Defocus Corrected Large Area Cryo-EM (DeCo-LACE) for label-free detection of molecules across entire cell sections
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Evaluation Summary:
The work details a new acquisition method of defocus corrected large area cryo-EM (DeCo-LACE). The data-acquisition approach is highly complementary to the research group's previous work of using high-resolution 2D template-matching (2DTM) to identify macromolecular complexes in dense and heterogeneous cellular specimens. Notably and importantly, the data-acquisition approach minimizes sampling bias. Overall, DeCo-LACE is a very interesting approach to locating large ribosomal subunits in FIB-lamella at scale.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
A major goal of biological imaging is localization of biomolecules inside a cell. Fluorescence microscopy can localize biomolecules inside whole cells and tissues, but its ability to count biomolecules and accuracy of the spatial coordinates is limited by the wavelength of visible light. Cryo-electron microscopy (cryo-EM) provides highly accurate position and orientation information of biomolecules but is often confined to small fields of view inside a cell, limiting biological context. In this study, we use a new data-acquisition scheme called Defocus-Corrected Large-Area cryo-EM (DeCo-LACE) to collect high-resolution images of entire sections (100- to 250-nm-thick lamellae) of neutrophil-like mouse cells, representing 1–2% of the total cellular volume. We use 2D template matching (2DTM) to determine localization and orientation of the large ribosomal subunit in these sections. These data provide maps of ribosomes across entire sections of mammalian cells. This high-throughput cryo-EM data collection approach together with 2DTM will advance visual proteomics and provide biological insight that cannot be obtained by other methods.
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Evaluation Summary:
The work details a new acquisition method of defocus corrected large area cryo-EM (DeCo-LACE). The data-acquisition approach is highly complementary to the research group's previous work of using high-resolution 2D template-matching (2DTM) to identify macromolecular complexes in dense and heterogeneous cellular specimens. Notably and importantly, the data-acquisition approach minimizes sampling bias. Overall, DeCo-LACE is a very interesting approach to locating large ribosomal subunits in FIB-lamella at scale.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript is likely of interest to cryo-electron microscopists working on cellular samples. It details a data-acquisition scheme for mapping large areas at a fine pixel size by cryo-electron microscopy for the purpose of macromolecular identification by high-resolution 2D template matching (2DTM). The authors succinctly describe the methodology, as well as detail the apparent effects of microscope aberrations on 2DTM results.
While other montaging approaches have been described recently, the one presented here differs in its approach to controlling defocus and avoids the need to sacrifice a biologically meaningful region of a sample. The authors investigate the compatibility of the data acquisition with their 2DTM method using cryoFIB-milled mouse neutrophil-like cells and the 60S ribosome as an example …
Reviewer #1 (Public Review):
The manuscript is likely of interest to cryo-electron microscopists working on cellular samples. It details a data-acquisition scheme for mapping large areas at a fine pixel size by cryo-electron microscopy for the purpose of macromolecular identification by high-resolution 2D template matching (2DTM). The authors succinctly describe the methodology, as well as detail the apparent effects of microscope aberrations on 2DTM results.
While other montaging approaches have been described recently, the one presented here differs in its approach to controlling defocus and avoids the need to sacrifice a biologically meaningful region of a sample. The authors investigate the compatibility of the data acquisition with their 2DTM method using cryoFIB-milled mouse neutrophil-like cells and the 60S ribosome as an example case. In order to minimize unnecessary exposures, the authors restrict illumination to a circle inscribed on the detector and use beam image-shift in lieu of stage shift. This approach introduces several optical aberrations for which the authors investigate the effects on the 2DTM results. The results of the investigated aberration effects may be of general interest to the cryoEM community, not just those using montaging methods.
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Reviewer #2 (Public Review):
The manuscript by Elferisch and colleagues extends the previously reported approach for localization of large ribosomal subunits to larger areas of the dimensions of cells (several square microns). A large area of imaging is achieved by recording minimally overlapping circular exposures followed by an assembly of them into a large image. The large ribosomal subunits are then searched in the 2D images taking the defocus into account. In order to make the approach functional the authors specifically tailored the algorithms to perform motion correction and validated that image shift does not seem to limit the precision of localization of large ribosomal subunits in images. Overall, DeCo-LACE is a very interesting approach to locating large ribosomal subunits in FIB-lamella at scale.
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