3D Super-resolution Imaging Research Articles

Tilted Light Sheet Microscopy with 3D Point Spread Functions for Single-Molecule Super-Resolution Imaging in Mammalian Cells.

To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

Primary fibroblasts derived from sporadic amyotrophic lateral sclerosis patients do not show ALS cytological lesions

Objective: Sporadic amyotrophic lateral sclerosis (sALS) is a fatal neurodegenerative disorder affecting upper and lower motor neurons. In view of the heterogeneous presentation of the disease, one of the current challenges is to identify diagnostic and prognostic markers in order to diagnose sALS at early stage and to stratify patients in trials. In this study, we sought to identify cytological hallmarks of sALS in patient-derived fibroblasts with the aim of finding new clinical-related markers of the disease. Methods: Primary fibroblasts were prospectively collected from patients affected with classical, rapid, and slow forms of sALS. TDP-43 localization, cytoskeleton distribution, mitochondrial network architecture, and stress granules formation were analyzed using 3D fluorescence microscopy and new super-resolution imaging. Intracellular reactive oxygen species (ROS) production was assessed using live imaging techniques. Results: Six sALS patients (two classical, two rapid, and two slow) and four age-matched controls were included. No difference in fibroblasts cell growth, morphology, and distribution was noticed. The analysis of TDP-43 did not reveal any mislocalization nor aggregation of the protein. The cytoskeleton was harmoniously distributed among the cells, without any inclusion noticed, and no difference was observed regarding the mitochondrial network architecture. Basal ROS production and response to induced stress were similar among patient and control fibroblasts. Conclusions: ALS cytological lesions are absent in patient-derived fibroblasts and thus cannot contribute as diagnostic nor prognostic markers of the disease.

3D single-molecule super-resolution microscopy with a tilted light sheet

Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.

3D mapping of nanoscale crosslink heterogeneities in microgels.

The majority of swollen polymer networks exhibit spatial variations in crosslink density. These spatial heterogeneities are particularly important in colloidal gel particles, or microgels, where they manifest themselves on the nanoscale and impact mechanical and transport properties. Despite their importance, the real space nanostructure of these heterogeneities at the individual particle level has remained elusive. Using state of the art super-resolution microscopy known as Whole cell 4Pi Single Molecule Switching Nanoscopy (W-4PiSMSN) we demonstrate 3D nanoscale mapping of spatial crosslink heterogeneities in a model system of poly(N-isopropylacrylamide) colloidal gel particles containing a novel fluorophore tagged crosslinker. We reveal the presence of higher crosslink density clusters embedded in a lower crosslink density matrix within the core of individual microgel particles, a phenomenon that has been predicted, but never been observed before in real space. The morphology of the clusters provides insight into the kinetics of microgel formation. This study also provides proof-of-concept 3D super-resolution imaging of spatial heterogeneities in bulk hydrogels.

Multiplexed 3D super-resolution imaging of whole cells using spinning disk confocal microscopy and DNA-PAINT

Single-molecule localization microscopy (SMLM) can visualize biological targets on the nanoscale, but complex hardware is required to perform SMLM in thick samples. Here, we combine 3D DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) with spinning disk confocal (SDC) hardware to overcome this limitation. We assay our achievable resolution with two- and three-dimensional DNA origami structures and demonstrate the general applicability by imaging a large variety of cellular targets including proteins, DNA and RNA deep in cells. We achieve multiplexed 3D super-resolution imaging at sample depths up to ~10 µm with up to 20 nm planar and 80 nm axial resolution, now enabling DNA-based super-resolution microscopy in whole cells using standard instrumentation.

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Myosin Va molecular motors manoeuvre liposome cargo through suspended actin filament intersections in vitro

Intracellular cargo transport relies on myosin Va molecular motor ensembles to travel along the cell’s three-dimensional (3D) highway of actin filaments. At actin filament intersections, the intersecting filament is a structural barrier to and an alternate track for directed cargo transport. Here we use 3D super-resolution fluorescence imaging to determine the directional outcome (that is, continues straight, turns or terminates) for an ∼10 motor ensemble transporting a 350 nm lipid-bound cargo that encounters a suspended 3D actin filament intersection in vitro. Motor–cargo complexes that interact with the intersecting filament go straight through the intersection 62% of the time, nearly twice that for turning. To explain this, we develop an in silico model, supported by optical trapping data, suggesting that the motors’ diffusive movements on the vesicle surface and the extent of their engagement with the two intersecting actin tracks biases the motor–cargo complex on average to go straight through the intersection.

Broadband superoscillation brings a wave into perfect three-dimensional focus

The fundamental properties of a wave precludes it from being localized to subwavelength distances in all dimensions of the wave's existence. The inability to focus electromagnetic waves to an all-direction subwavelength spot limits the 3D resolution of a conventional imaging system to about half the imaging wavelength. A plethora of super-resolution imaging systems have been designed which obtain super-resolution in one or two (but not all) dimensions, but they suffer various restrictions in working distance and the classes of objects they can image. In this paper, we report a first investigation into a wave that is focused to subwavelength dimensions in all directions. After reviewing the physics of wave dispersion and diffraction which seemingly preclude this phenomenon, we sidestep these preclusions using a broadband superoscillation waveform and synthesize an all-direction subwavelength focus. We report the salient spatial and temporal features of this wave, and apply it to achieve 3D super-resolution imaging.

Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking.

Single-molecule super-resolution fluorescence microscopy and single-particle tracking are two imaging modalities that illuminate the properties of cells and materials on spatial scales down to tens of nanometers or with dynamical information about nanoscale particle motion in the millisecond range, respectively. These methods generally use wide-field microscopes and two-dimensional camera detectors to localize molecules to much higher precision than the diffraction limit. Given the limited total photons available from each single-molecule label, both modalities require careful mathematical analysis and image processing. Much more information can be obtained about the system under study by extending to three-dimensional (3D) single-molecule localization: without this capability, visualization of structures or motions extending in the axial direction can easily be missed or confused, compromising scientific understanding. A variety of methods for obtaining both 3D super-resolution images and 3D tracking information have been devised, each with their own strengths and weaknesses. These include imaging of multiple focal planes, point-spread-function engineering, and interferometric detection. These methods may be compared based on their ability to provide accurate and precise position information on single-molecule emitters with limited photons. To successfully apply and further develop these methods, it is essential to consider many practical concerns, including the effects of optical aberrations, field dependence in the imaging system, fluorophore labeling density, and registration between different color channels. Selected examples of 3D super-resolution imaging and tracking are described for illustration from a variety of biological contexts and with a variety of methods, demonstrating the power of 3D localization for understanding complex systems.

Single-Molecule Real-Time 3D Imaging of the Transcription Cycle by Modulation Interferometry

Many essential cellular processes, such as gene control, employ elaborate mechanisms involving the coordination of large, multi-component molecular assemblies. Few structural biology tools presently have the combined spatial-temporal resolution and molecular specificity required to capture the movement, conformational changes, and subunit association-dissociation kinetics, three fundamental elements of how such intricate molecular machines work. Here, we report a 3D single-molecule super-resolution imaging study using modulation interferometry and phase-sensitive detection that achieves <2nm axial localization precision, well below thefew-nanometer-sized individual protein components. To illustrate the capability of this technique in probing the dynamics of complex macromolecular machines, we visualize the movement of individual multi-subunit E.coli RNA polymerases through the complete transcription cycle, dissect the kinetics of the initiation-elongation transition, and determine the fate of σ70 initiation factors during promoter escape. Modulation interferometry sets the stage for single-molecule studies of several hitherto difficult-to-investigate multi-molecular transactions that underlie genome regulation.

The Use of 3D Telomere FISH for the Characterization of the Nuclear Architecture in EBV-Positive Hodgkin's Lymphoma.

The 3D nuclear architecture is closely related to cellular functions and chromosomes are organized in distinct territories. Quantitative 3D telomere FISH analysis (3D Q-FISH) and 3D super-resolution imaging (3D-SIM) at a resolution up to 80 nm as well as the recently developed combined quantitative 3D TRF2-telomere immune FISH technique (3D TRF2/Telo-Q-FISH) have substantially contributed to elucidate molecular pathogenic mechanisms of hematological diseases. Here we report the methods we applied to uncover major molecular steps involved in the pathogenesis of EBV-associated Hodgkin's lymphoma. These methods allowed us to identify the EBV-encoded oncoprotein LMP1 as a key element in the formation of Hodgkin (H-cell) and multinucleated Reed-Sternberg cells (RS-cell), the diagnostic tumor cell of classical Hodgkin's lymphoma (cHL). LMP1 mediates multinuclearity through downregulation of shelterin proteins, in particular telomere repeat binding factor 2 (TRF2).

PSF engineering in multifocus microscopy for increased depth volumetric imaging.

Imaging and localizing single molecules with high accuracy in a 3D volume is a challenging task. Here we combine multifocal microscopy, a recently developed volumetric imaging technique, with point spread function engineering to achieve an increased depth for single molecule imaging. Applications in 3D single molecule localization-based super-resolution imaging is shown over an axial depth of 4 µm as well as for the tracking of diffusing beads in a fluid environment over 8 µm.

Structural and Dynamic Study of Caveolin-1 Membrane Nanodomains in Response to Inflammatory Pathway Signaling

Within the plasma membrane, the protein caveolin-1 (cav-1) forms various diffraction-limited microdomains that have yet to been precisely characterized in terms of structure and dynamics. Cav-1 is an important regulator of the GPCR protease activated receptor-1 (PAR-1), notably during the activation of cellular inflammation responses. The regulatory role of cav-1 during inflammatory responses is linked to the differential activation of RhoA and Rac1 signaling pathways and to cytoskeletal rearrangements that result in wide scale plasma membrane reorganization and cell migration. Using single molecule and super-resolution imaging techniques coupled with quantitative spatial clustering analyses, we study the membrane distribution and the dynamics of caveolae and flat cav-1 scaffolds in response to PAR-1 pathway activations. Cav-1 knock-out MEF and iMAEC cells were rescued with cav-1 fusions to SNAP tag and immunolabeled against endogenous PAR-1 for dual color 3D super-resolution imaging of individual cav-1 and PAR-1 proteins. PAR-1 was directly stimulated using site-specific activating peptides to trigger inflammatory response cascades. At the plasma membrane, 3D super-resolution imaging revealed the expected ∼100 nm 3D globular shape of caveolae as well as flat cav-1 rich nanodomains with structures matching the expected size and z-dimensional range of cav-1 scaffolds. Using robust auto- and cross-correlation statistical analyses of individual cav-1 and PAR-1 proteins spatially mapped with a precision <10 nm, we show that the distribution, the number and the size of cav-1 nanodomains change dynamically in response to either pro- or anti- inflammatory signaling as does the interconnectivity between caveolae, cav-1 scaffolds and PAR-1 receptors at the cell surface. We are able to couple the molecular re-organization of diffraction-limited cav-1 nanodomains with broader cytoskeletal and plasma membrane remodeling in response to defined signaling cues during cellular wound healing.

Myosin Va Motor Teams Navigate Vesicle Cargos through 3D Actin Filament Intersections

Myosin Va (MyoVa) motor teams collectively move intracellular cargo through a complex, three dimensional (3D), actin meshwork. To address how MyoVa motors navigate this physical challenge, we imaged fluorescently labeled 350nm synthetic lipid vesicles, bound with ∼10 MyoVa HMM motors, moving through 3D actin filament intersections strung between 3µm beads. Intersections were formed by filaments being ∼90 degrees to one another and separated by 30-250nm. As the vesicle surface contacted the intersecting actin filaments, two regions of potential motor engagement were created on the vesicle surface. Geometric constraints limited the engaged motor number at each contact point to no more than 3, as confirmed by laser trap stall force measurements. These points of vesicle-actin filament contact resulted in an effective tug of war between two motor teams that were physically separated on the vesicle surface. The vesicle's directional outcome at an intersection was visualized using high spatial and temporal 3D super resolution imaging. For vesicles that physically contacted both actin filaments at the intersection, 55% of vesicles continued straight through the intersection, 35% switched filaments, while only 10% terminated. These outcomes were successfully simulated by a model in which the directional outcome was dependent on the interfilament gap, the vesicle's azimuthal approach angle to the intersection, the stochastic attachment and detachment of motors within a team (i.e. a team's force generation), and the Brownian motion of the vesicle while attached to the actin filaments. All of these physical factors contributed to which motor ensemble won the tug of war at the intersection and thus the vesicle's directional outcome. This simplified biomimetic system and resultant data provide an experimental framework for understanding how MyoVa motor teams navigate their cargo through evermore complex 3D actin networks that exist within cells.

Fluorescence Investigations on the Attack of Cell-Wall-Deficient Bacteria by Antimicrobial Peptides

Antimicrobial resistance remains a serious global issue, with an increasing prevalence of multi-drug resistant bacteria appearing throughout the biosphere. Evidence of resistance towards our last-line-of-defense antibiotics is also developing, painting a grim outlook for future treatment outcomes. Antimicrobial peptides offer a promising therapeutic alternative to conventional antibiotics, which act by directly permeating and disrupting the bacterial cell membrane. Much contention persists surrounding the precise mechanism of action responsible for cell death, compounded by the complexity of the biological systems under study.One largely forgotten characteristic of bacteria is the L-Form transformation. Under stressful environmental conditions, such as antibiotic exposure, bacteria enhance their survival by shedding their exterior membrane structure, leaving the cytoplasmic membrane unharmed. In this cell-wall-deficient state, known as the L-Form, bacteria continue to metabolize and reproduce. Upon removal of the stressful stimuli, the bacteria can revert to their classical morphology, resuming previous infectious behavior. The relatively simple L-Form morphology is of particular interest, as directly exposing the bacterial cytoplasmic membrane may allow for new therapeutic strategies, bypassing many classical bacterial defenses.In our work, we have investigated the interaction of a model antimicrobial peptide with Pseudomonas aeruginosa L-Forms via several fluorescence microscopy techniques. P. aeruginosa is a notorious pathogen known for its significant resistance capacity towards antibiotics, but its L-Form state is potentially vulnerable to attack. Details of the interaction are provided using 3D super-resolution imaging correlated with independent experiments of spatially resolved fluorescence lifetime information. We use our prior studies on membrane systems of varying complexity to help interpret our recent findings.

3D Super-Resolution Imaging of Unperturbed Cells

Most optical imaging of individual cells has been conducted in 2D on flat glass or culture plastic. Not only is this very abstract from the biological environment and does not imitate physiological conditions, but it may lead to unwanted perturbations of cellular functions and structures due to surface contact. This effect is extremely important in cells whose biological function requires them to interact or sense their local environment, a canonical example of which are T cells, which are responsible for initiating adaptive immune responses to extracellular stimuli.We have developed an experimental protocol capable of using 3D single-molecule super-resolution microscopy with a double-helix point spread function that allows for the imaging of live or fixed cells away from a glass surface. We have empirically determined an ultimate spatial precision of ∼15nm in x and y and ∼30nm in z.We will present both multicolour 3D super-resolution images and single-molecule tracking of human T cells, labelling key proteins involved in their function, including CD45, TCR, Zap70 and CD86.

Three-Dimensional Super-Resolution Imaging of the RNA Degradation Machinery in Caulobacter Crescentus

Due the small size of bacteria and the diffraction limit (DL), obtaining precise information on the location of biomolecules through the use of conventional fluorescence imaging is difficult. However, the conventional resolution of optical microscopes (∼200 nm) can be circumvented by the time-sequential super-localization of single-molecule fluorescent labels, resulting in ∼5x resolution enhancement. We applied these super-resolution techniques, in combination with a double-helix point spread function microscope, to study the three-dimensional (3D) spatial organization of the RNA degradation machinery in Caulobacter crescentus (C. crescentus). The RNA degradosome plays a dominant role in mRNA degradation, a key step in gene expression. RNaseE is a key component in the RNA degradosome, and is thought to be the scaffold for this complex in C. crescentus. Previous diffraction-limited fluorescence imaging has shown that C. crescentus RNaseE exhibits a patchy localization throughout the nucleoid. We report the first 3D super-resolution images of RNaseE in live C. crescentus, with careful attention to remove any spurious clustering from over-counting of blinking fluorescent protein labels. We use the acquired localizations to quantify RNaseE's spatial distribution and find a non-random, clearly clustered distribution of this protein. Moreover, most RNaseE molecules are localized in regular clusters spaced along the cell axis, and it is possible to extract specific measures of cluster size and other parameters. Disturbing transcription perturbs the protein distribution, resulting in more diffuse RNaseE localization. Our results suggest that mRNA decay is spatially organized within C. crescentus, and that this organization is related to the function of RNaseE in mRNA homeostasis.

Correlated confocal and super-resolution imaging by VividSTORM.

Single-molecule localization microscopy (SMLM) is rapidly gaining popularity in the life sciences as an efficient approach to visualize molecular distribution with nanoscale precision. However, it has been challenging to obtain and analyze such data within a cellular context in tissue preparations. Here we describe a 5-d tissue processing and immunostaining procedure that is optimized for SMLM, and we provide example applications to fixed mouse brain, heart and kidney tissues. We then describe how to perform correlated confocal and 3D-superresolution imaging on these sections, which allows the visualization of nanoscale protein localization within labeled subcellular compartments of identified target cells in a few minutes. Finally, we describe the use of VividSTORM (http://katonalab.hu/index.php/vividstorm), an open-source software for correlated confocal and SMLM image analysis, which facilitates the measurement of molecular abundance, clustering, internalization, surface density and intermolecular distances in a cell-specific and subcellular compartment-restricted manner. The protocol requires only basic skills in tissue staining and microscopy.

Correction of a Depth-Dependent Lateral Distortion in 3D Super-Resolution Imaging.

Three-dimensional (3D) localization-based super-resolution microscopy (SR) requires correction of aberrations to accurately represent 3D structure. Here we show how a depth-dependent lateral shift in the apparent position of a fluorescent point source, which we term `wobble`, results in warped 3D SR images and provide a software tool to correct this distortion. This system-specific, lateral shift is typically > 80 nm across an axial range of ~ 1 μm. A theoretical analysis based on phase retrieval data from our microscope suggests that the wobble is caused by non-rotationally symmetric phase and amplitude aberrations in the microscope’s pupil function. We then apply our correction to the bacterial cytoskeletal protein FtsZ in live bacteria and demonstrate that the corrected data more accurately represent the true shape of this vertically-oriented ring-like structure. We also include this correction method in a registration procedure for dual-color, 3D SR data and show that it improves target registration error (TRE) at the axial limits over an imaging depth of 1 μm, yielding TRE values of < 20 nm. This work highlights the importance of correcting aberrations in 3D SR to achieve high fidelity between the measurements and the sample.

Multi-pulse time delay integration method for flexible 3D super-resolution range-gated imaging.

Constructing flexible regular-shaped range-intensity profiles by the convolution of illuminator laser pulse and sensor gate pulse is crucial for 3D super-resolution range-gated imaging. However, ns-scale rectangular-shaped laser pulse with tunable pulse width is difficult to be obtained, especially for pulsed solid-stated lasers. In this paper we propose a multi-pulse time delay integration (MPTDI) method to reshape range-intensity profiles (RIP) free from the above limitation of pulsed lasers. An equivalent laser pulse temporal shaping model is established to evaluate and optimize the MPTDI method. By using MPTDI, the RIP shape and depth of viewing can both be flexibly changed as desired. Here typical triangular and trapezoidal RIPs are established for 3D imaging under triangular and trapezoidal range-intensity correlation algorithms. In addition, a prototype experiment is demonstrated to prove the feasibility of MPTDI.