Project description:Electron ptychography has seen a recent surge of interest for phase sensitive imaging at atomic or near-atomic resolution. However, applications are so far mainly limited to radiation-hard samples, because the required doses are too high for imaging biological samples at high resolution. We propose the use of non-convex Bayesian optimization to overcome this problem, and show via numerical simulations that the dose required for successful reconstruction can be reduced by two orders of magnitude compared to previous experiments. As an important application we suggest to use this method for imaging single biological macromolecules at cryogenic temperatures and demonstrate 2D single-particle reconstructions from simulated data with a resolution up to 5.4?Å at a dose of 20e - /Å2. When averaging over only 30 low-dose datasets, a 2D resolution around 3.5?Å is possible for macromolecular complexes even below 100 kDa. With its independence from the microscope transfer function, direct recovery of phase contrast, and better scaling of signal-to-noise ratio, low-dose cryo electron ptychography may become a promising alternative to Zernike phase-contrast microscopy.
Project description:Cryo-electron microscopy is an essential tool for high-resolution structural studies of biological systems. This method relies on the use of phase contrast imaging at high defocus to improve information transfer at low spatial frequencies at the expense of higher spatial frequencies. Here we demonstrate that electron ptychography can recover the phase of the specimen with continuous information transfer across a wide range of the spatial frequency spectrum, with improved transfer at lower spatial frequencies, and as such is more efficient for phase recovery than conventional phase contrast imaging. We further show that the method can be used to study frozen-hydrated specimens of rotavirus double-layered particles and HIV-1 virus-like particles under low-dose conditions (5.7 e/Å2) and heterogeneous objects in an Adenovirus-infected cell over large fields of view (1.14 × 1.14 μm), thus making it suitable for studies of many biologically important structures.
Project description:Nanoparticles offer great potential in drug delivery and imaging, but shielding strategies are necessary to increase circulation time and performance. Structure-function studies are required to define the design rules to achieve effective shielding. With several formulations reaching clinical testing and approval, the ability to assess and detail nanoparticle formulations at the single particle level is becoming increasingly important. To address this need, we use cryo-electron tomography (cryo-ET) to investigate stealth-coated nanoparticles. As a model system, we studied the soft matter nanotubes formed by tobacco mosaic virus (TMV) coated with human serum albumin (SA) stealth proteins. Cryo-ET and subtomogram averaging allow for visualization of individual SA molecules and determination of their orientations relative to the TMV surface, and also for measurement of the surface coverage provided by added stealth proteins. This information fills a critical gap in the understanding of the structural morphology of stealth-coated nanoparticles, and therefore cryo-ET may play an important role in guiding the development of future nanoparticle-based therapeutics.
Project description:Electron ptychography has recently attracted considerable interest for high resolution phase-sensitive imaging. However, to date studies have been mainly limited to radiation resistant samples as the electron dose required to record a ptychographic dataset is too high for use with beam-sensitive materials. Here we report defocused electron ptychography using a fast, direct-counting detector to reconstruct the transmission function, which is in turn related to the electrostatic potential of a two-dimensional material at atomic resolution under various low dose conditions.
Project description:With the introduction of direct electron detectors (DED) to the field of electron cryo-microscopy, a wave of atomic-resolution structures has become available. As the new detectors still require comparative characterization, we have used tobacco mosaic virus (TMV) as a test specimen to study the quality of 3D image reconstructions from data recorded on the two direct electron detector cameras, K2 Summit and Falcon II. Using DED movie frames, we explored related image-processing aspects and compared the performance of micrograph-based and segment-based motion correction approaches. In addition, we investigated the effect of dose deposition on the atomic-resolution structure of TMV and show that radiation damage affects negative carboxyl chains first in a side-chain specific manner. Finally, using 450,000 asymmetric units and limiting the effects of radiation damage, we determined a high-resolution cryo-EM map at 3.35Å resolution. Here, we provide a comparative case study of highly ordered TMV recorded on different direct electron detectors to establish recording and processing conditions that enable structure determination up to 3.2Å in resolution using cryo-EM.
Project description:Both high resolution and high precision are required to quantitatively determine the atomic structure of complex nanostructured materials. However, for conventional imaging methods in scanning transmission electron microscopy (STEM), atomic resolution with picometer precision cannot usually be achieved for weakly-scattering samples or radiation-sensitive materials, such as 2D materials. Here, we demonstrate low-dose, sub-angstrom resolution imaging with picometer precision using mixed-state electron ptychography. We show that correctly accounting for the partial coherence of the electron beam is a prerequisite for high-quality structural reconstructions due to the intrinsic partial coherence of the electron beam. The mixed-state reconstruction gains importance especially when simultaneously pursuing high resolution, high precision and large field-of-view imaging. Compared with conventional atomic-resolution STEM imaging techniques, the mixed-state ptychographic approach simultaneously provides a four-times-faster acquisition, with double the information limit at the same dose, or up to a fifty-fold reduction in dose at the same resolution.
Project description:Correlative light and electron cryo-microscopy (cryo-CLEM) combines information from the specific labeling of fluorescence cryo-microscopy (cryo-FM) with the high resolution in environmental context of electron cryo-microscopy (cryo-EM). Exploiting super-resolution methods for cryo-FM is advantageous, as it enables the identification of rare events within the environmental background of cryo-EM at a sensitivity and resolution beyond that of conventional methods. However, due to the need for relatively high laser intensities, current super-resolution cryo-CLEM methods require cryo-protectants or support films which can severely reduce image quality in cryo-EM and are not compatible with many samples, such as mammalian cells. Here, we introduce cryogenic super-resolution optical fluctuation imaging (cryo-SOFI), a low-dose super-resolution imaging scheme based on the SOFI principle. As cryo-SOFI does not require special sample preparation, it is fully compatible with conventional cryo-EM specimens, and importantly, it does not affect the quality of cryo-EM imaging. By applying cryo-SOFI to a variety of biological application examples, we demonstrate resolutions up to ∼135 nm, an improvement of up to three times compared with conventional cryo-FM, while maintaining the specimen in a vitrified state for subsequent cryo-EM. Cryo-SOFI presents a general solution to the problem of specimen devitrification in super-resolution cryo-CLEM. It does not require a complex optical setup and can easily be implemented in any existing cryo-FM system.