Publications & technical resources
Explore how DHO technology is facilitating scientific discovery
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From molecules to classrooms: a comprehensive guide to single-molecule localization microscopy
Single-molecule localization microscopy (SMLM) has revolutionized our ability to visualize cellular structures, offering unprecedented detail. However, the intricate biophysical principles that underlie SMLM can be daunting for newcomers, particularly undergraduate and graduate students. To address this challenge, we introduce the fundamental concepts of SMLM, providing a solid theoretical foundation. In addition, we have developed an intuitive graphical interface APP that simplifies these core concepts and makes them more accessible for students. This APP clarifies how super-resolved images are fitted and highlights the crucial factors that determine image quality. Our approach deepens students’ understanding of SMLM by combining theoretical instruction with practical learning. This development equips them with the skills to carry out single-molecule super-resolved experiments and explore the microscopic world beyond the diffraction limit.
Similarly slow diffusion of BAM and Sec YEG complexes in live E. coli cells observed with 3D spt-PALM
The β-barrel assembly machinery (BAM) complex is responsible for inserting outer membrane proteins (OMPs) into the Escherichia coli outer membrane. The SecYEG translocon inserts inner membrane proteins into the inner membrane and translocates both soluble proteins and nascent OMPs into the periplasm. Recent reports describe Sec possibly playing a direct role in OMP biogenesis through interactions with the soluble polypeptide transport-associated (POTRA) domains of BamA (the central OMP component of BAM). Here we probe the diffusion behavior of these protein complexes using photoactivatable super-resolution localization microscopy and single-particle tracking in live E. coli cells of BAM and SecYEG components BamA and SecE and compare them to other outer and inner membrane proteins. To accurately measure trajectories on the highly curved cell surface, three-dimensional tracking was performed using double-helix point-spread function microscopy. All proteins tested exhibit two diffusive modes characterized by “slow” and “fast” diffusion coefficients. We implement a diffusion coefficient analysis as a function of the measurement lag time to separate positional uncertainty from true mobility. The resulting true diffusion coefficients of the slow and fast modes showed a complete immobility of full-length BamA constructs in the time frame of the experiment, whereas the OMP OmpLA displayed a slow diffusion consistent with the high viscosity of the outer membrane. The periplasmic POTRA domains of BamA were found to anchor BAM to other cellular structures and render it immobile. However, deletion of individual distal POTRA domains resulted in increased mobility, suggesting that these domains are required for the full set of cellular interactions. SecE diffusion was much slower than that of the inner membrane protein PgpB and was more like OMPs and BamA. Strikingly, SecE diffused faster upon POTRA domain deletion. These results are consistent with the existence of a BAM-SecYEG trans-periplasmic assembly in live E. coli cells.
Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD
To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer–promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&Run experiments revealed that NuRD modulates enhancer–promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer–promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer–promoter relationships.
Neurexin-3 subsynaptic densities are spatially distinct from Neurexin-1 and essential for excitatory synapse nanoscale organization in the hippocampus
Proteins critical for synaptic transmission are non-uniformly distributed and assembled into regions of high density called subsynaptic densities (SSDs) that transsynaptically align in nanocolumns. Neurexin-1 and neurexin-3 are essential presynaptic adhesion molecules that non-redundantly control NMDAR- and AMPAR-mediated synaptic transmission, respectively, via transsynaptic interactions with distinct postsynaptic ligands. Despite their functional relevance, fundamental questions regarding the nanoscale properties of individual neurexins, their influence on the subsynaptic organization of excitatory synapses and the mechanisms controlling how individual neurexins engage in precise transsynaptic interactions are unknown. Using Double Helix 3D dSTORM and neurexin mouse models, we identify neurexin-3 as a critical presynaptic adhesion molecule that regulates excitatory synapse nano-organization in hippocampus. Furthermore, endogenous neurexin-1 and neurexin-3 form discrete and non-overlapping SSDs that are enriched opposite their postsynaptic ligands. Thus, the nanoscale organization of neurexin-1 and neurexin-3 may explain how individual neurexins signal in parallel to govern different synaptic properties.
Imaging and positioning through scattering media with double-helix point spread function engineering
Significance: Double-helix point spread function (DH-PSF) microscopy has been developed for three-dimensional (3D) localization and imaging at super-resolution but usually in environments with no or weak scattering. To date, super-resolution imaging through turbid media has not been reported. Aim: We aim to explore the potential of DH-PSF microscopy in the imaging and localization of targets in scattering environments for improved 3D localization accuracy and imaging quality. Approach: The conventional DH-PSF method was modified to accommodate the scanning strategy combined with a deconvolution algorithm. The localization of a fluorescent microsphere is determined by the center of the corresponding double spot, and the image is reconstructed from the scanned data by deconvoluting the DH-PSF. Results: The resolution, i.e., the localization accuracy, was calibrated to 13 nm in the transverse plane and 51 nm in the axial direction. Penetration thickness could reach an optical thickness (OT) of 5. Proof-of-concept imaging and the 3D localization of fluorescent microspheres through an eggshell membrane and an inner epidermal membrane of an onion are presented to demonstrate the super-resolution and optical sectioning capabilities. Conclusions: Modified DH-PSF microscopy can image and localize targets buried in scattering media using super-resolution. Combining fluorescent dyes, nanoparticles, and quantum dots, among other fluorescent probes, the proposed method may provide a simple solution for visualizing deeper and clearer in/through scattering media, making in situ super-resolution
microscopy possible for various demanding applications.
3D super-resolution fluorescence imaging of microgels
Super-resolution fluorescence microscopy techniques are powerful tools to investigate polymer systems. In this review, we address how these techniques have been applied to hydrogel nano- and microparticles, so-called nano- or microgels. We outline which research questions on microgels could be ad- dressed and what new insights could be achieved. Studies of the morphology, shape, and deformation of microgels; their internal compartmentalization; the cross-linker distribution and polarity inside them; and their dynamics and diffusion are summarized. In particular, the abilities to super-resolve structures in three dimensions have boosted the research field and have also allowed researchers to obtain impressive 3D images of deformed microgels. Accessing information beyond 3D localization, such as spectral and lifetime properties and correlative imaging or the combination of data with other methods, shines new light onto polymer systems and helps us understand their complexity in detail. Such future trends and developments are also addressed.
All-optical inter-layers functional connectivity investigation in the mouse retina
We developed a multi-unit microscope for all-optical inter-layers circuits interrogation. The system performs two-photon (2P) functional imaging and 2P multiplexed holographic optogenetics at axially distinct planes. We demonstrated the capability of the system to map, in the mouse retina, the functional connectivity between rod bipolar cells (RBCs) and ganglion cells (GCs) by activating single or defined groups of RBCs while recording the evoked response in the GC layer with cell-type specificity and single-cell resolution. We then used a logistic model to probe the functional connectivity between cell types by deriving the ‘‘cellular receptive field’’ describing how RBCs impact each GC type. With the capability to simultaneously image and control neuronal activity at axially distinct planes, the system enables a precise interrogation of multi-layered circuits. Understanding this information transfer is a promising avenue to dissect complex neural circuits and understand the neural basis of computations.
Single-molecule microscopy methods to study mitochondrial processes
Mitochondria are essential organelles of eukaryotic cells with key functions in metabolism, apoptosis, and signaling. As a result, impaired mitochondrial function has been associated with numerous diseases. In order to understand mitochondrial processes, it is fundamental to gain knowledge about their structure and microcompartmentalization, including the function, organization, and dynamics of their protein, nucleic acid, and lipid components. A number of recent groundbreaking advances in fluorescence microscopy enable the study of mitochondrial biology with unprecedented detail. Among them, new methods based on single-molecule and super-resolution microscopy allow us to study mitochondrial structures, protein organizations, and dynamics. Here, we discuss the advantages and disadvantages of different single-molecule microscopy methods to study individual proteins in fixed and living cells in the background of mitochondrial processes, in situ.
Super-resolution cryogenic correlative light and electron microscopy reveals protein organization in the context of intact cellular ultrastructure
To understand how cells work, we need elucidate how proteins
interact inside cellular ultrastructure. Super-resolution
microscopy, e.g. stochastic optical reconstruction microscopy
(STORM) [1], underpins our understanding of interacting
molecular networks in cells at the resolution of dozens of
nanometres. However, to ascertain protein structure and function
relationship, cryogenic correlative light and electron microscopy
(cryo-CLEM) [2] is highly sought after because it combines the
functional information from molecular tagging in light
microscopy with the intact ultrastructure information in electron
microscopy. The challenge is the discrepancy in resolving power
and imaging volume between cryo-EM and conventional cryoFM. To address this challenge, we developed cryogenic STORM
(cryo-STORM) to achieve sub-10 nm localization precision [3],
and 3D Double Helix STORM with extended imaging volume to
a few microns in a single shot. We are developing superresolution cryo-CLEM workflow, aiming at unravelling the
structure-function relationship of proteins and their partners
throughout the cells with unprecedented precision.
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Visualizing the dynamic human genome with DHO 3D tracking and Light Sheet Microscopy
Visualizing the dynamic human genome with DHO 3D tracking and Light Sheet Microscopy
DHO-enabled 3D dSTORM unlocks insights into nanoscale structure of the brain
DHO-enabled 3D dSTORM unlocks insights into nanoscale structure of the brain
Imaging bacterial cell outer membranes in search of new antibiotics
Imaging bacterial cell outer membranes in search of new antibiotics
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