![]() Here, we present a deconvolution approach to achieve both enhanced signal-to-noise ratio and missing wedge compensation. These techniques include nonlinear anisotropic diffusion, convolutional neural networks based on detector noise models, wavelet-based filtering methods, different implementations of deconvolution, and model-based iterative reconstruction ( 22 – 31). Because of these issues with cryo-ET data, filters to improve contrast and compensate for the missing wedge are an area of ongoing research ( 21). This is due to cross-terms in the WBP coming from the tilt wedges, as well as distortions in the WBP arising from the missing wedge. These include significant intensity above and below the sample volume, where we expect vacuum with no signal. In addition to the missing wedges, TEM images require a significant defocus to generate adequate contrast, and the process of reconstruction by weighted back projection (WBP) introduces well-known problems. Attenuation of high frequencies due to radiation damage as the tilt series progresses is not depicted ( 15, 16). ( Right) The middle slice of the kxkz plane shows the missing wedge (MW) and baby missing wedges (BMWs) of information visualized in Fourier space. Sample projections are acquired over a range of tilt angles, typically from –60 ° to +60 °. ( Left) Schematic of the tilt series collection scheme. Tilt series collection and the missing wedge issue. Commonly recognized artifacts are elongation along the Z direction and streaks projecting from high contrast points into neighboring planes in the volume. As such, it is not surprising that different algorithms can generate somewhat different reconstructions from the same data. Since the reconstruction is equivalent to an inversion in Fourier space, it is obvious that some interpolation is required and that the data are incomplete. The gaps between discrete tilt angles also leave small missing wedges as seen in Fig. 1. The missing information is best recognized in the kxkz plane of Fourier space, corresponding the XZ plane in real space, where it is known as the missing wedge. The projected thickness of a slab also increases with tilt angle, resulting in degraded contrast and resolution from these contributions to the reconstruction. The tilt range is restricted by the slab geometry, typically to about 120 ° around the vertical. Third, the available raw data are never sufficient to produce an unambiguous reconstruction. The correction is inherently approximate and is especially challenging in tomography, where the defocus varies across the field of view for tilted specimens ( 20). Postprocessing is applied to correct this representation in the image intensities. Contrast is lost at low spatial frequencies and oscillates at high spatial frequencies, meaning that material density could be represented as intensity either darker or lighter than background ( 17 – 19). ![]() Second, the modality of wide-field TEM depends on defocus to generate useful phase contrast, but with a nontrivial dependence on spatial frequency that is expressed in a contrast transfer function (CTF). Additionally, higher-resolution information is degraded by radiation damage over the course of imaging ( 15), although approaches such as dose-symmetric acquisition have been developed to optimize recording of high frequencies ( 16). Constraints on the permissible exposure result in limited contrast and a low signal-to-noise ratio ( 14). First, vitrified biological samples are highly sensitive to damage by the electron irradiation required for imaging. While cryo-ET offers unparalleled resolution of cellular interiors, it is challenging for a number of reasons. This 3D reconstruction is rendered for display and analysis, which may entail segmentation to highlight extended structures or averaging of subvolumes for enhancement of molecular-scale resolution ( 11 – 13). A series of projection images is acquired, typically with 1 ∘ to 5 ∘ increments, and then reconstructed into a three-dimensional (3D) volume ( 10). They are then cryo-FIB milled to a suitable thickness of 100 to 350 nm for imaging with transmission electron microscopy (TEM). Cells are rapidly frozen to achieve a vitreous form of ice that preserves biological molecules in a near-native state. Recent advances in cryo-electron tomography (cryo-ET), most notably the ability to thin cryopreserved specimens using a focused ion beam (FIB), have opened windows for the direct visualization of the cell interior at nanometer-scale resolution ( 1 – 9).
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