Publications & technical resources

Refocusing after Scanning using Helical phase engineering (RESCH) microscopy has previously been demonstrated to provide volumetric information from a single 2D scan. However, the practical application of this method is challenging due to its limited image acquisition speed and spatial resolution. Here, we report on a combination of RESCH and multifocal structured illumination microscopy (MSIM) to improve the image acquisition speed and spatial resolution. A phase mask is introduced to modulate the conventional point spread function (PSF) to the double-helix PSF (DH-PSF), which provides volumetric information, and meanwhile, sparse multifocal illumination patterns are generated by a digital micromirror device (DMD) for parallel 3D subdiffractive imaging information acquisition. We also present a strategy for processing the collected raw data with a Richardson-Lucy deconvolution and pixel reassignment algorithm to improve the spatial resolution of the depth estimation and imaging performance. The proposed 3D image scanning microscopy can record 3D specimen information and the corresponding depth information from a single multi-spot 2D planar scan, which ensures faster data acquisition, larger field of view, and higher spatial resolution than RESCH. Finally, we demonstrate the capability of our system with actual experiments.

Super-resolution techniques that localize single molecules in three dimensions through point spread function (PSF) engineering are very sensitive to aberrations and optical alignment. Here we show how double-helix point spread function is affected by such mis-alignment and aberration. Specifically, we demonstrate through simulation and experiment how misplacement of phase masks in infinity corrected systems is a common source of significant loss of accuracy. We also describe an optimal alignment and calibration procedure to correct for these errors. In combination, these optimizations allow for a maximal field of view with high accuracy and precision. Though discussed with reference to double-helix point spread function (DHPSF), the optimization techniques are equally applicable to other engineered PSFs.

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.

Theoretical predictions have suggested that molecular motion at interfaces—which influences processes including heterogeneous catalysis, (bio)chemical sensing, lubrication and adhesion, and nanomaterial self-assembly—may be dominated by hypothetical “hops” through the adjacent liquid phase, where a diffusing molecule readsorbs after a given hop according to a probabilistic “sticking coefficient.” Here, we use three-dimensional (3D) single-molecule tracking to explicitly visualize this process for human serum albumin at solid-liquid interfaces that exert varying electrostatic interactions on the biomacromolecule. Following desorption from the interface, a molecule experiences multiple unproductive surface encounters before readsorption. An average of approximately seven surface collisions is required for the repulsive surfaces, decreasing to approximately two and a half for surfaces that are more attractive. The hops themselves are also influenced by long-range interactions, with increased electrostatic repulsion causing hops of longer duration and distance. These findings explicitly demonstrate that interfacial diffusion is dominated by biased 3D Brownian motion involving bulk-surface coupling and that it can be controlled by influencing short- and long-range adsorbate-surface interactions.

The signal-to-noise ratio (SNR) and three-dimensional localization precision of a double helix point spread function (DH-PSF) can be significantly improved by applying variable-angle illumination epifluorescence microscopy (VAI, also commonly known as “pseudo-TIRF” or “quasi-TIRF”). Here, we performed a quantitative analysis of the dependence of SNR and localization precision on the number of measured photons and the incident angle for static particles under both low (at a planar index-matched interface) and high (within a porous silica matrix) fluorescent background conditions. We found that under noisier imaging conditions, the SNR and localization precision obtained using VAI are up to fivefold and threefold greater, respectively, than those obtained using epi-illumination. Moreover, we demonstrate that the combination of DH-PSF and VAI can significantly improve the accuracy of the measured diffusion coefficient for mobile particles, even at a relatively large distance (50 lm) from the boundary of the optical cell.

Single-molecule localization microscopy, typically based on total internal reflection illumination, has taken our understanding of protein organization and dynamics in cells beyond the diffraction limit. However, biological systems exist in a complicated three-dimensional environment, which has required the development of new techniques, including the double-helix point spread function (DHPSF), to accurately visualize biological processes. The application of the DHPSF approach has so far been limited to the study of relatively small prokaryotic cells. By matching the refractive index of the objective lens immersion liquid to that of the sample media, we demonstrate DHPSF imaging of up to 15-mm-thick whole eukaryotic cell volumes in three to five imaging planes. We illustrate the capabilities of the DHPSF by exploring large-scale membrane reorganization in human T cells after receptor triggering, and by using single-particle tracking to image several mammalian proteins, including membrane, cytoplasmic, and nuclear proteins in T cells and embryonic stem cells.

RESCH (refocusing after scanning using helical phase engineering) microscopy is a scanning technique using engineered point spread functions which provides volumetric information. We present a strategy for processing the collected raw data with a multi-view maximum likelihood deconvolution algorithm, which inherently comprises the resolution gain of pixel-reassignment microscopy. The method, which we term MD-RESCH (for multi-view deconvolved RESCH), achieves in our current implementation a 20% resolution advantage along all three axes compared to RESCH and confocal microscopy. Along the axial direction, the resolution is comparable to that of image scanning microscopy. However, because the method inherently reconstructs a volume from a single 2D scan, a significantly higher optical sectioning becomes directly visible to the user, which would otherwise require collecting multiple 2D scans taken at a series of axial positions. Further, we introduce the use of a single-helical detection PSF to obtain an increased post-acquisition refocusing range. We present data from numerical simulations as well as experiments to confirm the validity of our approach.

Stress granules are mRNA-protein granules that form when translation initiation is limited, and they are related to pathological granules in various neurodegenerative diseases. Super-resolution microscopy reveals stable substructures, referred to as cores, within stress granules that can be purified. Proteomic analysis of stress granule cores reveals a dense network of protein-protein interactions and links between stress granules and human diseases and identifies ATP-dependent helicases and protein remodelers as conserved stress granule components. ATP is required for stress granule assembly and dynamics. Moreover, multiple ATP-driven machines affect stress granules differently, with the CCT complex inhibiting stress granule assembly, while the MCM and RVB complexes promote stress granule persistence. Our observations suggest that stress granules contain a stable core structure surrounded by a dynamic shell with assembly, disassembly, and transitions between the core and shell modulated by numerous protein and RNA remodeling complexes.

Super-resolution localization microscopy involves acquiring thousands of image frames of sparse collections of single molecules in the sample. The long acquisition time makes the imaging setup prone to drift, affecting accuracy and precision. Localization accuracy is generally improved by a posteriori drift correction. However, localization precision lost due to sample drifting out of focus cannot be recovered as the signal is originally detected at a lower peak signal. Here, we demonstrate a method of stabilizing a super-resolution localization microscope in three dimensions for extended periods of time with nanometer precision. Hence, no localization correction after the experiment is required to obtain super-resolved reconstructions. The method incorporates a closed-loop with a feedback signal generated from camera images and actuation on a 3D nanopositioning stage holding the sample.