3D STED Super-Resolution Imaging Strategy for Visualizing Synaptic Nano-architecture in Brain Cryosections.

Double-helix point spread function photoactivation-localization microscopy allows three-dimensional (3D) superresolution imaging of objects smaller than the optical diffraction-limit. We demonstrate polarization sensitive detection with 3D super-localization of single-molecules and unveil 3D polarization specific characteristics of single-molecules within the intracellular structure of PtK1 cells expressing photoactivatable green fluorescent protein. The system modulates orthogonal polarization components of single-molecule emissions with a single spatial light modulator and detects them separately with a single detector. Information obtained from the two polarization channels demonstrates polarization based contrast in 3D superresolution imaging. Further, we show that the 3D information from the two channels can be optimally combined to yield up to 30% improvement in localization precision relative to a single polarization channel system.

3D Imaging of Lipid-Guided Vesicle Trafficking Along the Cytoskeleton.

Super-resolution (SR) microscopy is a cutting-edge method that can provide detailed structural information with high resolution. However, the thickness of the specimen has been a major limitation for SR methods, and large biological structures have posed a challenge. To overcome this, the key step is to optimise sample preparation to ensure optical homogeneity and clarity, which can enhance the capabilities of SR methods for the acquisition of thicker structures. Oocytes are the largest cells in the mammalian body and are crucial objects in reproductive biology. They are especially useful for studying membrane proteins. However, oocytes are extremely fragile and sensitive to mechanical manipulation and osmotic shocks, making sample preparation a critical and challenging step. We present an innovative, simple and sensitive approach to oocyte sample preparation for 3D STED acquisition. This involves alcohol dehydration and mounting into a high refractive index medium. This extended preparation procedure allowed us to successfully obtain a unique two-channel 3D STED SR image of an entire mouse oocyte. By optimising sample preparation, it is possible to overcome current limitations of SR methods and obtain high-resolution images of large biological structures, such as oocytes, in order to study fundamental biological processes. Lay Abstract: Super-resolution (SR) microscopy is a cutting-edge tool that allows scientists to view incredibly fine details in biological samples. However, it struggles with larger, thicker specimens, as they need to be optically clear and uniform for the best imaging results. In this study, we refined the sample preparation process to make it more suitable for SR microscopy. Our method includes carefully dehydrating biological samples with alcohol and then transferring them into a mounting medium that enhances optical clarity. This improved protocol enables high-resolution imaging of thick biological structures, which was previously challenging. By optimizing this preparation method, we hope to expand the use of SR microscopy for studying large biological samples, helping scientists better understand complex biological structures.

Isotropic 3D Super Resolution Imaging with Self-Bending Point Spread Function

We report super-resolution fluorescence imaging of live cells with high spatiotemporal resolutions using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and three-dimensional (3D) super-resolution images of living cells, using clathrin-coated pits and the transferrin cargo as model systems. Bright, fast switching probes enabled us to achieve 2D imaging at spatial resolutions of ~25 nm and temporal resolutions as fast as 0.5 sec. We also demonstrated live-cell 3D volumetric super-resolution imaging. A 3D spatial resolution of ~30 nm in the lateral directions and ~50 nm in the axial direction was obtained at time resolutions down to 1 – 2 sec with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we further demonstrated two-color 3D super-resolution imaging in live cells. These imaging capabilities open a new window for characterizing cellular structures in living cells at the ultrastructural level.

The continuous evolution of light microscopy technologies has led to the development of several superresolution methods that surpass the diffraction limit of classical fluorescence microscopy. Superresolution microscopy, combined with advanced image processing algorithms, has become essential for uncovering biological mechanisms and analyzing cellular and sub-cellular morphology in both health and disease (Dzyubenko et al., 2023; Fuhrmann et al., 2022; Willig, 2022; Zhou et al., 2024). This research topic explores recent advances in superresolution imaging and analysis methods, which are crucial for understanding critical biological mechanisms in the healthy and the injured brain.Visualizing the organization of synaptic proteins requires extremely high imaging resolution. In this Research topic, Sachs et al. introduce a method that combines expansion microscopy with Airyscan microscopy, enhancing labelling efficiency and enabling multicolour fluorescence imaging with 20-40 nm spatial resolution. Their study reveals that synaptic proteins RIM 1/2, Munc13-1, and AMPA receptors are organized into trans-synaptic nanocolumns, which are critical for effective synaptic transmission. This method provides a powerful tool for exploring the nanoscale arrangement of synaptic proteins, thereby deepening our understanding of synaptic function.Conventional microscopy methods often face issues with targeted labelling, which can be inhomogeneous and incomplete, leading to gaps in data and analysis. The review by Inavalli et al. explores the potential of fluorescence microscopy shadow imaging to overcome these challenges. Shadow imaging offers homogeneous and exhaustive fluorescence labelling of the extracellular space, resulting in cellular structures appearing as dark silhouettes, or shadows, against a fluorescent background, thereby providing clear and comprehensive visualization. Key advantages of this method include reduced photobleaching and toxicity, as well as consistent labelling across the entire sample. Originally developed to assist patch-clamp electrophysiology, shadow imaging has since evolved and found applications across various microscopy modalities. Techniques such as super-resolution STED microscopy, two-photon microscopy, and light-sheet microscopy have all benefited from the incorporation of shadow imaging. This evolution has expanded its use beyond neuroscience, demonstrating broad potential for intravital imaging in other tissues, such as bone marrow and lymph nodes.Obtaining three-dimensional (3D) superresolution information about dynamic processes in live cells at high speed remains challenging. In this research topic, Yang et al. demonstrate how single-point edge-excitation sub-diffraction (SPEED) microscopy, combined with 2D-to-3D transformation algorithms, can achieve high temporal and sub-diffraction-limit 3D resolution in subcellular structures with rotational symmetry. The authors review how this novel approach elucidates vesicle-assisted transport mechanisms in intracellular trafficking to the primary cilium, which is critical for understanding developmental disorders. Based on the superresolution imaging data obtained with SPEED microscopy, the authors propose a comprehensive model of ciliary transport.Despite recent technological advances, applying superresolution microscopy for deep imaging in thick specimens, such as brain sections, remains challenging. To overcome the optical limitations of deep superresolution imaging, such as the deterioration of light convergence properties, Tsutsumi et al. propose a method called 2P-SRRF, which combines super-resolution radial fluctuation analysis with two-photon microscopy. The authors demonstrate that 2P-SRRF can achieve sub-diffraction-limit spatial resolution, effectively resolving features separated by 120 nm even at significant depths. The resolution enhancement was validated at depths exceeding several hundred micrometres, showcasing high-resolution in vivo imaging in the fifth layer of the cerebral cortex. This makes 2P-SRRF a versatile and powerful tool for neuroscience research.This Research Topic highlights the recent advancements in superresolution imaging techniques that shed light on critical biological mechanisms in the brain. These advancements overcome the challenges in conventional fluorescence labelling, high-speed 3D superresolution microscopy, and deep brain imaging, offering versatile applications in neuroscience research.Author contributionsED drafted the manuscript. KIW, ED, and JC edited and revised the manuscript. All authors contributed to the article and approved the submitted version.AcknowledgmentsWe thank the authors and reviewers for their contributions to this Research Topic.Conflict of interestThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We demonstrate three-dimensional (3D) super-resolution live-cell imaging through thick specimens (50-150 μm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D super-resolution imaging of cellular spheroids.

Airy beams maintain their intensity profiles over a large propagation distance without substantial diffraction and exhibit lateral bending during propagation1-5. This unique property has been exploited for micromanipulation of particles6, generation of plasma channels7 and guidance of plasmonic waves8, but has not been explored for high-resolution optical microscopy. Here, we introduce a self-bending point spread function (SB-PSF) based on Airy beams for three-dimensional (3D) super-resolution fluorescence imaging. We designed a side-lobe-free SB-PSF and implemented a two-channel detection scheme to enable unambiguous 3D localization of fluorescent molecules. The lack of diffraction and the propagation-dependent lateral bending make the SB-PSF well suited for precise 3D localization of molecules over a large imaging depth. Using this method, we obtained super-resolution imaging with isotropic 3D localization precision of 10-15 nm over a 3 μm imaging depth from ∼2000 photons per localization.

We introduce a self-bending point spread function (SB-PSF) based on Airy beams for three-dimensional (3D) super-resolution fluorescence imaging. We designed a side-lobe-free SB-PSF for the incoherent fluorescence emission and implemented a two-channel detection scheme for the SB-PSF to enable unambiguous 3D localization of fluorescent molecules. The lack of diffraction and the propagation-dependent lateral bending make the SB-PSF ideal for precise 3D localization of molecules over a large imaging depth. Using this SB-PSF, we demonstrated super-resolution imaging with isotropic localization precisions of 10-15 nm in all three dimensions over a 3 μm imaging depth without sample scanning.

Recently, three-dimensional (3D) super resolution imaging of cellular structures in thick samples has been enabled with the wide-field super-resolution fluorescence microscopy based on double helix point spread function (DH-PSF). However, when the sample is Epi-illuminated, much background fluorescence from those excited molecules out-of-focus will reduce the signal-to-noise ratio (SNR) of the image in-focus. In this paper, we resort to a selective-plane illumination strategy, which has been used for tissue-level imaging and single molecule tracking, to eliminate out-of-focus background and to improve SNR and the localization accuracy of the standard DH-PSF super-resolution imaging in thick samples. We present a novel super-resolution microscopy that combine selective-plane illumination and DH-PSF. The setup utilizes a well-defined laser light sheet which theoretical thickness is 1.7μm (FWHM) at 640nm excitation wavelength. The image SNR of DH-PSF microscopy between selective-plane illumination and Epi-illumination are compared. As we expect, the SNR of the DH-PSF microscopy based selective-plane illumination is increased remarkably. So, 3D localization precision of DH-PSF would be improved significantly. We demonstrate its capabilities by studying 3D localizing of single fluorescent particles. These features will provide high thick samples compatibility for future biomedical applications.

4Pi stimulated emission depletion (STED) microscopy shows outstanding three-dimensional (3D) isotropic super-resolution imaging performance. However, this technology is still difficult for achieving long-term studying of the synapses that are deeply embedded inside brain tissue. Metalens, which can realize arbitrary nanoscale amplitude, phase, and polarization modulations, is a very useful tool to solve this limitation. In this paper, an ultracompact two-photon 4Pi STED microscopy involved two multifunctional metalenses patterned on the two fiber facets respectively for focusing the excitation and depletion laser beams to the same position was proposed to realize the 3D isotropic super-resolution imaging. The designed complementary structure of two metalenses and the optimized pupil ratio β assured the symmetry of the STED spot. Furthermore, the isotropic super-resolution of 27 nm was theoretically implemented based on the two-photon STED theoretical model. Our approach will greatly increase the viability of the 3D super-resolution morphological imaging in the deep brain.

Stimulated emission depletion (STED) microscopy enables the three-dimensional (3D) visualization of dynamic nanoscale structures in living cells, offering unique insights into their organization. However, 3D-STED imaging deep inside biological tissue is obstructed by optical aberrations and light scattering. We present a STED system that overcomes these challenges. Through the combination of two-photon excitation, adaptive optics, red-emitting organic dyes, and a long-working-distance water-immersion objective lens, our system achieves aberration-corrected 3D super-resolution imaging, which we demonstrate 164 µm deep in fixed mouse brain tissue and 76 µm deep in the brain of a living mouse.

Super-resolution imaging with photo-activatable or photo-switchable probes is a promising tool in biological applications to reveal previously unresolved intra-cellular details with visible light. This field benefits from developments in the areas of molecular probes, optical systems, and computational post-processing of the data. The joint design of optics and reconstruction processes using double-helix point spread functions (DH-PSF) provides high resolution three-dimensional (3D) imaging over a long depth-of-field. We demonstrate for the first time a method integrating a Fisher information efficient DH-PSF design, a surface relief optical phase mask, and an optimal 3D localization estimator. 3D super-resolution imaging using photo-switchable dyes reveals the 3D microtubule network in mammalian cells with localization precision approaching the information theoretical limit over a depth of 1.2 µm.

While super-resolution fluorescence microscopy is a powerful tool for biological research, obtaining multiplexed images for a large number of distinct target species remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT, point accumulation for imaging in nanoscale topography) for simple and easy-to-implement multiplexed 3D super-resolution imaging inside fixed cells and achieve sub-10 nm spatial resolution in vitro using synthetic DNA structures. We also report a novel approach for multiplexing (Exchange-PAINT) that allows sequential imaging of multiple targets using only a single dye and a single laser source. We experimentally demonstrate ten-“color” super-resolution imaging in vitro on synthetic DNA structures and four-“color” imaging of proteins in a fixed cell.

Compared to conventional widefield-illumination microscopy, multifocal structured illumination microscopy (MSIM) achieves a twofold enhancement in spatial resolution while exhibiting greater imaging penetration depth and superior optical sectioning capabilities. These technical advantages position MSIM as a prominent solution for three-dimensional (3D) super-resolution (SR) imaging of thick biological specimens. However, the intrinsically low signal-to-noise ratio (SNR) of raw MSIM data introduces reconstruction artifacts during SR processing. Such artifacts not only degrade image resolution and fidelity but also constrain imaging speed, thereby limiting the applications of MSIM in live-cell and tissue SR imaging. To address these challenges, this study proposes a novel approach termed ZSDN-MSIM: Zero-shot Deconvolution Networks (ZSDN) are implemented to perform denoising and deconvolution preprocessing on raw MSIM data, coupled with pixel reassignment post-processing for MSIM SR reconstruction. This methodology significantly enhances MSIM imaging quality under low-illumination conditions, demonstrating substantial value for advancing low-photodamage in vivo imaging applications.