EMC Abstracts

Official abstracts site for the European Microscopy Congress

MENU 
  • Home
  • Meetings Archive
    • The 16th European Microscopy Congress 2016
  • Keyword Index
  • Your Favorites
    • Favorites
    • Login
    • Register
    • View and Print All Favorites
    • Clear all your favorites
  • Advanced Search

A versatile high-vacuum cryo transfer system for cryo microscopy and analytics

Abstract number: 6577

Session Code: IM03-251

DOI: 10.1002/9783527808465.EMC2016.6577

Meeting: The 16th European Microscopy Congress 2016

Session: Instrumentation and Methods

Topic: New Instrumentation

Presentation Form: Poster

Corresponding Email: sebastian.tacke@scopem.ethz.ch

Sebastian Tacke (1, 2), Vladislav Krzyzanek (3), Harald Nüsse (1), Alexander Rosenthal (4), Jürgen Klingauf (1), Roger Albert Wepf (5), Rudolf Reichelt (1)

1. Institute of Medical Physics and Biophysics, University of Münster, Münster, Allemagne 2. Scientific Center for Optical and Electron Microscopy, ETH Zürich, Zürich, Suisse 3. Institute of Scientific Instruments of the CAS, The Czech Academy of Sciences, Brno, République tchèque 4. Microscopy Improvements e.U., Eisenstadt, Autriche 5. Centre for Microscopy & Microanalysis, University of Queensland, Brisbane, Autriche

Keywords: cryo-EM, High-Vacuum cryo Transfer

The conservation of the native state during sample preparation is mandatory for a correct interpretation of any micrograph. Particularly for EM, the preservation of the pristine architecture is challenging. If not imaged in situ (1), two different strategies can be followed to prepare hydrated samples for electron microscopy: the conventional and the cryogenic routine. Conventional preparation protocols typically rely on the chemical fixation and staining of the sample material, whereby both steps are known to induce artefacts (2). The second preparation routine, introduced by Moor (3) and Dubochet (4), circumvents chemical artefacts. The basic principle of every cryogenic (cryo) preparation protocol is the physical immobilization of the sample in a frozen-hydrated solid state, by vitrifying the sample within milliseconds. Due to the excellent structural preservation cryo imaging techniques have gained increasing popularity (5, 6).

However, the handling of a vitrified sample becomes rather delicate. Two prerequisites must be fulfilled during the entire handling in order to maintain the artefact free conservation of the specimen: 1) the sample must be kept well below the de-vitrification temperature of water (approximately -137°C) in order to avoid structural rearrangements due to ice crystal growth (5). As a consequence of the permanent cooling, the sample material acts as a cold trap and is therefore prone to contamination. 2) Thus, the sample must be transferred within an anhydrous environment all the time. In the case of the latest preparation protocols or imaging strategies this has been proven to be particularly challenging, since these methods include several transfer steps, either due to their extensive post-processing or complex workflow (7).

In the past, several cryo-transfer concepts were introduced mainly for cryo SEM, with bulk add-ons and cryo-stages inside the microscope to allow e.g. high-vacuum cryo-transfer (8), which is not feasible for “in-lens” systems like S/TEM’s. Standard cryo-transfer systems for cryo-TEM, however, work under ambient pressure from liquid nitrogen direct into the load lock of cryo-TEM.

Here, we present a high-vacuum cryo-transfer system that overcomes the limitations of existing systems. The system includes four parts: 1) sample cartridge (Fig 1a), 2) storage device (Fig 1b), 3) high-vacuum cryo shuttle (Fig 1c) and 4) a side-entry TEM cryo-stage. Moreover, our solution offers connectivity between different kind of instruments not limited to post-lens systems (8), enabling new types of multimodal imaging approaches by transferring cryo-samples between different imaging and manipulation devices (Fig 1d). The performance of the developed system is demonstrated on an “in-lens” cryo-STEM. In order to determine the quality of the transfer process, the temperature and pressure level were recorded during the entire transfer. Moreover, prior and subsequent to the cryo-transfer, the mass of the TMV as well as the thickness of a carbon film were measured and compared. Here, any possible contamination would falsify the scattering characteristics of the sample material, and consequently cause an apparent increase in mass or thickness, see also (9).

REFERENCES

(1) M. J. Dukes et al, Microsc. Microanal. 20 (2014) p.338.

(2) M. Pilhofer et al, Environ. Microbiol.16 (2014) p.417.

(3) H. Moor, and K. Mühlethaler, J. Cell Biol. 17 (1963) p.609.

(4) J. Dubochet, and A.W. McDowall, J. Microsc. 124 (1983) p.3.

(5) A. Al-Amoudi et al, EMBO J., 23 (2004) p. 3583.

(6) W. Kühlbrandt, Science 6178 (2014) p. 1443.

(7) A. Rigort and J. M. Plitzko, Arch. Biochem. Biophys. 581 (2015), p. 122.

(8) M. Ritter et al, Microsc. Microanal. 5 Suppl. 2 (1999) p. 424.

(9) S.Tacke et al, Biophys. J. 110 (2016), p. 758.

(10) Rudolf Reichelt initiated this project. Unfortunately, he passed away on 2nd October 2010, too early to see the final results. This research was supported by the DFG Grant RE 782/11. V. Krzyzanek acknowledges the support by the grant 14-20012S (GACR).

Figures:

Figure 1 a) Sample cartridge. b) Storage device. c) High-vacuum cryo transfer. d) Proposed working routine.

To cite this abstract:

Sebastian Tacke, Vladislav Krzyzanek, Harald Nüsse, Alexander Rosenthal, Jürgen Klingauf, Roger Albert Wepf, Rudolf Reichelt; A versatile high-vacuum cryo transfer system for cryo microscopy and analytics. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/a-versatile-high-vacuum-cryo-transfer-system-for-cryo-microscopy-and-analytics/. Accessed: January 26, 2021
  • Tweet
  • Email
  • Print
Save to PDF

« Back to The 16th European Microscopy Congress 2016

EMC Abstracts - https://emc-proceedings.com/abstract/a-versatile-high-vacuum-cryo-transfer-system-for-cryo-microscopy-and-analytics/

Most Viewed Abstracts

  • mScarlet, a novel high quantum yield (71%) monomeric red fluorescent protein with enhanced properties for FRET- and super resolution microscopy
  • 3D structure and chemical composition reconstructed simultaneously from HAADF-STEM images and EDS-STEM maps
  • Pixelated STEM detectors: opportunities and challenges
  • Atomic relaxation in ultrathin fcc metal nanowires
  • Layer specific optical band gap measurement at nanoscale in MoS2 and ReS2 van der Waals compounds by high resolution electron energy loss spectroscopy

Your Favorites

You can save and print a list of your favorite abstracts by clicking the “Favorite” button at the bottom of any abstract. View your favorites »

Visit Our Partner Sites

The 16th European Microscopy Congress

The official web site of the 16th European Microscopy Congress.

European Microscopy Society

European Microscopy Society logoThe European Microscopy Society (EMS) is committed to promoting the use and the quality of advanced microscopy in all its aspects in Europe.

International Federation of Societies for Microscopy

International Federation of Societies for Microscopy logoThe IFSM aims to contribute to the advancement of microscopy in all its aspects.

Société Française des Microscopies

Société Française des MicroscopiesThe Sfµ is a multidisciplinary society which aims to improve and spread the knowledge about Microscopy.

Connect with us

Imaging & Microscopy
Official Media Partner of the European Microscopy Society.

  • Help & Support
  • About Us
  • Cookies & Privacy
  • Wiley Job Network
  • Terms & Conditions
  • Advertisers & Agents
Copyright © 2021 John Wiley & Sons, Inc. All Rights Reserved.
Wiley
loading Cancel
Post was not sent - check your email addresses!
Email check failed, please try again
Sorry, your blog cannot share posts by email.
This site uses cookies: Find out more.