3D Single-molecule Localization Microscopy Research Articles

Temporal-Focusing Multiphoton Excitation Single-Molecule Localization Microscopy Using Spontaneously Blinking Fluorophores.

Single-molecule localization microscopy (SMLM) based on temporal-focusing multiphoton excitation (TFMPE) and single-wavelength excitation is used to visualize the three-dimensional (3D) distribution of spontaneously blinking fluorophore-labeled subcellular structures in a thick specimen with a nanoscale-level spatial resolution. To eliminate the photobleaching effect of unlocalized molecules in out-of-focus regions for improving the utilization rate of the photon budget in 3D SMLM imaging, SMLM with single-wavelength TFMPE achieves wide-field and axially confined two-photon excitation (TPE) of spontaneously blinking fluorophores. TPE spectral measurement of blinking fluorophores is then conducted through TFMPE imaging at a tunable excitation wavelength, yielding the optimal TPE wavelength for increasing the number of detected photons from a single blinking event during SMLM. Subsequently, the TPE fluorescence of blinking fluorophores is recorded to obtain a two-dimensional TFMPE-SMLM image of the microtubules in cancer cells with a localization precision of 18±6 nm and an overall imaging resolution of approximately 51 nm, which is estimated based on the contribution of Nyquist resolution and localization precision. Combined with astigmatic imaging, the system is capable of 3D TFMPE-SMLM imaging of brain tissue section of a 5XFAD transgenic mouse with the pathological features of Alzheimer's disease, revealing the distribution of neurotoxic amyloid-beta peptide deposits.

Large-FOV 3D localization microscopy by spatially variant point spread function generation.

Accurate characterization of the microscopic point spread function (PSF) is crucial for achieving high-performance localization microscopy (LM). Traditionally, LM assumes a spatially invariant PSF to simplify the modeling of the imaging system. However, for large fields of view (FOV) imaging, it becomes important to account for the spatially variant nature of the PSF. Here, we propose an accurate and fast principal components analysis-based field-dependent 3D PSF generator (PPG3D) and localizer for LM. Through simulations and experimental three-dimensional (3D) single-molecule localization microscopy (SMLM), we demonstrate the effectiveness of PPG3D, enabling super-resolution imaging of mitochondria and microtubules with high fidelity over a large FOV. A comparison of PPG3D with a shift-variant PSF generator for 3D LM reveals a threefold improvement in accuracy. Moreover, PPG3D is approximately 100 times faster than existing PSF generators, when used in image plane-based interpolation mode. Given its user-friendliness, we believe that PPG3D holds great potential for widespread application in SMLM and other imaging modalities.

Quantifying nanoscopic alterations associated with mitochondrial dysfunction using three-dimensional single-molecule localization microscopy.

Mitochondrial morphology provides unique insights into their integrity and function. Among fluorescence microscopy techniques, 3D super-resolution microscopy uniquely enables the analysis of mitochondrial morphological features individually. However, there is a lack of tools to extract morphological parameters from super-resolution images of mitochondria. We report a quantitative method to extract mitochondrial morphological metrics, including volume, aspect ratio, and local protein density, from 3D single-molecule localization microscopy images, with single-mitochondrion sensitivity. We validated our approach using simulated ground-truth SMLM images of mitochondria. We further tested our morphological analysis on mitochondria that have been altered functionally and morphologically in controlled manners. This work sets the stage to quantitatively analyze mitochondrial morphological alterations associated with disease progression on an individual basis.

High-throughput single-molecule localization microscopy: Potential clinical applications.

High-throughput single-molecule localization microscopy: Potential clinical applications.

Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy.

Extracellular vesicles (EVs) play a key role in cell-cell communication and thus have great potential to be utilized as therapeutic agents and diagnostic tools. In this study, we implemented single-molecule microscopy techniques as a toolbox for a comprehensive characterization as well as measurement of the cellular uptake of HEK293T cell-derived EVs (eGFP-labeled) in HeLa cells. A combination of fluorescence and atomic force microscopy revealed a fraction of 68% fluorescently labeled EVs with an average size of ∼45 nm. Two-color single-molecule fluorescence microscopy analysis elucidated the 3D dynamics of EVs entering HeLa cells. 3D colocalization analysis of two-color direct stochastic optical reconstruction microscopy (dSTORM) images revealed that 25% of EVs that experienced uptake colocalized with transferrin, which has been linked to early recycling of endosomes and clathrin-mediated endocytosis. The localization analysis was combined with stepwise photobleaching, providing a comparison of protein aggregation outside and inside the cells.

Deep learning driven adaptive optics for single molecule localization microscopy.

Single molecule localization microscopy (SMLM) forms super-resolution images with a resolution of several to tens of nanometers, by detecting isolated single molecule emission patterns and localizing the centers of individual probes. However, the inhomogeneous refractive indices distort and blur single molecule emission patterns, reduce the information content carried by each detected photon, increase localization uncertainty, and thus cause significant resolution loss, which is irreversible by post-processing. To compensate tissue induced aberrations, conventional sensorless adaptive optics methods rely on iterative mirror-changes and image-quality metrics to compensate aberrations. However, these metrics result in inconsistent, and sometimes opposite, metric responses which fundamentally limited the efficacy of these approaches for aberration correction in tissues. Bypassing the previous iterative trial-then-evaluate processes, we developed deep learning driven adaptive optics (DL-AO) for SMLM to allow direct inference of wavefront distortion and near real-time compensation. Our trained deep neural network (DNN) monitors the individual emission patterns from single molecule experiments, infers their shared wavefront distortion, feeds the estimates through a dynamic filter (Kalman), and drives a deformable mirror to compensate sample induced aberrations. The method simultaneously estimates and compensates 28 types of wavefront deformation shapes, restores single molecule emission patterns approaching the conditions untouched by specimen, and improves the resolution and fidelity of 3D SMLM through brain tissues over 130 µm, with as few as 3-20 mirror changes.

Single molecule imaging simulations with advanced fluorophore photophysics

Advanced fluorescence imaging techniques such as single-molecule localization microscopy (SMLM) fundamentally rely on the photophysical behavior of the employed fluorophores. This behavior is generally complex and impacts data quality in a subtle manner. A simulation software named Single-Molecule Imaging Simulator (SMIS) is introduced that simulates a widefield microscope and incorporates fluorophores with their spectral and photophysical properties. With SMIS, data collection schemes combining 3D, multicolor, single-particle-tracking or quantitative SMLM can be implemented. The influence of advanced fluorophore characteristics, imaging conditions, and environmental parameters can be evaluated, facilitating the design of real experiments and their proper interpretation.

SOLEIL: single-objective lens inclined light sheet localization microscopy.

High-NA light sheet illumination can improve the resolution of single-molecule localization microscopy (SMLM) by reducing the background fluorescence. These approaches currently require custom-made sample holders or additional specialized objectives, which makes the sample mounting or the optical system complex and therefore reduces the usability of these approaches. Here, we developed a single-objective lens-inclined light sheet microscope (SOLEIL) that is capable of 2D and 3D SMLM in thick samples. SOLEIL combines oblique illumination with point spread function PSF engineering to enable dSTORM imaging in a wide variety of samples. SOLEIL is compatible with standard sample holders and off-the-shelve optics and standard high NA objectives. To accomplish optimal optical sectioning we show that there is an ideal oblique angle and sheet thickness. Furthermore, to show what optical sectioning delivers for SMLM we benchmark SOLEIL against widefield and HILO microscopy with several biological samples. SOLEIL delivers in 15 μm thick Caco2-BBE cells a 374% higher intensity to background ratio and a 54% improvement in the estimated CRLB compared to widefield illumination, and a 184% higher intensity to background ratio and a 20% improvement in the estimated CRLB compared to HILO illumination.

Direct-laser writing for subnanometer focusing and single-molecule imaging

Two-photon direct laser writing is an additive fabrication process that utilizes two-photon absorption of tightly focused femtosecond laser pulses to implement spatially controlled polymerization of a liquid-phase photoresist. Two-photon direct laser writing is capable of nanofabricating arbitrary three-dimensional structures with nanometer accuracy. Here, we explore direct laser writing for high-resolution optical microscopy by fabricating unique 3D optical fiducials for single-molecule tracking and 3D single-molecule localization microscopy. By having control over the position and three-dimensional architecture of the fiducials, we improve axial discrimination and demonstrate isotropic subnanometer 3D focusing (<0.8 nm) over tens of micrometers using a standard inverted microscope. We perform 3D single-molecule acquisitions over cellular volumes, unsupervised data acquisition and live-cell single-particle tracking with nanometer accuracy.

Direct supercritical angle localization microscopy for nanometer 3D superresolution

3D single molecule localization microscopy (SMLM) has emerged as a powerful method for structural cell biology, as it allows probing precise positions of proteins in cellular structures at a resolution of tens of nanometers in both 2D and 3D. Popular approaches to 3D imaging include PSF engineering to encode the position of single-emitters in its shape. However, such methods lead to worse localization precision axially than laterally. Complex localization microscopy techniques, such as iPALM, can provide isotropic resolution at the cost of a complex instrument.

Numerical simulation study of three-dimensional high-density single molecule localization microscopy based on orthogonal astigmatism

Single molecule localization microscopy (SMLM) detects and locates sparsely luminous single fluorescent molecules to achieve super-resolution imaging at nanoscale spatial resolution. In order to improve the temporal resolution, it is necessary to increase the density of the simultaneously emitting molecules. However, with the increase of the density, the point spread function (PSF) of different molecules will overlap severely on the detector, resulting in reduced spatial resolution, especially for three-dimensional (3D) SMLM. To solve this problem, a high density 3D-SMLM imaging method based on orthogonal astigmatism is proposed. Analysis and numerical simulation study for the method are carried out and presented. The main idea of the proposed orthogonal astigmatic method is to split the collected fluorescence in a SMLM microscope into two beams, each of which passes through a separate channel with a cylindrical lens and arrives at a specific region on the same detector. The two cylindrical lenses have the same optical parameters, but their orientations are set to be orthogonal to each other. They are used to obtain both positive and negative astigmatic PSF images of the same fluorescent molecule. Then, a linear projection model of the imaging process is established, and the 3D localization of the fluorescent molecules is realized by using a compression sensing algorithm. The results show that the two orthogonal cylindrical lenses produce a pair of astigmatic PSFs for one single molecule so that different PSF pairs between different molecules have lower mutual correlation, and thus the 3D localization accuracy for high density imaging can be significantly improved as compared with traditional astigmatic method, in which one single cylindrical lens is used. The larger the defocusing degree, the greater the shape difference between the two astigmatic PSFs is, and the more obvious this advantage.

Studying the Dynamics of Chromatin-Binding Proteins in Mammalian Cells Using Single-Molecule Localization Microscopy.

Single-molecule localization microscopy (SMLM) allows the super-resolved imaging of proteins within mammalian nuclei at spatial resolutions comparable to that of a nucleosome itself (~20nm). The technique is therefore well suited to the study of chromatin structure. Fixed-cell SMLM has already allowed temporal "snapshots" of how proteins are arranged on chromatin within mammalian nuclei. In this chapter, we focus on how recent developments, for example in selective plane illumination, 3D SMLM, and protein labeling, have led to a range of live-cell SMLM studies. We describe how to carry out single-particle tracking (SPT) of single proteins and, by analyzing their diffusion parameters, how to determine whether proteins interact with chromatin, diffuse freely, or do both. We can study the numbers of proteins that interact with chromatin and also determine their residence time on chromatin. We can determine whether these proteins form functional clusters within the nucleus as well as whether they form specific nuclear structures.

3D Structure From 2D Microscopy Images Using Deep Learning.

Understanding the structure of a protein complex is crucial in determining its function. However, retrieving accurate 3D structures from microscopy images is highly challenging, particularly as many imaging modalities are two-dimensional. Recent advances in Artificial Intelligence have been applied to this problem, primarily using voxel based approaches to analyse sets of electron microscopy images. Here we present a deep learning solution for reconstructing the protein complexes from a number of 2D single molecule localization microscopy images, with the solution being completely unconstrained. Our convolutional neural network coupled with a differentiable renderer predicts pose and derives a single structure. After training, the network is discarded, with the output of this method being a structural model which fits the data-set. We demonstrate the performance of our system on two protein complexes: CEP152 (which comprises part of the proximal toroid of the centriole) and centrioles.

Fast 3D localization algorithm for high-density molecules based on multiple measurement vector compressed sensing

Single-molecule localization microscopy (SMLM) achieves super-resolution based on a series of images of sparsely distributed molecules, which makes it time-consuming. To improve the temporal resolution, several high-density localization algorithms have been developed, and the computational speed is considered one of the important factors for evaluating their performance. In this study, for the first time, a three-dimensional (3D) multiple measurement vector compressed sensing (3D-MMV-CS) model was constructed and then solved using a multiple sparse Bayesian learning algorithm to realize 3D single-molecule localization and obtain 3D super-resolved fluorescence images. The 3D-MMV-CS successfully recovered a 3D super-resolved image from a series of simulated high-density fluorescence images. The computation time for reconstruction only increases marginally with an increase in the number of raw images and approximately stays constant with an increase in the density of fluorescent molecules. Simulation results demonstrated that, when the number of raw images reaches 1900, compared with the other two algorithms L1-Homotopy and compressed sensing STORM (CSSTORM) with CVX, the 3D-MMV-CS method is 100 times faster than the former and five orders of magnitude faster than the latter. This outstanding performance in computation speed would allow the 3D-MMV-CS approach to be routinely applicable to SMLM. Validation test of the 3D-MMV-CS method was performed successfully with experimental data from an astigmatism-based 3D SMLM system.

Real-time 3D single-molecule localization microscopy analysis using lookup tables.

Herein, we present a new algorithm for real-time analysis of 3D single molecule localization microscopy images with a small impact on fitting accuracy using lookup-tables with discrete xyz-positions. The algorithm realizes real-time visualization during acquisition. We demonstrate its performance on simulated and measured data. Additionally, combining real-time fitting with a feedback loop controlling the activation laser pulse keeps the number of emitters per image frame constant.

Super-resolution visualization and modeling of human chromosomal regions reveals cohesin-dependent loop structures

BackgroundThe 3D organization of the chromatin fiber in cell nuclei plays a key role in the regulation of gene expression. Genome-wide techniques to score DNA-DNA contacts, such as Hi-C, reveal the partitioning of chromosomes into epigenetically defined active and repressed compartments and smaller “topologically associated” domains. These domains are often associated with chromatin loops, which largely disappear upon removal of cohesin. Because most Hi-C implementations average contact frequencies over millions of cells and do not provide direct spatial information, it remains unclear whether and how frequently chromatin domains and loops exist in single cells.ResultsWe combine 3D single-molecule localization microscopy with a low-cost fluorescence labeling strategy that does not denature the DNA, to visualize large portions of single human chromosomes in situ at high resolution. In parallel, we develop multi-scale, whole nucleus polymer simulations, that predict chromatin structures at scales ranging from 5 kb up to entire chromosomes. We image chromosomes in G1 and M phase and examine the effect of cohesin on interphase chromatin structure. Depletion of cohesin leads to increased prevalence of loose chromatin stretches, increased gyration radii, and reduced smoothness of imaged chromatin regions. By comparison to model predictions, we estimate that 6–25 or more purely cohesin-dependent chromatin loops coexist per megabase of DNA in single cells, suggesting that the vast majority of the genome is enclosed in loops.ConclusionOur results provide new constraints on chromatin structure and showcase an affordable non-invasive approach to study genome organization in single cells.

Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections

Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 µm, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 µm thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons.

Accelerating 3D single-molecule localization microscopy using blind sparse inpainting.

.Significance: Single-molecule localization-based super-resolution microscopy has enabled the imaging of microscopic objects beyond the diffraction limit. However, this technique is limited by the requirements of imaging an extremely large number of frames of biological samples to generate a super-resolution image, thus requiring a longer acquisition time. Additionally, the processing of such a large image sequence leads to longer data processing time. Therefore, accelerating image acquisition and processing in single-molecule localization microscopy (SMLM) has been of perennial interest.Aim: To accelerate three-dimensional (3D) SMLM imaging by leveraging a computational approach without compromising the resolution.Approach: We used blind sparse inpainting to reconstruct high-density 3D images from low-density ones. The low-density images are generated using much fewer frames than usually needed, thus requiring a shorter acquisition and processing time. Therefore, our technique will accelerate 3D SMLM without changing the existing standard SMLM hardware system and labeling protocol.Results: The performance of the blind sparse inpainting was evaluated on both simulation and experimental datasets. Superior reconstruction results of 3D SMLM images using up to 10-fold fewer frames in simulation and up to 50-fold fewer frames in experimental data were achieved.Conclusions: We demonstrate the feasibility of fast 3D SMLM imaging leveraging a computational approach to reduce the number of acquired frames. We anticipate our technique will enable future real-time live-cell 3D imaging to investigate complex nanoscopic biological structures and their functions.

Direct supercritical angle localization microscopy for nanometer 3D superresolution

3D single molecule localization microscopy (SMLM) is an emerging superresolution method for structural cell biology, as it allows probing precise positions of proteins in cellular structures. In supercritical angle localization microscopy (SALM), z-positions of single fluorophores are extracted from the intensity of supercritical angle fluorescence, which strongly depends on their distance to the coverslip. Here, we realize the full potential of SALM and improve its z-resolution by more than four-fold compared to the state-of-the-art by directly splitting supercritical and undercritical emission, using an ultra-high NA objective, and applying fitting routines to extract precise intensities of single emitters. We demonstrate nanometer isotropic localization precision on DNA origami structures, and on clathrin coated vesicles and microtubules in cells, illustrating the potential of SALM for cell biology.

Versatile, High-Resolution, and Large Field-of-View Single-Molecule Imaging by Oblique Plane Microscopy

Building on recent advancements in oblique plane microscopy (OPM), also known as single objective light sheet, we demonstrate a versatile, high resolution, and large field-of-view OPM with single-molecule resolution. We achieve 300-nm lateral resolution and 800-nm axial resolution, without deconvolution, through the use of a bespoke solid immersion objective for remote focusing. We extend the imaging volume by rapidly translating the sample through the oblique light sheet using optimized stage scanning and digital control of the light sheet to eliminate motion blur. Because OPM requires only one objective at the sample, we can perform high-resolution light sheet imaging using standard sample mounting methods, including microfluidics for flow cytometry and fluidic chambers for iterative multiplexed labeling. We quantitatively evaluated and verified the instrument's volume performance using calibration samples and multiple imaging-based resolution metrics. As constructed, we can image 1-cm x 1-cm x 60-micron (x,y,z) per color per hour, with single-molecule resolution, in real samples. To demonstrate the instrument's capability, we quantified the co-expression of RNA and protein markers for the SARS-CoV-2 entry genes ACE2 and TMPRSS2 in alveolar epithelial type II cells across centimeter-scale fixed human lung tissue slices. In contrast to currently used imaging methods for spatial transcriptomics, our OPM approach provides the correct spatial sampling required to perform rigorous 3D single-molecule localization. Importantly, we achieve this high sampling rate and light collection efficiency at volume imaging rates that are orders of magnitude faster than existing approaches. Beyond spatial -omics, we additionally performed 3D single-molecule localization microscopy in fixed cells and live-cell imaging with with no modifications to the instrument. We envision the increase in resolution, sampling, and speed of high numerical aperture OPM versus traditional high numerical aperture microscopes will greatly aid high-throughput single-molecule imaging.