this process result in substantial variations in the local signal-to-noise ratio within the resulting reconstruction, complicating the interpretation of molecular structure. An effective local de-noising filter could therefore improve interpretability and maximise the amount of useful information obtained from cryo-EM maps.LAFTER is a local de-noising algorithm based on a pair of serial real-space filters. It compares independent half-set reconstructions to identify and retain shared features that have power greater than the noise. It is capable of recovering features across a wide range of signal-to-noise ratios, and we demonstrate recovery of the strongest features at Fourier shell correlation (FSC) values as low as 0.144 over a 2563-voxel cube. A fast and computationally efficient implementation of LAFTER is freely available.We also propose a new way to evaluate the effectiveness of real-space filters for noise suppression, based on the correspondence between two FSC curves: 1) the FSC between the filtered and unfiltered volumes, and 2) Cref, the FSC between the unfiltered volume and a hypothetical noiseless volume, which can readily be estimated from the FSC between two half-set reconstructions.

4Pi Microscopy of the Nuclear Pore Complex

STED Microscopy with Optimized Labeling Density Reveals 9-Fold Arrangement of a Centriole Protein

Precise knowledge of the effective spatial resolution in a stimulated emission depletion (STED) microscopy experiment is essential for reliable interpretation of the imaging results. STED microscopy theoretically provides molecular resolution, but practically different factors limit its resolution. Because these factors are related to both the sample and the system, a reliable estimation of the resolution is not straightforward. Here we show a method based on the Fourier ring correlation (FRC), which estimates an absolute resolution value directly from any STED and, more in general, point-scanning microscopy image. The FRC-based resolution metric shows terrific sensitivity to the image signal-to-noise ratio, as well as to all sample and system dependent factors. We validated the method both on commercial and on custom-made microscopes. Since the FRC-based metric can be computed in real time, without any prior information of the system/sample, it can become a fundamental tool for (i) microscopy users to optimize the experimental conditions and (ii) microscopy specialists to optimize the system conditions.

Super-resolution far-field fluorescence microscopy (optical nanoscopy) is a mature set of methods which enable visualization of the nanometer-scale distribution of objects such as organic molecules, photoswitchable proteins, point-like defects in the diamond lattice, upconversion nanoparticles, semiconductor quantum dots, etc. Utilization of internal emitter states in the imaging scheme has made it possible to create contrast at the nanoscale in conventional lens-based optical imaging systems. Spatial resolutions down to the single nanometer scale have been reported in some cases. Nonetheless, routine biological experiments, which employ the molecular probes in physiological conditions, are hampered by photodestructive chemical reactions of fluorophores (photobleaching) which limit signal levels and the attainable resolution to ~20-50 nm. Substantial effort has been devoted to both finding more photostable markers and minimizing the light-induced damage by novel experimental strategies. This thesis is concerned with coordinate-targeted super-resolution microscopy techniques applied to imaging of common molecular probes and semiconductor heterostructured nanowires at room temperature. These techniques have already demonstrated single nanometer resolution in far-field optical microscopy for ultra-stable emitters: namely color-defects in the diamond lattice. Similar resolution levels have not been reported for standard fluorophores imaged at room temperature. Therefore, the question arises how to increase the imaging capabilities of super-resolution microscopy under biologically relevant conditions. The first aim of this thesis was to gain new insights into the photobleaching of organic dyes under photon fluxes typically applied in stimulated emission depletion (STED) microscopy. The impact of STED-light photons on the photobleaching of several organic molecules was studied with the goal to identify optimal imaging conditions. To this end, an optical system and experimental strategy were developed to systematically assess the key parameters in STED microscopy: transient de-excitation, irreversible photobleaching and STED-light-induced fluorescence resulting from undesirable excitation events caused by absorption of the STED-light photons. These parameters determine to what extent the STED concept works in practice with a specific dye. We varied the STED pulse duration from 0.13 ps to 500 ps and the time-averaged STED power up to 200 mW at 80 MHz repetition rate at the popular wavelength of 750 nm, examining common fluorescent compounds (ATTO590, STAR580, ATTO647N, STAR635P) in bulk experiments in thiodiglycol. The magnitude of photobleaching was different for different dyes. In general, two characteristic photobleaching regimes at a given STED pulse energy were found: intensity-dependent (high-order) and intensity-independent (low-order) bleaching. Surprisingly, for ATTO647N we observed a single effective photobleaching scaling over a wide range of STED peak powers (~0.1-200 W). Based on this observation, we developed an intuitive model for this dye which provides a quantitative prediction of the influence of STED-light photons on the resolution and bleaching. We inferred the spatial distribution of photobleaching probability, the role of detection time gating and the impact of residual STED intensity at the targeted coordinate on the resulting image at different STED pulse energies. The dominant bleaching mechanism determines the optimal STED pulse duration to acquire a super-resolved image with minimal photodamage of the marker. High-order photobleaching can be efficiently reduced by increasing STED pulse duration up to roughly the fluorescence lifetime. For low-order photobleaching, chemical triplet quenchers and optical strategies allowing dark-state relaxation hold more promise. Overall, this is the first systematic study of molecular photobleaching in STED microscopy, aimed at finding the optimal optical conditions which minimize STED-light-induced damage. The second project within this thesis investigated the inherent photoluminescence of heterostructured gallium phosphide–gallium indium phosphide (GaP-GaInP) nanowires (NWs) to improve the resolution of far-field optical microscopy of these emitters. Due to their small diameter (<100 nm) but significantly larger length, and their tunable electro-optical properties, semiconductor nanowires are gaining interest in intra- and extracellular biological research. They hold potential as local probes of, for example, electric field or forces. Many of these applications require precise localization and identification of NWs featuring different geometries and surface coatings in a scattering biological environment. Traditional fluorescence microscopy, while suitable for nanowires and live-cell imaging, is hampered by limited spatial resolution. We addressed this issue and found that ground state depletion (GSD) microscopy can resolve heterostructured nanowires with a 5-fold resolution enhancement over confocal microscopy. This resolution improvement allowed us to image nanowires with diameters of 20-80 nm characterized by different geometries of photoluminescent GaInP segments of lengths 50-200 nm spaced by 50-150 nm. The influence of the GaInP segment sizes and positions within a single NW on the GSD image contrast is discussed in detail. The relative simplicity of this method and its moderate laser power requirements make it relevant for further biological studies.

In the last decade, several different structured illumination microscopy (SIM) approaches have been developed. Precise determination of the effective spatial resolution in a live cell SIM reconstructed image is essential for reliable interpretation of reconstruction results. Theoretical resolution improvement can be calculated for every SIM method. In practice, the final spatial resolution of the cell structures in the reconstructed image is limited by many different factors. Therefore, assessing the resolution directly from the single image is an inherent part of the live cell imaging. There are several commonly used resolution measurement techniques based on image analysis. These techniques include full-width at half maximum (FWHM) criterion, or Fourier ring correlation (FRC). FWHM measurement requires fluorescence beads or sharp edge/line in the observed image to determine the point spread function (PSF). FRC method requires two stochastically independent images of the same observed sample. Based on our experimental findings, the FRC method does not seem to be well suited for measuring the resolution of SIM live cell video sequences. Here we show a method based on the Fourier transform analysis using power spectral density (PSD). In order to estimate the cut-off frequency from a noisy signal, we use PSD estimation based on Welch's method. This method is widely used in non-parametric power spectra analysis. Since the PSD-based metric can be computed from a single SIM image (one video frame), without any prior knowledge of the acquiring system, it can become a fundamental tool for imaging in live cell biology.