Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

Cryo-electron Microscopy01:28

Cryo-electron Microscopy

3.2K
Conventional electron microscopy (EM) involves dehydration, fixation, and staining of biological samples, which distorts the native state of biological molecules and results in several artifacts. Also, the high-energy electron beam damages the sample and makes it difficult to obtain high-resolution images. These issues can be addressed using cryo-EM, which uses frozen samples and gentler electron beams. The technique was developed by Jacques Dubochet, Joachim Frank, and Richard Henderson, for...
3.2K
Confocal Fluorescence Microscopy01:16

Confocal Fluorescence Microscopy

13.1K
Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
13.1K

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Ultrathin liquid cells for microsecond time-resolved cryo-EM.

Nature communications·2026
Same author

Microsecond Time-Resolved Cryo-EM Based on Jet Vitrification.

bioRxiv : the preprint server for biology·2025
Same author

Overcoming Preferred Orientation in Cryo-EM With Ultrasonic Excitation During Vitrification.

bioRxiv : the preprint server for biology·2025
Same author

Laser flash melting cryo-EM samples to overcome preferred orientation.

Nature methods·2025
Same author

Ultrathin Liquid Cells for Microsecond Time-Resolved Cryo-EM.

bioRxiv : the preprint server for biology·2025
Same author

Mitochondrial dysfunction drives a neuronal exhaustion phenotype in methylmalonic aciduria.

Communications biology·2025

Related Experiment Video

Updated: Jun 6, 2025

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam
10:54

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Published on: July 26, 2014

26.6K

Laser Flash Melting Cryo-EM Samples to Overcome Preferred Orientation.

Monique S Straub1, Oliver F Harder1, Nathan J Mowry1

  • 1Ecole Polytechnique Fédérale de Lausanne (EPFL), Laboratory of Molecular Nanodynamics, CH-1015 Lausanne, Switzerland.

Biorxiv : the Preprint Server for Biology
|November 28, 2024
PubMed
Summary

Sample preparation for cryo-electron microscopy (cryo-EM) is challenging due to protein orientation issues. A new laser flash melting and revitrification method effectively scrambles protein orientation, improving cryo-EM data quality.

More Related Videos

Nanoscale Characterization of Liquid-Solid Interfaces by Coupling Cryo-Focused Ion Beam Milling with Scanning Electron Microscopy and Spectroscopy
11:03

Nanoscale Characterization of Liquid-Solid Interfaces by Coupling Cryo-Focused Ion Beam Milling with Scanning Electron Microscopy and Spectroscopy

Published on: July 14, 2022

3.4K
Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells
11:55

Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells

Published on: May 28, 2021

4.1K

Related Experiment Videos

Last Updated: Jun 6, 2025

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam
10:54

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Published on: July 26, 2014

26.6K
Nanoscale Characterization of Liquid-Solid Interfaces by Coupling Cryo-Focused Ion Beam Milling with Scanning Electron Microscopy and Spectroscopy
11:03

Nanoscale Characterization of Liquid-Solid Interfaces by Coupling Cryo-Focused Ion Beam Milling with Scanning Electron Microscopy and Spectroscopy

Published on: July 14, 2022

3.4K
Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells
11:55

Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells

Published on: May 28, 2021

4.1K

Area of Science:

  • Structural Biology
  • Biophysics
  • Biochemistry

Background:

  • Protein adsorption to the air-water interface during sample preparation is a major challenge in cryo-electron microscopy (cryo-EM).
  • This preferred orientation limits the ability to obtain high-resolution structures and can lead to project failure.
  • Previous work showed laser flash melting and revitrification reduced preferred orientation for large, symmetric particles.

Purpose of the Study:

  • To demonstrate the broad applicability of laser flash melting and revitrification for scrambling protein orientation across various sizes and symmetries.
  • To investigate methods for enhancing the orientation scrambling effect.
  • To elucidate the underlying mechanism of orientation scrambling.

Main Methods:

  • Application of laser flash melting and subsequent revitrification to cryo-EM samples containing proteins of diverse characteristics.
  • Systematic variation of laser heating rates during flash melting.
  • Deposition of amorphous ice onto samples prior to revitrification.
  • Analysis of resulting cryo-EM data to assess particle orientation distribution.

Main Results:

  • The laser flash melting and revitrification method successfully scrambled the orientation of proteins across a range of sizes and symmetries.
  • Increasing the heating rate during flash melting enhanced the scrambling effect for certain proteins.
  • Pre-deposition of amorphous ice also improved orientation scrambling for some samples.
  • These modifications provide insights into the mechanism of orientation scrambling.

Conclusions:

  • Laser flash melting and revitrification is a versatile tool for overcoming preferred orientation in cryo-EM sample preparation.
  • Adjustable parameters like heating rate and amorphous ice deposition offer further optimization.
  • These techniques can be readily integrated into existing cryo-EM workflows to improve data quality and success rates.