Harnessing artificial intelligence to reduce phototoxicity in live imaging.

Fluorescence microscopy is essential for studying living cells, tissues and organisms. However, the fluorescent light that switches on fluorescent molecules also harms the samples, jeopardizing the validity of results - particularly in techniques such as super-resolution microscopy, which demands extended illumination. Artificial intelligence (AI)-enabled software capable of denoising, image restoration, temporal interpolation or cross-modal style transfer has great potential to rescue live imaging data and limit photodamage. Yet we believe the focus should be on maintaining light-induced damage at levels that preserve natural cell behaviour. In this Opinion piece, we argue that a shift in role for AIs is needed - AI should be used to extract rich insights from gentle imaging rather than recover compromised data from harsh illumination. Although AI can enhance imaging, our ultimate goal should be to uncover biological truths, not just retrieve data. It is essential to prioritize minimizing photodamage over merely pushing technical limits. Our approach is aimed towards gentle acquisition and observation of undisturbed living systems, aligning with the essence of live-cell fluorescence microscopy.

High-fidelity 3D live-cell nanoscopy through data-driven enhanced super-resolution radial fluctuation.

Live-cell super-resolution microscopy enables the imaging of biological structure dynamics below the diffraction limit. Here we present enhanced super-resolution radial fluctuations (eSRRF), substantially improving image fidelity and resolution compared to the original SRRF method. eSRRF incorporates automated parameter optimization based on the data itself, giving insight into the trade-off between resolution and fidelity. We demonstrate eSRRF across a range of imaging modalities and biological systems. Notably, we extend eSRRF to three dimensions by combining it with multifocus microscopy. This realizes live-cell volumetric super-resolution imaging with an acquisition speed of ~1 volume per second. eSRRF provides an accessible super-resolution approach, maximizing information extraction across varied experimental conditions while minimizing artifacts. Its optimal parameter prediction strategy is generalizable, moving toward unbiased and optimized analyses in super-resolution microscopy.

Closed mitosis requires local disassembly of the nuclear envelope.

At the end of mitosis, eukaryotic cells must segregate the two copies of their replicated genome into two new nuclear compartments1. They do this either by first dismantling and later reassembling the nuclear envelope in an 'open mitosis' or by reshaping an intact nucleus and then dividing it into two in a 'closed mitosis'2,3. Mitosis has been studied in a wide variety of eukaryotes for more than a century4, but how the double membrane of the nuclear envelope is split into two at the end of a closed mitosis without compromising the impermeability of the nuclear compartment remains unknown5. Here, using the fission yeast Schizosaccharomyces pombe (a classical model for closed mitosis5), genetics, live-cell imaging and electron tomography, we show that nuclear fission is achieved via local disassembly of nuclear pores within the narrow bridge that links segregating daughter nuclei. In doing so, we identify the protein Les1, which is localized to the inner nuclear envelope and restricts the process of local nuclear envelope breakdown to the bridge midzone to prevent the leakage of material from daughter nuclei. The mechanism of local nuclear envelope breakdown in a closed mitosis therefore closely mirrors nuclear envelope breakdown in open mitosis3, revealing an unexpectedly high conservation of nuclear remodelling mechanisms across diverse eukaryotes.

Fast4DReg - fast registration of 4D microscopy datasets.

Unwanted sample drift is a common issue that plagues microscopy experiments, preventing accurate temporal visualization and quantification of biological processes. Although multiple methods and tools exist to correct images post acquisition, performing drift correction of three-dimensional (3D) videos using open-source solutions remains challenging and time consuming. Here, we present a new tool developed for ImageJ or Fiji called Fast4DReg that can quickly correct axial and lateral drift in 3D video-microscopy datasets. Fast4DReg works by creating intensity projections along multiple axes and estimating the drift between frames using two-dimensional cross-correlations. Using synthetic and acquired datasets, we demonstrate that Fast4DReg can perform better than other state-of-the-art open-source drift-correction tools and significantly outperforms them in speed. We also demonstrate that Fast4DReg can be used to register misaligned channels in 3D using either calibration slides or misaligned images directly. Altogether, Fast4DReg provides a quick and easy-to-use method to correct 3D imaging data before further visualization and analysis.

Physical mechanisms of ESCRT-III-driven cell division.

Living systems propagate by undergoing rounds of cell growth and division. Cell division is at heart a physical process that requires mechanical forces, usually exerted by assemblies of cytoskeletal polymers. Here we developed a physical model for the ESCRT-III-mediated division of archaeal cells, which despite their structural simplicity share machinery and evolutionary origins with eukaryotes. By comparing the dynamics of simulations with data collected from live cell imaging experiments, we propose that this branch of life uses a previously unidentified division mechanism. Active changes in the curvature of elastic cytoskeletal filaments can lead to filament perversions and supercoiling, to drive ring constriction and deform the overlying membrane. Abscission is then completed following filament disassembly. The model was also used to explore how different adenosine triphosphate (ATP)-driven processes that govern the way the structure of the filament is changed likely impact the robustness and symmetry of the resulting division. Comparisons between midcell constriction dynamics in simulations and experiments reveal a good agreement with the process when changes in curvature are implemented at random positions along the filament, supporting this as a possible mechanism of ESCRT-III-dependent division in this system. Beyond archaea, this study pinpoints a general mechanism of cytokinesis based on dynamic coupling between a coiling filament and the membrane.

The rise of data-driven microscopy powered by machine learning.

Optical microscopy is an indispensable tool in life sciences research, but conventional techniques require compromises between imaging parameters like speed, resolution, field of view and phototoxicity. To overcome these limitations, data-driven microscopes incorporate feedback loops between data acquisition and analysis. This review overviews how machine learning enables automated image analysis to optimise microscopy in real time. We first introduce key data-driven microscopy concepts and machine learning methods relevant to microscopy image analysis. Subsequently, we highlight pioneering works and recent advances in integrating machine learning into microscopy acquisition workflows, including optimising illumination, switching modalities and acquisition rates, and triggering targeted experiments. We then discuss the remaining challenges and future outlook. Overall, intelligent microscopes that can sense, analyse and adapt promise to transform optical imaging by opening new experimental possibilities.

Author Correction: Quantitative mapping and minimization of super-resolution optical imaging artifacts.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

vLUME: 3D virtual reality for single-molecule localization microscopy.

vLUME is a virtual reality software package designed to render large three-dimensional single-molecule localization microscopy datasets. vLUME features include visualization, segmentation, bespoke analysis of complex local geometries and exporting features. vLUME can perform complex analysis on real three-dimensional biological samples that would otherwise be impossible by using regular flat-screen visualization programs.

Super-beacons: Open-source probes with spontaneous tuneable blinking compatible with live-cell super-resolution microscopy.

Localization-based super-resolution microscopy relies on the detection of individual molecules cycling between fluorescent and non-fluorescent states. These transitions are commonly regulated by high-intensity illumination, imposing constrains to imaging hardware and producing sample photodamage. Here, we propose single-molecule self-quenching as a mechanism to generate spontaneous photoswitching. To demonstrate this principle, we developed a new class of DNA-based open-source super-resolution probes named super-beacons, with photoswitching kinetics that can be tuned structurally, thermally and chemically. The potential of these probes for live-cell compatible super-resolution microscopy without high-illumination or toxic imaging buffers is revealed by imaging interferon inducible transmembrane proteins (IFITMs) at sub-100 nm resolutions.

The cell biologist's guide to super-resolution microscopy.

Fluorescence microscopy has become a ubiquitous method to observe the location of specific molecular components within cells. However, the resolution of light microscopy is limited by the laws of diffraction to a few hundred nanometers, blurring most cellular details. Over the last two decades, several techniques - grouped under the 'super-resolution microscopy' moniker - have been designed to bypass this limitation, revealing the cellular organization down to the nanoscale. The number and variety of these techniques have steadily increased, to the point that it has become difficult for cell biologists and seasoned microscopists alike to identify the specific technique best suited to their needs. Available techniques include image processing strategies that generate super-resolved images, optical imaging schemes that overcome the diffraction limit and sample manipulations that expand the size of the biological sample. In this Cell Science at a Glance article and the accompanying poster, we provide key pointers to help users navigate through the various super-resolution methods by briefly summarizing the principles behind each technique, highlighting both critical strengths and weaknesses, as well as providing example images.

Publisher Correction: Nuclear pores as versatile reference standards for quantitative superresolution microscopy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nuclear pores as versatile reference standards for quantitative superresolution microscopy.

Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here, we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use (1) as three-dimensional resolution standards for calibration and quality control, (2) to quantify absolute labeling efficiencies and (3) as precise reference standards for molecular counting. These cell lines will enable the broader community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software.

With the widespread uptake of two-dimensional (2D) and three-dimensional (3D) single-molecule localization microscopy (SMLM), a large set of different data analysis packages have been developed to generate super-resolution images. In a large community effort, we designed a competition to extensively characterize and rank the performance of 2D and 3D SMLM software packages. We generated realistic simulated datasets for popular imaging modalities-2D, astigmatic 3D, biplane 3D and double-helix 3D-and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D SMLM software and provides a holistic view of how the latest 2D and 3D SMLM packages perform in realistic conditions. This resource allows researchers to identify optimal analytical software for their experiments, allows 3D SMLM software developers to benchmark new software against the current state of the art, and provides insight into the current limits of the field.

Publisher Correction: Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software.

In the version of this paper originally published, Figure 4a contained errors that were introduced during typesetting. The bottom 11° ThunderSTORM image is an xz view but was incorrectly labeled as xy, and the low x-axis value in the four line profiles was incorrectly set as -60 instead of -50. These errors have been corrected in the PDF and HTML versions of the paper.

Quantitative mapping and minimization of super-resolution optical imaging artifacts.

Super-resolution microscopy depends on steps that can contribute to the formation of image artifacts, leading to misinterpretation of biological information. We present NanoJ-SQUIRREL, an ImageJ-based analytical approach that provides quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same acquisition volume, this approach generates a quantitative map of super-resolution defects and can guide researchers in optimizing imaging parameters.

Content-aware image restoration: pushing the limits of fluorescence microscopy.

Fluorescence microscopy is a key driver of discoveries in the life sciences, with observable phenomena being limited by the optics of the microscope, the chemistry of the fluorophores, and the maximum photon exposure tolerated by the sample. These limits necessitate trade-offs between imaging speed, spatial resolution, light exposure, and imaging depth. In this work we show how content-aware image restoration based on deep learning extends the range of biological phenomena observable by microscopy. We demonstrate on eight concrete examples how microscopy images can be restored even if 60-fold fewer photons are used during acquisition, how near isotropic resolution can be achieved with up to tenfold under-sampling along the axial direction, and how tubular and granular structures smaller than the diffraction limit can be resolved at 20-times-higher frame rates compared to state-of-the-art methods. All developed image restoration methods are freely available as open source software in Python, FIJI, and KNIME.

Fluctuation-Based Super-Resolution Traction Force Microscopy.

Cellular mechanics play a crucial role in tissue homeostasis and are often misregulated in disease. Traction force microscopy is one of the key methods that has enabled researchers to study fundamental aspects of mechanobiology; however, traction force microscopy is limited by poor resolution. Here, we propose a simplified protocol and imaging strategy that enhances the output of traction force microscopy by increasing i) achievable bead density and ii) the accuracy of bead tracking. Our approach relies on super-resolution microscopy, enabled by fluorescence fluctuation analysis. Our pipeline can be used on spinning-disk confocal or widefield microscopes and is compatible with available analysis software. In addition, we demonstrate that our workflow can be used to gain biologically relevant information and is suitable for fast long-term live measurement of traction forces even in light-sensitive cells. Finally, using fluctuation-based traction force microscopy, we observe that filopodia align to the force field generated by focal adhesions.

Between life and death: strategies to reduce phototoxicity in super-resolution microscopy.

Super-resolution microscopy (SRM) enables non-invasive, molecule-specific imaging of the internal structure and dynamics of cells with sub-diffraction limit spatial resolution. One of its major limitations is the requirement for high-intensity illumination, generating considerable cellular phototoxicity. This factor considerably limits the capacity for live-cell observations, particularly for extended periods of time. Here, we give an overview of new developments in hardware, software and probe chemistry aiming to reduce phototoxicity. Additionally, we discuss how the choice of biological model and sample environment impacts the capacity for live-cell observations.