3D Single-molecule Localization Research Articles

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.

Optimal sampling rate for 3D single molecule localization.

Resolution of single molecule localization microscopy (SMLM) depends on the localization accuracy, which can be improved by utilizing engineered point spread functions (PSF) with delicate shapes. However, the intrinsic pixelation effect of the detector sensor will deteriorate PSFs under different sampling rates. The influence of the pixelation effect to the achieved 3D localization accuracy for different PSF shapes under different signal to background ratio (SBR) and pixel dependent readout noise has not been investigated in detail so far. In this work, we proposed a framework to characterize the 3D localization accuracy of pixelated PSF at different sampling rates. Four different PSFs (astigmatic PSF, double helix (DH) PSF, Tetrapod PSF and 4Pi PSF) were evaluated and the pixel size with optimal 3D localization performance were derived. This work provides a theoretical guide for the optimal design of sampling rate for 3D super resolution imaging.

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.

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.

Three-dimensional diffusion coefficient measurement by a large depth-of-field rotating point spread function.

A prominent challenge in single-molecule localization microscopy is the real-time, fast, and accurate localization of nano-objects moving in three-dimensional (3D) samples. A well-established method for 3D single-molecule localization is the double-helix pointspread-function (DH-PSF) engineering, which uses additional optical elements to make the PSF exhibit different rotation angles with different nanoparticle depths. However, the compact main lobe size, effective detection depth, and precise conversion between rotation angle and depth are necessary, posing challenges to the DH-PSF generation method. Here we generate a more compact DH-PSF using Fresnel-zone-based spiral phases, and the pure phase mask achieves high transmission efficiency. The final generated DH-PSFs have a linear rotation rate at each axial position, showing a more accurate rotation angle and depth conversion. The Cramer-Rao lower limit calculation results show that the axial depth of DH-PSF extends to ∼11µm with an axial localization precision of ∼45nm at 3000 photons and average background noise of 15. We measured the diffusion coefficient of nanospheres in different concentrations of glycerol using the generated DH-PSF. The measured results are within 6% error from the theoretical values, indicating the superior performance of the DH-PSF for nanoparticle diffusion coefficient measurements.

Robust emitter localization with enhanced harmonic analysis.

We present a non-iterative and model-free algorithm for three-dimensional (3D) single emitter localization. Our algorithm decodes the axial position and the emitter width via the ratio of the first and second Fourier harmonic. The retrieved width information is further used for dynamic extraction of the proper region of interest to robustly eliminate the outer noisy background, thus improving the localization precision over existing non-iterative algorithms. Using simulated and experimental datasets, we demonstrate that our algorithm achieves localization precision approaching the state-of-the-art iterative fitting-based methods in all three dimensions at two orders of magnitude faster speed, applicable in various 3D single-molecule localization techniques.

High-axial-resolution single-molecule localization under dense excitation with a multi-channel deep U-Net.

Single-molecule localization microscopy (SMLM) can bypass the diffraction limit of optical microscopes and greatly improve the resolution in fluorescence microscopy. By introducing the point spread function (PSF) engineering technique, we can customize depth varying PSF to achieve higher axial resolution. However, most existing 3D single-molecule localization algorithms require excited fluorescent molecules to be sparse and captured at high signal-to-noise ratios, which results in a long acquisition time and precludes SMLM's further applications in many potential fields. To address this problem, we propose a novel 3D single-molecular localization method based on a multi-channel neural network based on U-Net. By leveraging the deep network's great advantages in feature extraction, the proposed network can reliably discriminate dense fluorescent molecules with overlapped PSFs and corrupted by sensor noise. Both simulated and real experiments demonstrate its superior performance in PSF engineered microscopes with short exposure and dense excitations, which holds great potential in fast 3D super-resolution microscopy.

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.

Fast and robust multiplane single-molecule localization microscopy using a deep neural network

Single-molecule localization microscopy is a widely used technique in biological research for measuring the nanostructures of samples smaller than the diffraction limit. This study uses multifocal plane microscopy and addresses the three-dimensional (3D) single-molecule localization problem, where lateral and axial locations of molecules are estimated. However, when multifocal plane microscopy is used, the estimation accuracy of 3D localization is easily deteriorated by the small lateral drifts of camera positions. A 3D molecule localization problem was presented along with the lateral drift estimation as a compressed sensing problem. A deep neural network (DNN) was applied to solve this problem accurately and efficiently. The results show that the proposed method is robust to lateral drift and achieves an accuracy of 20 nm laterally and 50 nm axially without an explicit drift correction.

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.

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.

Fundamental precision bounds for three-dimensional optical localization microscopy using self-interference digital holography

Localization based microscopy using self-interference digital holography (SIDH) provides three-dimensional (3D) positional information about point sources with nanometer scale precision. To understand the performance limits of SIDH, here we calculate the theoretical limit to localization precision for SIDH when designed with two different configurations. One configuration creates the hologram using a plane wave and a spherical wave while the second configuration creates the hologram using two spherical waves. We further compare the calculated precision bounds to the 3D single molecule localization precision from different Point Spread Functions. SIDH results in almost constant localization precision in all three dimensions for a 20 µm thick depth of field. For high signal-to-background ratio (SBR), SIDH on average achieves better localization precision. For lower SBR values, the large size of the hologram on the detector becomes a problem, and PSF models perform better.

Whole-cell imaging of plasma membrane receptors by 3D lattice light-sheet dSTORM

The molecular organization of receptors in the plasma membrane of cells is paramount for their functionality. We combined lattice light-sheet (LLS) microscopy with three-dimensional (3D) single-molecule localization microscopy (dSTORM) and single-particle tracking to quantify the expression and distribution, and mobility of CD56 receptors on whole fixed and living cells, finding that CD56 accumulated at cell–cell interfaces. For comparison, we investigated two other receptors, CD2 and CD45, which showed different expression levels and distributions in the plasma membrane. Overall, 3D-LLS-dSTORM enabled imaging and single-particle tracking of plasma membrane receptors with single-molecule sensitivity unperturbed by surface effects. Our results demonstrate that receptor distribution and mobility are largely unaffected by contact to the coverslip but the measured localization densities are in general lower at the basal plasma membrane due to partial limited accessibility for antibodies.

In Depth 3D Single Molecule Localization Microscopy with Time Modulated Excitation

In Single Molecule Localization Microscopy, the position of the emitters is obtained by fitting the Point Spread Function (PSF). This spatially based localization precision thus strongly relies on the PSF shape, which is degraded by defocusing or aberrations. In particular, this strongly degrades 3D imaging, where axial resolution is always worse than lateral one, and imaging depth limited to few microns. We propose a new localization strategy based on a time signature to retrieve fluorophores’ position over the whole field of view.