Automated vitrification of cryo-EM samples with controllable sample thickness using suction and real-time optical inspection

Research Square

Abstract

Abstract Speed and efficiency of data collection and image processing in cryo electron microscopy have increased over the last decade. However, cryo specimen preparation techniques have lagged behind and faster, more reproducible specimen preparation devices are needed. Here we present a new vitrification device with highly automated sample handling, requiring only limited user interaction. Moreover, the device allows inspection of thin films using light microscopy, since excess liquid is removed through suction by tubes, not blotting paper. In combination with dew-point control, this enables thin film preparation in a controlled and reproducible manner. The advantage is that quality of the prepared cryo specimen is characterized prior to electron microscopy data acquisition. Practicality and performance of the device are illustrated by experimental results obtained by vitrification of protein suspensions, lipid vesicles, bacterial and human cells, followed by imaged using single particle analysis, cryo electron tomography and cryo correlated light and electron microscopy.

Authors 9

1. Roman I. Koning corresponding Aachen Electron Microscopy

   Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Center

   Electron Microscopy, Cell and Chemical Biology, Leiden University Medical Center, P.O.Box 9600, 2300 RC Leiden The Netherlands

2. Hildo Vader
   Affiliation as printed

   Linkam Scientific Instruments Ltd

   Linkam Scientific Instruments Ltd, Tadworth, Surrey, KT20 5LR, United Kingdom

3. Martijn van Nugteren
   Affiliation as printed

   Linkam Scientific Instruments Ltd

   Linkam Scientific Instruments Ltd, Tadworth, Surrey, KT20 5LR, United Kingdom

4. Peter A. Grocutt
   Affiliation as printed

   Linkam Scientific Instruments Ltd

   Linkam Scientific Instruments Ltd, Tadworth, Surrey, KT20 5LR, United Kingdom

5. Wen Jing Yang Aachen NeCEN Institute of Biology Leiden

   Leiden University

   Affiliation as printed

   Leiden University

   NeCEN, Institute of Biology Leiden, Leiden University, Gorlaeus Building, Einsteinweg 55, 2333 CC Leiden, The Netherlands

6. Ludovic L. R. Renault Aachen NeCEN Institute of Biology Leiden

   Leiden University

   Affiliation as printed

   Leiden University

   NeCEN, Institute of Biology Leiden, Leiden University, Gorlaeus Building, Einsteinweg 55, 2333 CC Leiden, The Netherlands

7. Abraham J. Koster Aachen Electron Microscopy

   Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Center

   Electron Microscopy, Cell and Chemical Biology, Leiden University Medical Center, P.O.Box 9600, 2300 RC Leiden The Netherlands

8. A. C. F. Kamp
   Affiliation as printed

   Linkam Scientific Instruments Ltd

   Linkam Scientific Instruments Ltd, Tadworth, Surrey, KT20 5LR, United Kingdom

9. Michael Schwertner
   Affiliation as printed

   Linkam Scientific Instruments Ltd

   Linkam Scientific Instruments Ltd, Tadworth, Surrey, KT20 5LR, United Kingdom