Cryo-electron tomography : a journey from sample preparation to data mining / edited by Dorit Hanein, Departments of Bioengineering, and of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, CA, United States, Niels Volkmann, Departments of Bioengineering, and of Electrical and Computer Engineering, Interdisciplinary Program for Quantitative Biosciences, University of California Santa Barbara, Santa Barbara, CA, United States.

Format:

Book

Contributor:

Hanein, Dorit, editor, contributor.

Volkmann, Niels, editor, contributor.

Language:

English

Subjects (All):

Cryoelectronics.

Tomography.

Physical Description:

1 online resource (360 pages) : illustrations (chiefly color)

Other Title:

Journey from sample preparation to data mining

Place of Publication:

Amsterdam, Netherlands : Academic Press, an imprint of Elsevier, [2025].

Biography/History:

"Prof. Dorit Hanein received her doctoral degree from the Weizmann Institute in Israel. She completed her training as a Fulbright postdoctoral fellow at Brandeis University under the mentorship of Professor David DeRosier, a pioneer in three-dimensional image reconstruction techniques via electron microscopy. Prof. Hanein is affiliated with the University of California, Santa Barbara, and serves as a PEW Innovation Fund Investigator. She holds a “Habilitation à Diriger des Recherches” (HDR, Accreditation to Direct Research) from Sorbonne University, France.Prof. Hanein’s research lies at the intersection of structural biology, cell biology, systems biology, and engineering science. Her work focuses on the quantitative integration of high-resolution imaging technologies, designed to visualize the molecular architecture and dynamic conformational landscape of biological nanomachines in three dimensions within their native environments and under mechanical perturbations. She has made seminal contributions to our understanding of the cytoskeleton and macromolecular assemblies, advanced the field of quantitative electron microscopy, pioneered the use of correlative light and cryo-electron microscopic tomography and functionalized substrates. The strategic employment of these techniques has revolutionized our ability to define the building blocks of large dynamic macromolecular complexes in three dimensions with high fidelity and high resolution, while contextualizing their function within whole cells."--Provided by publisher.

"Professor Niels Volkmann received his Ph.D. in Physics from the University of Hamburg, Germany, conducting his doctoral research at the Max Planck Institute under the mentorship of Ada Yonath, who was awarded the Nobel Prize in Chemistry in 2009. Following his doctoral studies, Dr. Volkmann trained as a postdoctoral fellow at Brandeis University with David DeRosier, a pioneer in three-dimensional image reconstruction techniques. He is currently a Professor at the University of California, Santa Barbara. Professor Volkmann’s research group focuses on advancing computational, artificial intelligence, and data science approaches to bridge the gap between atomic and cellular scales, spanning over six orders of magnitude from Ångstroms to tens of microns. His lab has introduced groundbreaking algorithms in cryogenic electron microscopy and tomography, including automated correlation-based docking and the watershed transform, significantly enhancing structural analysis and interpretation."--Provided by publisher.

Summary:

"Cryo-Electron Tomography: A Journey from Sample Preparation to Data Mining providing a holistic overview of this rapidly advancing field and equipping researchers with the knowledge and tools necessary to initiate their own investigations. The book begins with a section on advanced cryogenic sample preparation for in situ cellular tomography, covering a range of sample types including viruses, bacteria, eukaryotic cells, and perspectives for organoids and tissues. Specific chapters focus on essential approaches such as vitrification, sample thinning, and bimodal correlative techniques. The book then transitions to a section on data mining and validation. Key Features: Offers a comprehensive overview of cryo-ET sample preparation and data mining protocols, consolidating approaches in a single resource. Includes foundational knowledge underpinning the field Explores the latest methods, techniques and recent developments in cryo-ET. Offers guidance to researchers for key procedures. Written by an international range of experts."--Provided by publisher.

Contents:

1. Characteristics of sample preparation for electron microscopy / Seth A. Villarreal

2. Cryo-FIB developments to enable nanoscale biopsies / Alexander Rigort and Andrew Leis

3. Preparing bacterial cells for cryo-electron tomography / Mohammed Kaplan, William J. Nicolas and Grant J. Jensen

4. Revealing the hidden world of microbes in situ: Sample preparation workflows for cryo-electron tomography / Samira Heydari and Jun Liu

5. Mastering the preparation of eukaryotic cell samples for cryo-ET: Tips and techniques / Cécile Sauvanet and Dorit Hanein

6. Interpretation of cellular tomograms / Niels Volkmann

7. Denoising in cryoET / Jose-Jesus Fernandez

8. Feature detection in cryo-electron tomography image analysis / Mostofa Rafid Uddin, Sajib Acharjee Dip, Rajat Aayush Jha, Xiangrui Zeng and Min Xu

9. Validation methods in cryo-electron tomography / F.P. de Isidro-Gómez, J.L. Vilas, E. Fernández-Giménez, O.L. Zarrabeitia, D. Marchán, M. Iceta, D. Herreros, P. Conesa, Y.C. Fonseca, J. Jiménez de la Morena, A. Cuervo, P. Losana, C.O.S. Sorzano and J.M. Carazo.

1 - Characteristics of sample preparation for electron microscopy

Introduction

Methods of Imaging by Electron Microscopy

Sample stability

Cryofixation

Workflow comparisons

Conclusions

References

2 - Cryo-FIB developments to enable nanoscale biopsies

The rise of cryo-FIB in structural biology

Micromachining cryo-lamellae

Technological advancements

Properties of plasma FIB beams

Assessing beam damage on cryo-lamellae

Toward more challenging samples

Another dimension - from 2D to 3D cell culture

Importance of correlative cryo-fluorescence microscopy

Bulk sample processing

Role of automation for in situ tomography

Streamlining cryo-lamella preparation

Outlook

Acknowledgments

3 - Preparing bacterial cells for cryo-electron tomography

Sample preparation

Bacterial growth

Imaging individual cells

Imaging cell-cell interactions

Other tips

Preparing grids for cryo-ET

Gold fiducials

Freezing samples

Thinning samples

Collecting data

Analysis and data storage (and significance of cryo-ET database)

Concluding remarks

4 - Revealing the hidden world of microbes in situ: Sample preparation workflows for cryo-electron tomography

An overview of the cryo-ET workflow

Sample preparation for direct cryo-ET imaging

Intact bacteria with thin diameter

Preparing bacterial culture

Collecting bacteria

Bacterial minicells

Bacteria-host interactions: Extracellular pathogens

Frozen-hydrated specimen preparation

Grid type and preparation.

Vitrification

Clipping

Sample preparation for cryo-FIB-ET imaging

Bacteria-host interactions: Intracellular pathogens

Grid preparation

Target localization

Cryo-FIB milling

Mapping

Milling

Summary and outlook

5 - Mastering the preparation of eukaryotic cell samples for cryo-ET: Tips and techniques

Summary

Preparation of cell-culture-amenable cryo-ET substrates

Grids

What size mesh, hole, and distribution?

Holey grids

Making grids cell culture compatible

Grids sterilization

Increasing efficient cell seeding

Seeding cells

Vitrification

Manual plunger versus automated (Vitrobot/EMGP)

Sample thickness

Prior to vitrification

Cryo-CLEM

Fiducials

Gold fiducial

Dual-purpose fluorescent fiducial

Cell integrity and sample quality assessment

Perspectives

Cell suspension

Grid functionalization

6 - Interpretation of cellular tomograms

The need for holistic approaches to tomogram interpretation

Tomogram enhancements

Membrane segmentation

Tracing of filamentous structures

Analysis of cellular architecture

Outlook and conclusion

7 - Denoising in cryoET

Basic concepts of filtering in the real and Fourier space

Handling noise during tomographic reconstruction

Denoising tomograms

Linear methods

Gaussian filter

Nonlinear methods

Median filter

Beltrami flow

Other nonlinear methods

Anisotropic methods

Bilateral filtering

Nonlocal means

Anisotropic nonlinear diffusion

Deep-learning approaches to denoising tomograms

Contrast improvement through CTF deconvolution

Software

8 - Feature detection in cryo-electron tomography image analysis

Introduction.

Feature enhancement

Denoising

Wavelet-based denoising methods

Neural-network-based denoising methods

Missing wedge compensation

MW compensation during reconstruction

MW compensation after reconstruction

Saliency detection

Feature object detection

Particle picking

Membrane-bounded particle detection

Ultrastructure detection

Feature semantic segmentation

Deep semantic segmentation of subtomograms

Feature representation learning and classification

Subtomogram classification and clustering

Feature representation learning and pattern mining

Feature structural recovery

Subtomogram alignment and averaging

Continuous conformational variability analysis

Discussion and conclusion

9 - Validation methods in cryo-electron tomography

The cryoET image processing workflow

Movie alignment

Validation methods

Tilt-series alignment

Contrast transfer function (CTF)

Tomogram reconstruction

Tomogram postprocessing

Proposed validation methods

Subtomogram averaging: Alignment and classification

Quality of the average map

Workflow validation strategies

Emerging topics in cryoET validation

Acknowledgment

Index.

Notes:

Includes bibliographical references and index.

Description based on online resource, publisher supplied metadata and other sources.

Other Format:

Print version:

ISBN:

9780443188305

0443188300

OCLC:

1521234160