# Advancing Super-Resolution Microscopy: Recent Innovations in Commercial Instruments

Louisa Mezache, Christophe Leterrier

► To cite this version:

Louisa Mezache, Christophe Leterrier. Advancing Super-Resolution Microscopy: Recent Innovations in Commercial Instruments. Microscopy and Microanalysis, 2025, 31 (2), 10.1093/mam/ozaf004. hal-05379733

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# Advancing Super-Resolution Microscopy: Recent Innovations in Commercial Instruments

Louisa Mezache¹,* and Christophe Leterrier¹ ID

¹Aix Marseille Université, CNRS, INP UMR7051, NeuroCyto, 27 Blvd Jean Moulin, 13005 Marseille, France

*Corresponding author: Louisa Mezache, E-mail: louisa.mezache@univ-amu.fr

## Abstract

Super-resolution microscopy techniques have accelerated scientific progress, enabling researchers to explore cellular structures and dynamics with unprecedented detail. This review highlights the most recent developments in commercially available super-resolution microscopes, focusing on the most widely used techniques: confocal laser scanning systems, structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, and single-molecule localization microscopy (SMLM). We detail the technological advancements of Confocal.NL's GAIA, Nikon's NSPARC, CSR Biotech's MI-SIM, Zeiss's Lattice SIM 5, Leica's STELLARIS STED, and abberior's STED and MINFLUX systems, as well as Abbelight's SAFe MN360 and Bruker's Vutara VXL SMLM platforms. These advancements address the need for enhanced resolution, reduced phototoxicity, and improved imaging capabilities in a range of sample types, while also aiming to enhance user friendliness.

**Key words:** commercial, innovations, instruments, microscopy, super-resolution

## Introduction

Since the advent of super-resolution microscopy techniques, advancements in the field have accelerated, expanding the breadth of biological questions that can be addressed. While there are numerous review articles describing the differences between various super-resolution imaging techniques and their respective applications (Sigal et al., 2018; Vangindertael et al., 2018; Schermelleh et al., 2019; Jacquemet et al., 2020; Valli et al., 2021; Bond et al., 2022; Balasubramanian et al., 2023; D'Este et al., 2024), this review article aims to showcase the latest technological advancements in the most widely used commercialized microscopy techniques (Thorley et al., 2014; Worden, 2024). It is specifically designed for readers who are already familiar with the fundamental principles of each technique, allowing us to focus on highlighting new developments and innovations rather than providing introductory explanations. The goal is to provide a comprehensive update on the cutting-edge tools and methods now available in these systems, benefiting those with a working knowledge of microscopy. Here, we discuss the current landscape of the latest technological improvements in confocal laser scanning systems, structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, and single-molecule localization microscopy (SMLM). This review presents detailed insights into the technological advancements across these four microscopy techniques, gathered through interviews with companies that have recently introduced these innovations.

## Confocal Laser Scanning Microscopy

Despite the development and push toward dedicated super-resolution techniques in the last 10 years, confocal-based systems remain heavily sought after and used, and for obvious reasons. Confocal microscopy today is based on a relatively simple

concept, is easy to use even for nonexperienced users, and produces good-quality images for many applications (Elliott, 2020). However, confocal microscopy has also evolved to adapt to the ever-growing need for achieving subdiffraction resolution, incorporating pixel reassignment strategies in point scanning, spinning disk, and camera-based systems (Jacquemet et al., 2020). The Zeiss Airyscan was one of the first super-resolution technologies integrated into confocal microscopes (Weisshart, 2014; Wu & Hammer, 2021), replacing the traditional single-point detector with an array of 32 elements, thereby capturing and using the information from the entire Airy disk. The Yokogawa SoRa spinning disk module employs a disk containing thousands of tiny pinholes through which the laser passes, and a microlens array that optically reduces the size of the focal points projected through the pinholes, creating multiple focused points of illumination that scan the sample (Nakano, 2002; Oreopoulos et al., 2014; Azuma & Kei, 2015). Here, we discuss the latest innovations in commercially available confocal-based super-resolution microscopes offered by Confocal.NL and Nikon.

### Confocal.NL

#### REscan Technology

GAIA is Confocal.NL's latest confocal microscope based on point rescanning technology (De Luca et al., 2013), which involves scanning the sample twice. The first scan is the traditional confocal scan, where a focused laser beam is used to illuminate the sample, before the emitted fluorescence is detected through a pinhole to reject out-of-focus light (Fig. 1). The emission light passing through the pinhole is then rescanned onto a scientific Complementary Metal-Oxide-Semiconductor (sCMOS) camera. The two synchronized scanners ensure that each part of the emission point spread function (PSF) is correctly aligned with the corresponding camera pixel, allowing a wider pinhole
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Schematic depicting Confocal.NL GAIA point rescan technology. (1) The sample is scanned as in conventional point scanning microscopy, here using a high-precision mirror. (2) The emitted light from the sample passes through a series of lenses that magnify the sample image (2a, 2c), and a pinhole (2b). (3) The signal is then rescanned onto a camera with a second mirror synchronized to the first. Image courtesy of Confocal.NL.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Confocal.NL GAIA's switchable pinhole. Left: Schematic of GAIA'S motorized switchable pinhole, providing several pinhole size options to maintain confocality for a wide range of objectives. Right: Pinhole size chart provides guidance on pinhole diameter settings for optimal imaging conditions for objectives with any given numerical aperture and magnification. Image courtesy of Confocal.NL.</figcaption></figure>

[1.5 Airy Units (AU)] to collect more light and achieving 1.4× improved resolution over conventional point-scanning confocal microscopy. Lenses in the optical path are configured to expand and refocus the emitted light to create a smaller, demagnified illumination spot on the camera, allowing for improved resolution (170 nm raw image, 120 nm deconvolved) over a larger field of view (330 × 330 µm with 40× objective). This technology eliminates the need for high laser power, thus enabling gentler imaging using down to nanowatt laser power and minimizing phototoxicity.

## System Specifications

In addition to the dual scanning mechanism and optical system, the GAIA point rescanning confocal microscope is equipped with a motorized switchable pinhole to retain confocality and achieve subdiffraction resolution with objectives from 30× to 100× (Fig. 2). These features facilitate super-resolution imaging over a large field of view at depths <500 µm, making GAIA suitable for live-cell imaging and thick samples alike (Fig. 3). GAIA is an add-on device, adaptable to any commercially available microscope body, and integrates Microvolution (Bruce &

Butte, 2013) real-time deconvolution to enhance image clarity, beneficial for observing cellular processes.

## Applications

By combining the strengths of confocal scanning and camera-based detection, GAIA enables detailed imaging with minimal phototoxicity, suitable for a wide range of biological applications, particularly live-cell imaging. The point rescan technology in Confocal.NL's GAIA has demonstrated significant benefits for deep-tissue imaging of zebrafish larvae to investigate host-pathogen interactions (Jim et al., 2022). In another application, bacterial spores were live-imaged to examine protein organization and dynamics during dormancy and germination (Wen et al., 2022).

## Nikon

The Nikon SPatial ARray Confocal (NSPARC) detector is the latest imaging technology integrated into the Nikon AX/AX R confocal microscope series. This super-resolution detector employs image scanning microscopy (ISM) technology in conjunction with a single-photon avalanche detector (SPAD)
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Imaging thick samples with Confocal.NL's GAIA. Zebrafish embryo optic tectum (60X 1.3NA), acquired at ~120 µm of depth. Image courtesy of Bram Willems (Brinks Lab, TU Delft) via Confocal.NL website, with permission from Confocal.NL.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 4.** NSPARC detector. Left: The NSPARC is a 25-element array detector, with each element representing a 0.2 AU pinhole. Zooming optics enable optimal projection of the full PSF onto the NSPARC detector. Right: Timelapse imaging of zebrafish vasculature with NSPARC detector (top) versus traditional GaAsP detector (bottom). Kymographs (zoomed insets) show greater resolution with the NSPARC detector in visualizing vessel endothelium position over time. Image courtesy of Nikon.</figcaption></figure>

array composed of 25 elements, or pixels (Fig. 4, left). In contrast to conventional confocal microscopy, where a single-element detector captures all signals, the NSPARC's 5 × 5 matrix array captures different lateral PSFs, generating a distinct 2D image at each of the 25 elements. Rather than passing through a pinhole, the emission light from the scan point fills the entire array detector, representing 1 AU, where each pixel, representing a 0.2 AU pinhole, records an image of the scanned area. The 25 pixels are then summed to allow a brightness of 1 AU with an enhanced resolution of 0.2 AU (Delattre, 2023).

## System Specifications

The NSPARC detector can reach a claimed resolution of 100 nm when paired with the validated deconvolution method, Richardson-Lucy (Richardson, 1972; Lucy, 1974), an iterative algorithm that estimates the original, unblurred image through repeated adjustments based on the blurred image and the PSF. The NSPARC's high sensitivity allows for gentle imaging with low laser power, making it suitable for dynamic live-cell imaging. The NSPARC also offers an improved signal-to-noise ratio across a range of magnifications via zooming optics, comprising a movable lens group to correctly project the PSF and fill the
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Cultured cells and neurons imaged with NSPARC. Left: Rat2-cells stained for DNA (blue), actin (gray), and microtubules (orange), imaged with the NSPARC module and deconvolved. Right: Comparison between the conventional confocal image (top) and the NSPARC deconvolved image (bottom) of rat hippocampal neurons in culture stained for dendrites (map2, blue), axons (β2-spectrin, gray), presynapses (munc13, red), and postsynapses (PSD-95, green). The NSPARC image distinguishes the presynapse from the postsynapse (red and green, respectively, double arrowheads). Data courtesy of C. Leterrier, NeuroCyto, INP, CNRS-AMU UMR 7051.</figcaption></figure>

entire array detector (Fig. 4, right). This low noise is beneficial for imaging challenging samples, such as those with dim signal or with wide dynamic range (Fig. 5), and it works effectively in conjunction with the high-speed resonant scanner for live-cell imaging, now at a resolution of 2,048 × 2,048 pixels. This improved pixel density over a wide field of view (25 mm diagonal) enhances the resolution, and together with the sensitive detector technology, supports efficient imaging of large samples without sacrificing detail.

## Applications

With its recent launch, NSPARC's capabilities have been showcased in studying new modes of cell migration in metastatic cancer cells (Morishita et al., 2024). Here, the authors investigated the implication of actinin expression in lamellipodia of cultured epithelial cells. In another study, Nikon's NSPARC was used to facilitate the high-resolution imaging of a full Drosophila brain (Chen et al., 2024).

## Structured Illumination Microscopy

SIM utilizes patterned illumination to create moiré patterns that encode high-frequency spatial information within the sample (Gustafsson, 2000). By computationally analyzing the interference patterns generated between the structured illumination and the sample, SIM achieves resolutions approximately twice the diffraction limit; however, recent advancements in image processing described here have been implemented, claiming to attain sub-100 nm resolution. Such computationally based enhancement in the resolution must be carefully validated experimentally, as the capacity of deconvolution-based methods to retrieve meaningful higher frequencies depends on the validity of the *a priori* constraints assumed for the imaged object (Heintzmann, 2007).

## CSR Biotech

### MI-SIM Technology

CSR Biotech's Machine Intelligent Structured Illumination Microscope (MI-SIM) is a technology that uniquely combines two deconvolution methods to address challenges associated

with SIM image reconstruction. First, the MI-SIM employs a Hessian matrix-based deconvolution (Hessian-SIM) algorithm to provide information about the curvature of image structures that helps enhance edges and fine structures to filter out noise without increasing the light exposure to the sample (Huang et al., 2018). This method leverages the spatial-temporal continuity of dynamic biological processes, presuming smooth structural changes over space and time, distinct from high-frequency noise. Second, the MI-SIM utilizes sparse deconvolution to enhance image resolution by applying sparsity constraints (Zhao et al., 2022), which assume the image consists of mostly zero-value pixels with only a few pixels having significant values that represent actual features. This method significantly narrows the PSF, reducing the blurring effect and producing sharper images.

### System Specifications

By combining spatial continuity with sparse deconvolution, the MI-SIM claims an approximate lateral resolution enhancement down to 60 nm with reduced artifacts common in SIM image reconstruction. To ensure optimal spatial and temporal sampling, the system's hardware design comprises a ferroelectric liquid crystal spatial light modulator (SLM) for fast switching between different diffraction gratings, a polarization rotator to minimize switching times for illumination polarization changes, and an sCMOS camera (ORCA-Flash4.0 V2, Hamamatsu) for efficient emission detection (Huang et al., 2018) (Fig. 6). MI-SIM's optical system allows for high-resolution and photon-efficient imaging, optimized for long live-cell acquisitions and fixed sensitive samples alike. This microscope also features a number of complementary imaging modes, including TIRF, 2D and 3D SIM, and TIRF-SIM, combining selective illumination with super-resolution capabilities for various applications. The MI-SIM image acquisition software includes GPU-accelerated reconstruction and automatically sets reconstruction parameters, allowing real-time visualization while changing the field of view and focus. This system also has an intelligent live-cell TRACKER that automatically adjusts for axial defocus and lateral shifts during long-term live-cell imaging (up to 12 h). The TRACKER technology also enables long-term multisite imaging for cell migration applications (Fig. 7). Together, these elements allow for
<figure><img src="image_7.png" /><figcaption>**Fig. 6.** CSR Biotech MI-SIM. MI-SIM's hardware design comprises a high dynamic SLM, a polarization rotator, an sCMOS camera (ORCA-Flash4.0 V2, Hamamatsu), and a control system designed for real-time visualization while rapidly changing the field of view and focus. Image courtesy of CSR Biotech.</figcaption></figure>

high-resolution, long-term live-cell imaging while minimizing photodamage and optimal use of available light.

## Applications

The MI-SIM technology has been used to address a broad range of scientific questions. In mitochondrial studies, the MI-SIM has been employed to identify morphological changes in mitochondria and nuclei within cultured primary B cells isolated from mouse spleen (Chi et al., 2024). It has also been used to visualize the simultaneous targeting of both the inner and outer mitochondrial membranes by synthetic photosensitizers (Liu et al., 2024). The MI-SIM was used to acquire high-resolution 3D images of HeLa cells, revealing the spatial distribution of special vesicles within the cytoplasm (Han et al., 2024). To understand cell membrane repair, whole *Caenorhabditis elegans* were live-imaged with the MI-SIM to observe membrane morphology after wounding (Meng et al., 2023). In another application, mouse brain tissue sections were imaged to study synaptic generation (Tian et al., 2022).

## ZEISS

### Lattice SIM Technology

Zeiss's Lattice SIM 5 uses a 3D lattice spot pattern of light to illuminate the sample. The lattice pattern is generated by grating that diffracts the excitation light in such a way that five orders of light enter the objective and constructively interfere to create 13 phases, which are used to capture different spatial frequencies (Betzig, 2005). Thus, the lattice dot pattern modulates the light in

multiple directions and angles simultaneously, in contrast to the traditional approach that requires a line pattern to be rotated to improve resolution. Here, the lattice pattern is only shifted laterally to generate Moiré fringes in all directions at once, capturing either 9 images in 2D and 13 images in 3D (Zheng et al., 2022), allowing decreased sample illumination to protect from photodamage.

### System Specifications

The Lattice SIM 5 is equipped with five different lattice grating frequencies tailored to each laser wavelength and configured for 63× oil or water immersion objectives. Each grating spacing (corresponding to a given spatial frequency as determined by the Fourier transform) generates a different pattern of light used to illuminate the sample. This system also has one common ApoTome grating for acquisitions with 40× down to 10× objectives, enabling imaging with a range of objectives for high-speed acquisitions of dynamic processes. Lattice SIM 5 can use Leap mode, a feature designed for fast 3D live-cell imaging (Zheng et al., 2022). It reduces the number of images acquired in a z-stack by capturing every third slice instead of each one. Missing data between these slices are computationally reconstructed (Jost et al., 2015) to maintain image integrity. Although this feature requires longer processing times than acquiring all z-stack slices, it decreases the illumination exposure and phototoxicity. This makes it advantageous for long acquisitions of sensitive samples, balancing speed and sample preservation. Burst mode, or rolling SIM (Guo et al., 2018), is another feature designed for high-speed 3D imaging. It reconstructs images using a rolling temporal
<figure><img src="image_8.png" /><figcaption>**Fig. 7.** CSR Biotech's MI-SIM TRACKER technology. Long-term live acquisitions of bone marrow stem cell migration across multiple fields of view. Figure shows 20 h simultaneous live acquisitions of 20 regions of interest. Data courtesy of Zhang Lab, Xi'an Dental Hospital via CSR Biotech's website, with permission from CSR Biotech.</figcaption></figure>

window, allowing the quick capture of several images in rapid succession (up to 255 fps). This is particularly useful for visualizing fast dynamic processes in live cells (Boualam & Rowlands, 2021). By acquiring images at a high frame rate, burst mode helps reduce motion blur.

The captured images are processed using either the regular SIM processing or the newly updated SIM² deconvolution, an algorithm based on the Richardson–Lucy iterative deconvolution method, with a claimed sub-100 nm resolution (Löschberger et al., 2021). One advantage of SIM² is its ability to perform iterative deconvolution in real-time for live-cell imaging applications. Additionally, it automatically adapts to different imaging conditions and is thus compatible with various objectives.

## Applications

Since its launch in December 2023, collaborative research with scientists has demonstrated its potential for several biological applications such as studying cellular dynamics and multicolor imaging to visualize subcellular structures. The Lattice SIM in conjunction with SIM2 has proved advantageous in exploring the trafficking of membrane proteins in HeLa M cells

(Robinson et al., 2024). The long-term live-cell imaging capabilities of the Lattice SIM technology have also been exploited to understand how axonal structures respond dynamically to induced mechanical forces. The high-resolution images captured by Lattice SIM allowed for the analysis of axon diameter changes and the quantification of axonal beading (Pan et al., 2024).

## STimulated Emission Depletion Microscopy

In STED microscopy, resolutions beyond the diffraction limit of light are achieved by employing a donut-shaped depletion beam that selectively deactivates fluorescence emission, thereby minimizing the illumination area at the focal point (Okhonin, 1986; Hell and Wichmann, 1994; Klar and Hell, 1999). This approach can yield spatial resolutions on the order of tens of nanometers, allowing for detailed visualization of cellular organelles and molecular complexes in both fixed and live samples. The latest advancements in STED microscopy presented below have targeted the limitations associated with this technique, such as photodamage induced by the high-intensity STED beam and its operational complexity.
<figure><img src="image_9.png" /><figcaption>**Fig. 8.** STED and fluorescence lifetime readouts. (a) schematics for confocal, STED donut and the effective STED PSF with the resulting lifetime-coded STED PSF. (b) STED process and the resulting fluorescence lifetime gradient. (c) TauSTED concept blue shows shorter lifetimes and red longer lifetimes adapted from Alvarez et al. (2021). Image and caption courtesy of Leica Microsystems (Alvarez et al., 2024).</figcaption></figure>

## Leica Microsystems

The philosophy behind Leica's STED solution is to combine nanoscopy capabilities with the STELLARIS confocal platform, thus providing flexibility for imaging various samples over a wide spectrum.

## STELLARIS System Specifications

The STELLARIS system features a pulsed white light laser (spanning from 440 to 790 nm), and up to three STED lasers (pulsed 589 nm, continuous wave 660 nm, and pulsed 775 nm). One of the strengths of the STELLARIS STED systems is due to Leica's Power HyD detector family, designed for enhanced photon counting, sensitive imaging, and flexibility across a broad spectrum: Power Hyd S detector for better blue-green spectrum sensitivity and Power HyD R for infrared spectrum up to 850. This Power HyD R and the Power Hyd S are also designed to combine imaging with fluorescence lifetime imaging microscopy (FLIM), fluorescence correlation spectroscopy (FCS), and fluorescence cross-correlation spectroscopy (FCCS). One noticeable property of these detectors is their improved photon counting capability thanks to the discrimination of overlapping photon arrival at high signal intensities.

Another feature of Leica's STED platform is TauSTED, which merges STED information with fluorescence lifetime data to achieve enhanced-resolution imaging in live specimens (Alvarez, 2021). TauSTED is based on the concept that the effective PSF, generated from the STED donut and confocal PSF, results in a lifetime-coded STED PSF between the photons emitted at the center of the effective PSF and those emitted at the periphery (Fig. 8). With access to the photon arrival time in a phasor plot, this information appears as an elongated cluster extending from long to shorter lifetimes (Lanzano et al., 2015). This is the STED trajectory, which, when considered alongside the photon arrival times, enables a precise calculation of the

<figure><img src="image_10.png" /><figcaption>**Fig. 9.** Leica's TauSTED Xtend. The graph illustrates the resolution achievable with confocal, TauSTED, and TauSTED Xtend. Using TauSTED at low STED laser power (<1% STED) enhances image resolution compared to confocal imaging, improving from 250 nm (confocal) to 150 nm. At the same STED laser power, TauSTED Xtend delivers twice the resolution, reaching 75 nm. Image courtesy of Leica Microsystems.</figcaption></figure>

expected lifetimes across the lifetime-based STED trajectory. Furthermore, noise from the sample or e.g., embedding medium can be easily distinguished from the signal emanating from the STED process. By exploiting changes in fluorescence lifetime induced by STED, TauSTED enhances the spatial resolution beyond conventional STED, in particular at low STED power (<1% of available STED light dose) (Fig. 9).

The latest development building on TauSTED is TauSTED Xtend (Alvarez, 2024), which uses the fluorescence lifetime
information to extract an on-the-fly photophysical map of the effective STED PSF fingerprint, paired with an accelerated Richardson-Lucy algorithm for real-time deconvolution considering both lifetime and noise information, thereby further reducing the required STED laser power and achieving resolutions in the tens of nanometers range. Additionally, the STED separation tool in the phasor analysis module (FALCON) allows the separation of spectrally overlapping fluorophores, enabling further multiplexing (Lanzano et al., 2015; Gonzalez Pisfil et al., 2022).

## Applications

Leica's STELLARIS STED platforms have been used in a number of publications. Recent work using TauSTED revealed detailed spatial distribution and dynamics of labile iron in cultured primary hippocampal neurons (Kittilukkana et al., 2024). This level of detail is crucial for understanding how iron homeostasis is maintained in the brain and how disruptions might contribute to neurological disorders such as Alzheimer's and Parkinson's. In another recent publication, both fixed and live cultured lung pleural mesothelial cells were imaged using this system to observe the dynamics of actin cytoskeleton and mitochondria within tunneling nanotubes (Benard et al., 2024). Other applications include analyzing genome organization in individual cell nuclei during DNA replication and transcription (Privitera et al., 2024), and exploring the formation of plant vacuoles (Scheuring et al., 2024).

## abberior Instruments

abberior offers three dedicated STED instruments—the FACILITY, STEDYCON, and INFINITY lines—tailored to meet the specific requirements of various users.

## FACILITY Line Specifications

The STED FACILITY Line is equipped with two pulsed depletion lasers (595 and 775 nm), and five pulsed excitation lasers (405, 488, 518, 561, and 640 nm). When combined with spectral detection filters (RAINBOW detection), which enable defining the spectral detection window with 1 nm precision, and abberior's lifetime imaging (TIMEBOW), users gain the flexibility to precisely adjust the wavelength to meet their specific requirements. In addition to employing avalanche photodiode (APD) detectors, which have higher sensitivity and yield very low noise, this platform offers several mechanisms used either alone or in combination to minimize aberrations, reduce background, and improve the image quality of a given sample.

First, the 2D and 3D STED beam is produced using a SLM designed to maintain a single beam that carries both XY- and Z-STED (Fig. 10). In generating the 2D STED donut, the depletion laser is reflected onto one side of the SLM, then onto the other via a mirror to create the 3D STED effect. By tuning the relative weight on either side of the SLM, this beam-shaping device (EASY3D) can be used to adjust resolutions between the best lateral resolution of 20 nm and the full 3D isometric resolution of 75 nm. This enables users to flexibly tune the optimal resolution compromise for their application. Further, the single beam design enforces the stable superposition of the 2D and 3D STED PSFs, providing a more robust alignment than beam-splitting optical paths.

Due to the high power of the STED beam for high resolutions, a common challenge in STED imaging is managing photobleaching and phototoxicity. To address this, abberior's FLEXPOSURE adaptive illumination techniques, such as DYMIN, dynamically adjust the STED and excitation beam

<figure><img src="image_11.png" /><figcaption>**Fig. 10.** abberior's STED beam-shaping technology. The 2D and 3D STED beams are produced using SLMs placed in the laser light path and designed to maintain a single beam that carries both xy- and z-STED depletion. Image courtesy of abberior Instruments.</figcaption></figure>

powers in response to signal detection, highly reducing the total light dose on the sample.

To address background issues common in fluorescence microscopy, abberior employs adaptive optics using deformable mirrors (RAYSHAPE) to correct aberrations that originate particularly from thick or dense samples. Conventional approaches to aberration correction involve (optionally motorized) correction collar objective lenses, which can adjust over an axial range of ~20 µm. However, for thicker samples, the RAYSHAPE mirror presents the advantage of preserving resolution and signal at depths of up to roughly the working distance of the objective lenses (Fig. 11). abberior's deformable mirror consists of 140 actuators that shape the mirror membrane to counteract the beam distortions caused by the sample, dynamically adjusting as the microscope focus moves through the sample, which a motorized correction collar cannot do. The RAYSHAPE mirror can manipulate both the excitation and STED beams to achieve an optimal focal point, while also adjusting the emission wavefront to correct for any sample-induced aberrations. This is especially beneficial for 3D STED, as the STED PSF is more susceptible to deformation at greater depths, enabling users to overcome the inherent challenges of deep-tissue imaging.

abberior's MATRIX detection, constituting a 23-element array detector, presents yet another unique solution to reducing background and improving image quality. While the
<figure><img src="image_12.png" /><figcaption>**Fig. 11.** abberior's RAYSHAPE technology. Top: Deep-tissue imaging of a stage 17 Drosophila embryo stained for chitin with abberior LIVE 610, acquired using RAYSHAPE deformable mirrors to achieve greater imaging depth than conventional STED (scale bar is 100 µm). Bottom: Cross-section (dashed line in top image) without (left) and with (right) RAYSHAPE, recorded at depth of 70 µm (scale bar is 10 µm). Image courtesy of abberior Instruments.</figcaption></figure>

purpose of the confocal pinhole in laser scanning microscopes is to block out-of-focus light, some unwanted signal neverthe- less reaches the detector. In contrast to a single detector, which is incapable of distinguishing structures at the focal plane ver- sus those that are out of focus but remain in the region of ex- citation, the 23 APDs comprising the array detector enable measuring and subtracting background signal without the need for less precise postprocessing algorithms, thereby further squaring the effect of a confocal pinhole and enabling superior optical sectioning.

abberior's latest addition to the STED lines is a novel adapta-
tion of the Richardson-Lucy deconvolution algorithm, integrat-
ing two key pieces of information: the background and the PSF,
accounting for changes induced by the STED effect (Fig. 12).

STEDYCON Specifications

The STEDYCON microscope is a module that can be attached to
any widefield microscope frame, transforming it into an STED
and confocal microscope (Fig. 13). By attaching to the camera
port, the STEDYCON incorporates lasers, detectors, and a scan-
ner, thus enabling STED imaging capabilities though with fewer
features than the FACILITY line. The STEDYCON is designed
as an entry-level system well-suited for beginners with limited mi-
croscopy experience, with minimal training required to obtain
super-resolution images of a wide range of sample types. It features
one STED laser at 775 nm, four excitation lasers, and two fixed
detectors. One of the key features of the STEDYCON is the stable
alignment between excitation and depletion lines ensured by

overlapping the beam paths through the same optical fiber. The STEDYCON's browser-based and user-friendly software affords users the flexibility to operate the system remotely and from any device with the same minimal input as the LiGHTBOX software to capture high-resolution images. Image files and metadata can be exported or transferred via the system's direct transfer to Huygens Software, for further processing and analysis.

INFINITY Line Specifications

For users needing customization, the INFINITY line offers an open platform adaptable to different sample geometries and applications, incorporating new features as required. The INFINITY microscope can be custom-built to house any combination of pulsed or multiphoton lasers from 375 to 1,320 nm, as well as detectors and other custom components according to the user's specifications. It can integrate with other imaging modalities to facilitate advanced scientific research.

Applications

The comprehensive capabilities of abberior's STED technology are showcased in numerous publications across a broad spectrum of research applications from live-cell imaging to materials characterization. Of note in recent publications, abberior's STED systems were employed to investigate the biphasic organization of RNA-protein condensates in Drosophila germ granules (Ramat et al., 2024), and to examine the nanoscale organization of dendritic spine subcompartments and protein trafficking behavior of related proteins in cultured mouse brain tissue sections
<figure><img src="image_13.png" /><figcaption>**Fig. 12.** abberior's deconvolution. (a) Mouse neocortex axon initial segment stained for βIV-spectrin with Alexa 594, acquired without (top) and with (bottom) abberior's new deconvolution method. Cryo-section cut at 20 µm (scale bar is 1 µm). The sample is kindly provided by Dr. Maren Engelhardt, Institute of Neuroanatomy, Medical Faculty Mannheim, Heidelberg University. (b) Golgi apparatus stained for GM130 and Giantin with abberior STAR 580 and abberior STAR 635P, acquired without (left) and with (right) abberior's new deconvolution method. Cryo-section cut at 20 µm (scale bar is 1 µm). Images courtesy of abberior Instruments.</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Fig. 13.** abberior STEDYCON, abberior's STEDYCON STED microscope, designed as an entry-level system suited for novice microscopists, is a module that can be attached to any widefield microscope frame. Image courtesy of abberior Instruments.</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Fig. 14.** Abbelight's ASTER technology. Abbelight's ASTER technology employs a two-galvanometer raster scanning system to generate a homogeneous field of illumination. The system includes four lenses (L1–L4) with specific focal lengths and a dielectric mirror (M1). A small Gaussian input beam is scanned between lenses L1 and L2, creating a flat-top illumination profile. This beam then passes through a TIRF translation stage and is magnified between lenses L3 and L4. Once focalized at the back focal plane, the scanned Gaussian beam results in a flat-top excitation profile at the sample. Image adapted from Mau et al. (2021), with permission from Abbelight.</figcaption></figure>

(Phadke et al., 2024). Using correlative STED and atom force tomography (AFM) in a custom-built INFINITY, researchers observed the impact of gap-junction protein Cx30 on the surface topology and cortical stiffness of primary mouse astrocytes (Ghezali et al., 2024).

## Single Molecule Localization Microscopy

SMLM techniques harness the stochastic blinking behavior of fluorescent molecules, as seen in stochastic optical reconstruction microscopy (STORM), or photoswitching properties of fluorophores, used in point accumulation for imaging in nanoscale topography (PAINT), to precisely localize individual fluorophores within the sample (Betzig et al., 2006; Hess et al., 2006; Rust et al., 2006; Heilemann et al., 2008; Lelek et al., 2021). By iteratively imaging sparse subsets of fluorophores and determining their precise positions with high accuracy, SMLM techniques can achieve resolutions 10–20 nm, revealing fine structural details of cellular components that were previously inaccessible. Since the 2008 launch of Nikon's N-STORM, the first commercially available SMLM microscope, several companies have developed and released their versions. Notably, Zeiss's Elyra 7 uses phase ramp illumination imaging localization microscopy (PRILM) (Baddeley, 2011), allowing for imaging at greater sample depths than astigmatism-based systems. Another commercialized system, the Nanoimager developed by Oxford Nanoimaging (ONI), is the first benchtop SMLM platform designed with a damping enclosure to minimize the need for accessories, thereby making advanced super-resolution microscopy accessible to a broader scientific community. Here, we introduce and discuss Abbelight's SAFeMN360 and Bruker's Vutara VXL,

two systems offering the latest advancements in commercial SMLM systems.

### Abbelight

#### ASTER Technology

Abbelight's SAFe MN360 is a specialized spectral demixing astigmatism-based SMLM system designed to achieve uniform localization precision over large fields of view, up to 150 µm × 150 µm with a 100 × objective and 250 µm × 250 µm at 60×. In classical SMLM methods, Gaussian illumination can yield nonuniform excitation across large fields of view, with fluorophores at the center receiving more light and thus displaying different blinking properties than those at the edges of the Gaussian irradiance. This can affect the accuracy of molecule localization and result in reconstruction artifacts, restricting the usable area of the microscope's field of view to the most homogeneous part of the Gaussian beam. To address this, Abbelight's Adaptable Scanning for Tunable Excitation Regions (ASTER) technology (Mau et al., 2021) decreases the size of the Gaussian beam and rapidly scans the entire field of view faster than the camera's acquisition time, resulting in a more homogeneous intensity distribution for each camera frame (Fig. 14).

This uniform illumination ensures consistent excitation conditions for more accurate localization and quantification. Moreover, particularly in STORM, the ASTER technology facilitates shorter acquisition times for smaller fields of view by tuning the irradiance to a specific region of interest. Another advantage of this technology, when combined with TIRF imaging, is the reduction of speckle noise in SMLM by temporal averaging. During the rapid scanning, each point in the field of
<figure><img src="image_16.png" /><figcaption>**Fig. 15.** Bruker's Vutara biplane detection technology. Schematic of biplane beam path. The emitted fluorescence from the sample passes through a dichroic mirror, and is split into two separate optical planes. These two planes are then projected onto different regions of the camera sensor (planes 1 and 2). Image courtesy of Bruker Corporation.</figcaption></figure>

view is irradiated from multiple positions of the scanning beam, thus averaging out any speckle patterns.

## Dual Camera Module for Spectral Demixing and Enhanced 3D SMLM

In addition to Abbelight's ASTER technology, the SAFe MN360 spectral demixing capabilities enable simultaneous two-color imaging, overcoming challenges generally associated with multi-color SMLM such as chromatic aberrations and variations in fluorophore photophysical properties. This system employs a dual camera setup with a beam splitter and dichroic mirrors, allowing the emission spectra from each of two far red fluorophores to be split and directed into two separate cameras (Friedl et al., 2023). For each detected emission, the system calculates a photon ratio, based on the signals captured simultaneously by the two cameras, that helps determine the identity of the fluorophore in real-time. This dual camera system can also be used in single-channel mode, splitting the emitted photos with a 50:50 beam splitter, where one pathway captures undistorted PSFs for XY localization while the other captures astigmatic PSFs for Z-range detection. This allows for greater astigmatism, and thus access to a larger Z-range without compromising lateral localization precision during single-color SMLM acquisitions. This is supported by SAFe MN360's integrated cubic spline (C-spline) interpolation, which more accurately models the shape of PSFs across greater depths compared to traditional Gaussian models. Briefly, C-splines use a series of cubic polynomials to model the PSF (Li et al., 2018) and more accurately capture the variations in the PSF shape. By accurately modeling the PSF across different Z-positions, the C-spline interpolation extends the range of depths that can accurately be imaged—the improved fit yields more precise 3D localization.

## Applications

Abbelight's SAFe MN360 microscope is suitable for a wide range of applications and has made significant contributions across

different biological fields. The SAFe MN360 was used to directly visualize microtubule repair events after micro-injection of fluorescent tubulin in cells (Gazzola et al., 2023). To elucidate mechanisms of cell adhesion and mechanosensing, the SAFe MN360 was used to interrogate the enzyme-dependent clustering behavior of fibronectin in cultured mouse embryonic fibroblasts (Melamed et al., 2023). This microscope was also employed to observe and quantify the clustering of TRPV2 cation channels in cultured metastatic melanoma cells, exposing the role of these channels in melanoma metastasis and presenting them as potential therapeutic targets (Shoji et al., 2023). The SAFe MN360 has recently been used to help describe peculiar multisynaptic boutons within the CA1 subregion of the hippocampus. Here, the 3D nanoscale arrangement of active zone proteins was determined using two-color STORM of mouse brain slices and revealed multiple active zones within a single presynapse (Rigby et al., 2023). In our laboratory, we used the SAFe MN360 to image nanostructures of actin together with the actin nucleator Arp2/3 within isolated presynapses induced by beads (Bingham et al., 2023).

## Bruker Corporation

### Biplane Detection Technology

Bruker's Vutara VXL employs a double-plane, or biplane, detection technology developed in the laboratory of Joerg Bewersdorf in 2008 (Juette et al., 2008). This method uses a 50:50 beam splitter to divide the focused light into two separate light paths directed toward the camera, facilitating simultaneous detection from two planes separated along the axis (Fig. 15), thus achieving 3D subdiffraction resolution. In the biplane detection method, variation in intensity for an emitting dye molecule on the two imaging planes determines its z-position. Rather than using a Gaussian function, a 3D PSF is measured by imaging a small fluorescent bead and fitted to each dataset. The Airy patterns of every blinking event within the biplane module are recorded by the two detection planes, then used to calculate the center of
## Bi-Plane to Detect Z-Position

<figure><img src="image_17.png" /><figcaption>**Fig. 16.** Bruker's Vutara biplane to detect z-position. Schematic of biplane detection, which captures light from an emitter at two different focal planes simultaneously. The PSF of each emitter is projected onto these two imaging planes set ~1 µm apart, creating two slightly different images of the same molecule. The intensity difference between the two planes translates directly to each individual emitter's z-position. Image courtesy of Bruker Corporation.</figcaption></figure>

the PSF and thus the z-localization of the emitter (Fig. 16). The duration of each blinking event, photon count, position, and the accuracy of the PSF fitting can be used to reconstruct high-resolution images of the sample and perform quantitative and statistical analyses.

## System Specifications

The Vutara VXL's has a lightproof enclosed design and is equipped with a 60X/1.30NA silicone oil objective paired with a Hamamatsu sCMOS camera (Orca Flash 4.0 V3 or Fusion BT). Additionally, its enclosed setup makes it suitable for long experiments, protected from external factors. The system's magnification is adjusted to match the camera pixel size, ensuring Nyquist sampling over a large field of view (50 µm × 50 µm for SMLM and 200 µm × 200 µm for multiplexed omics applications). It features three excitation laser lines (405 nm 120 mW, 555 nm 2,000 mW, and 638 nm 1,200 mW) with two possible additional lines (488 nm 2,000 mW and 750 nm 1,500 mW). Coupled with biplane technology, this configuration enables up to five-color sequential 3D imaging at depths of up to 50 µm with z-stacking for all acquisitions, from cell cultures to tissue slices and organoids. A two-camera option allows to image two colors simultaneously. Chromatic aberrations inherent to multispectral imaging are mitigated by calibrating the focal shifts and lateral displacements between the two-color channels, then computationally correcting for these shifts during postacquisition alignment of localizations. In addition to its capability for deep-tissue fixed and live-cell imaging, the Vutara VXL, with

the integration of Bruker's automated microfluidics unit, is equipped to support multiplexed DNA-PAINT and fluorescence *in situ* hybridization (FISH), chromatin tracing, and particle tracking. Current enhancements to the Vutara VXL include spectral demixing, thereby expanding this microscope's multiplexed imaging capabilities.

## Applications

In addition to its unique capability for deep-tissue fixed (Veeraraghavan et al., 2018; Mezache et al., 2020) and live-cell imaging, current and previous models of the Vutara have been employed in various other research domains. This system has been used to visualize chromatin structure at the nanometer scale, revealing details about chromosomal organization and interactions within the cell nucleus previously unknown (Nir et al., 2018). By tracking human chromosome 19 and observing genomic features with OligoSTORM, the authors were able to more clearly delineate the segregation of genomes into active and inactive regions (Beliveau et al., 2015, 2017; Nir et al., 2018). The Vutara biplane system has been exploited to explore the spatial localization of various proteins within special bullet-shaped RNA virions, further illuminating the final stages of genome packaging during viral budding (Hodges et al., 2013). The Vutara has also been used to characterize viral particles, map their positions in 3D, and quantify virus replication across an entire cycle (Alonas et al., 2014).

## Hybrid Techniques

Emerging techniques, involving combinations of existing concepts, continue to push the boundaries of spatial and temporal resolution (Gu et al., 2019; Reymond et al., 2019; Cnossen et al., 2020; Gu et al., 2021; Jouchet, 2021). Here, we discuss abberior's MINFLUX, developed in 2017 and the first commercialized hybrid system.

### MINFLUX

The MINimal photon FLUXes (MINFLUX) technology is based on combining concepts from two super-resolution microscopy techniques, SMLM and STED. In MINFLUX, a donut-shaped light beam is used to excite the fluorescent molecules. The crucial innovation is to place the dark minimum of the donut center as close as possible to the target molecule, instead of searching for maximal emission, as in other SMLM methods. If the emitting molecule is near the excitation donut's zero-intensity center, fewer photons are produced because the excitation intensity is weakened. Moving the beam and measuring the photon fluxes emitted for numerous positions allows a highly precise localization of the molecule's position (Fig. 17) without the need for high photon counts. This is an iterative process. After the initial approximation, the beam is further adjusted, refining the position until the donut minimum is precisely localized at the same position as the molecule. This approach results in much higher resolution, in the single-digit nanometer range, with minimal photon expenditure and thus low photobleaching.

## System Specifications

The 2D and 3D excitation beams are generated as in abberior's STED, using a reflective SLM placed in the beam path. Beam scanning is mediated laterally by electro-optics and axially with deformable mirrors designed for rapid scanning, yielding microsecond temporal resolution. An automated subnanometer stabilization system, which includes a piezo component, adjusts for
<figure><img src="image_18.png" /><figcaption>**Fig. 17.** abberior's MINFLUX technology. MINFLUX uses a donut-shaped excitation beam, with a central intensity zero, to target specific points in space. The emitter's position is localized by moving the center of the donut to different positions near the fluorophore and measuring the emission at each step. When the fluorescent molecule is near the zero-intensity center of the donut beam, fewer photons are emitted, thus achieving highly accurate localization of the molecule. Image courtesy of abberior Instruments.</figcaption></figure>

drift ensuring the sample remains stable during long acquisitions. This stabilization system, together with beamline monitoring for laser drift correction, eliminates errors due to external factors (e.g., room temperature fluctuations, environmental vibrations) during the long-term acquisition of localization data. This positions MINFLUX technology as a unique tool for detecting and tracing dynamic events in living cells (Deguchi et al., 2023) and separating single-digit nanometer structures and protein conformations in fixed cells (Gwosch et al., 2020; Pape et al., 2020; Schmidt et al., 2021).

## Applications

Since its inception in 2017, MINFLUX has been used to explore biological questions previously restricted by achievable spatial and temporal resolutions. Retinal sections were imaged with MINFLUX to visualize presynaptic active zones of rod photoreceptors in 3D with single-digit nanometer isometric resolution (Grabner et al., 2022). The authors demonstrated that the synaptic vesicle release site in rod photoreceptors is a complex molecular structure arranged with longitudinal periodicity along the ribbon. In Deguchi et al. (2023), MINFLUX was used to describe the exact movement of kinesin-1 motor proteins in living cells, showing their step size and dynamic interaction while transporting cellular cargo. Multiple steps of motor proteins were visualized with ~2 nm spatial and submillisecond temporal resolution, providing molecular-level insights into their function and mechanics in a cellular environment.

The super-resolving capabilities of MINFLUX now allow imaging of the structural dynamics of the PIEZO1 ion channel within living cells in response to mechanical stimuli (Mulhall et al., 2023). Here, the authors showed that PIEZO1 senses mechanical forces and revealed previously unattainable details regarding conformational changes.

## Conclusion

Super-resolution techniques have enhanced our ability to study biological structures and processes at the molecular level, providing insights into cellular organization, protein interactions, and dynamics. These novel developments to now familiar super-resolution microscopy techniques highlight the boundless possibilities for bypassing the innate physical properties of light and present a glimpse of what may lie ahead. Besides the technological advancements in overcoming the fundamental limitations imposed by the diffraction of light presented in this article, a common thread across these companies is accessibility. Ease of use is a crucial aspect of these advanced microscopy techniques to continue pushing research forward. Streamlining workflows and developing user-friendly software interfaces expand access to a broader range of researchers, beyond those with extensive technical expertise. Several systems have made significant improvements in this respect. abberior Instruments, for example, have developed two software interfaces, LiGHTBOX and iMSPECTOR, to accommodate users with varying levels of experience. Zeiss's ZEN software aims at allowing easy control and setup of imaging parameters, including an integrated artificial intelligence sample finder and automatically selecting the appropriate lattice grid based on the objective and excitation wavelength used. Software developments like these, as in Vutara's SRX, guide users through the image acquisition workflow and offer a range of analysis tools, including real-time image processing. As advancements in imaging technology expands, so must user accessibility. One of the current challenges with advanced microscopy techniques, however, is the substantial volume of data they generate. As imaging resolutions improve and more complex analyses are performed, there remains an ever-growing need to develop robust data-storage strategies.

## Availability of Data and Materials

The authors have declared that no datasets apply for this piece.

## Acknowledgments

The authors would like to thank Confocal.NL, Nikon, Zeiss, CSR Biotech, Leica Microsystems, abberior, Bruker Corporation, and Abbelight for providing information on their respective microscope technologies. The authors would also like to acknowledge Ciarán Butler-Hallissey for his insights based on his experience as a core facility manager.

## Financial Support

The current study hasn't received any fund from any organizations or institutions.

## Conflict of Interest

The authors declare that they have no competing interest.

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