Low-dose cryo-electron ptychography of Apoferritin at 4.0 mrad
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ABSTRACT:
SUBMITTER: Henning Stahlberg
PROVIDER: EMPIAR-12236 | biostudies-other |
REPOSITORIES: biostudies-other
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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.
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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.
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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.
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Project description:High-throughput three-dimensional cryogenic imaging of thick biological specimens is valuable for identifying biologically- or pathologically-relevant features of interest, especially for subsequent correlative studies. Unfortunately, high-resolution imaging techniques at cryogenic conditions often require sample reduction through sequential physical milling or sectioning for sufficient penetration to generate each image of the 3-D stack. This study represents the first demonstration of using ptychographic hard X-ray tomography at cryogenic temperatures for imaging thick biological tissue in a chemically-fixed, frozen-hydrated state without heavy metal staining and organic solvents. Applied to mammalian brain, this label-free cryogenic imaging method allows visualization of myelinated axons and sub-cellular features such as age-related pigmented cellular inclusions at a spatial resolution of ~100 nanometers and thicknesses approaching 100 microns. Because our approach does not require dehydration, staining or reduction of the sample, we introduce the possibility for subsequent analysis of the same tissue using orthogonal approaches that are expected to yield direct complementary insight to the biological features of interest.
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