<i>In situ</i>structural analysis of the flagellum attachment zone in<i>Trypanosoma brucei</i>using cryo-scanning transmission electron tomography

Trépout, Sylvain

doi:10.1101/2020.02.14.949115

Cited by 2 publications

(2 citation statements)

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“…Considering the multi-scattering effect of electrons, the resolution and contrast of STEM tomography of thick section could be further improved by changing dark field to bright field (Hohmann-Marriott et al, 2009). The attempts of applying STEM tomography to frozen-hydrated biological sample were also performed in recently years (Rez et al, 2016;Trepout, 2020;Wolf and Elbaum, 2019;Wolf et al, 2014), which turned to be useful to detect metabolic ions in biomacromolecules (Elad et al, 2017), study mitochondrial calcium stores physiology (Wolf et al, 2017) and dynamics of ferritin crystallization (Houben et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Imaging biological samples by integrated differential phase contrast (iDPC) STEM technique

Lazić

Huang

et al. 2021

Preprint

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Scanning transmission electron microscopy (STEM) is a powerful imaging technique and has been widely used in current material science research. The attempts of applying STEM into biological research have been going on for decades while applications have still been limited because of the existing bottlenecks in dose efficiency and non-linearity in contrast. Recently, integrated differential phase contrast (iDPC) STEM technique emerged and achieved a linear phase contrast imaging condition, while resolving signals of light elements next to heavy ones even at low electron dose. This enables successful investigation of beam sensitive materials. Here, we investigate iDPC-STEM advantages in biology, in particular, chemically fixed and resin embedded biological tissues. By comparing results to the conventional TEM, we have found that iDPC-STEM not only shows better contrast but also resolves more structural details at molecular level, including conditions of extremely low dose and minimal heavy-atom staining. For thick sample sections, iDPC-STEM is particularly advantageous. Unlike TEM, it avoids contrast inversion canceling effects, and by adjusting the depth of focus, fully preserves the contrast of relevant features along with the sample. In addition, using depth-sectioning, iDPC-STEM enables resolving in-depth structural variation. Our work suggests that promising, wide and attractive applications of iDPC-STEM in biological research are opening.

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Section: Introductionmentioning

confidence: 99%

Imaging biological samples by integrated differential phase contrast (iDPC) STEM technique

Lazić

Huang

et al. 2021

Preprint

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“…At equivalent electron dose, STEM induces less radiation damage to the sample than classical transmission electron microscopy (TEM) [Wolf et al 2014] and is able to image micrometer thick cryo-fixed biological samples in 3D (see Fig. 1) [Trépout 2020]. However, in order to image thicker specimens or to reach higher magnifications we need to increase the dwell time or to sample the specimen more densely, respectively.…”

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confidence: 99%

Sparse cryo-STEM tomography for biological samples

Cossa

Arluison

2021

Microsc Microanal

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Cryo-electron tomography (Cryo-ET) enables to recover 3D information of cryo-fixed samples in a near-native state [Lučić et al. 2013] with a thickness up to ~250 nm [Aoyama et al. 2008]. Thicker samples (up to 1 µm) can be studied in scanning transmission electron microscopy (STEM), which consists in the raster scanning of a (sub-)nanometric convergent electron beam focused at the sample plane [Midgley &Weyland2003][Pennycook 2012]. At equivalent electron dose, STEM induces less radiation damage to the sample than classical transmission electron microscopy (TEM) [Wolf et al. 2014] and is able to image micrometer thick cryo-fixed biological samples in 3D (see Fig. 1) [Trépout 2020]. However, in order to image thicker specimens or to reach higher magnifications we need to increase the dwell time or to sample the specimen more densely, respectively. This will inevitably increase the electron dose and the beam damage proportionally. The need to further reduce the electron dose emerges. In this work, we present a sparse cryo-STET acquisition scheme. Instead of collecting a full frame for each tilt-angle, we collect only a fraction of the pixels. This permits to dramatically reduce the radiation dose received by the sample on each frame and across the full tilt-series. Multiple sparse data collection schemes have been previously investigated at our lab [Trépout 2019] (see Fig. 2). Before aligning the tilt-series, a preprocessing step is necessary to recover the missing information due to the data sparsity. Multiple inpainting methods are commonly used such as discrete cosine transform (DCT) [Garcia 2009], and shearlets transform [Kutynioket al. 2014]. Then the 3D volume can be reconstructed using classical methods like weighted backprojection (WBP) [Radermacher 2006] implemented in IMOD [Kremer et al. 1996] or iterative algorithms such as simultaneous iterative reconstruction technique (SIRT) [Gilbert 1972] in Tomo3D [Agullero& Fernandez 2011]. To demonstrate the advantages of a sparse cryo-STET acquisition scheme, we applied our method on a sample that is too thick ( > 1 µm) to be studied using conventional TEM methods.

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In situstructural analysis of the flagellum attachment zone inTrypanosoma bruceiusing cryo-scanning transmission electron tomography

Cited by 2 publications

References 69 publications

Imaging biological samples by integrated differential phase contrast (iDPC) STEM technique

Imaging biological samples by integrated differential phase contrast (iDPC) STEM technique

Sparse cryo-STEM tomography for biological samples

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