Obtaining atomically resolved structural information
on individual bio molecules using electron holography
The context of the SIBMAR project was located in the general area of structural biology. However, in contrast to the current state of the art,
SIBMAR explored the conditions for imaging just one single molecule instead of averaging over many molecules as it is common and needed
while unavoidable in all other structural biology tools known today, be it X-ray diffraction, conventional high energy electron microscopy or
NMR (Nuclear Magnetic Resonance). To be able to drop the need for averaging over millions of molecules, entirely new methods are needed.
Pre-conditions for such endeavors are to be able to fix a single molecule in space and to present this molecule to a coherent beam of low
energy electrons for obtaining an electron hologram of just one single entity without destroying it during the imaging process.
Top: Schematic of the Low Energy Electron Holography setup indicating graphene as a possible sample support for presenting a single protein
to the electron wave front
Bottom le: Low energy electron transmission through graphene flakes supported by an 8 micron square copper grid
Bottom right: Low energy electron hologram of freestanding DNA
© European Union, 2011. This document should not be considered as representative of the Commission’s official position.
The approach of SIBMAR was to either fix a molecule to a free-standing graphene sheet or to attach it to a template DNA molecule,
stretched over a micro-machined hole in a thin film. Either way, a coherent electron wave subsequently interacts with this single
molecule in order to generate an electron hologram of the structure. The shape and structure of the molecule can then be reconstructed
by employing newly developed numerical routines for hologram reconstruction.
Example of the numerical scheme for reconstructing electron holograms
Achievements
• The main achievements in the course of the SIBMAR project were
- The development of freeze-drying methods to stretch single DNA molecules over tiny holes milled by a focused ion beam into
a thin support film.
- The development and characterization of free-standing graphene sheets as sample support. Graphene, the first two-dimensional
crystal, discovered by the Manchester group, seems an ideal sample support for imaging individual proteins. Various technologies
for preparing free-standing graphene films have successfully been developed and tested. The unique possibility of the SLEEM
(Scanning Low Energy Electron Microscope) invented by the Brno group allowed imaging with low energy electrons combined
with high spatial resolution in the nanometer-range.
- The development of a variety of novel routines for hologram reconstruction, including the solution of the long standing twin
image problem in holography which could finally be solved in the course of the project.
- Careful studies have been designed to examine the amount of radiation damage present in imaging with low energy electrons.
It turns out that the permissible amount of electron dose at energies around 100eV is at least 5 orders of magnitude higher
in comparison to high energy electrons or X-rays. In fact, at sub-nanometer resolution, no sign for decomposition of DNA molecules could be detected despite a total accumulated dose of about 108 electrons/nm2. This remarkably high dose is to be
compared to the maximal permissible dose in high energy cryo-electron microscopy on biological objects which amounts to
1000 electrons nm2. Since in X-ray scattering processes, the number of inelastic scattering events is even greater and thus damage even more severe than with high energy electrons, we end up with the following statement: The resistance of freestanding
biological molecules to low energy electron radiation, as observed in our LEEPS microscope, suggests that coherent low energy
electrons are the best choice of radiation to carry out high resolution microscopy of fragile biomolecules.
Contract
SIBMAR
Coordinator
Universität Zürich (UNIZH), Switzerland
Partners
ISI ASCR - Ustav Pistrojove Techniky, Akademie vd Eske Republiky Verejna Vyzkumna Instituce, Czech Republic
UNIMAN - University of Manchester, Great Britain
EMBL - European Molecular Biology Laboratory, United Kingdom
EC-contribution
1.309.787,93 €
Full partner and project information available on http://cordis.europa.eu/fp6/projects.htm
The coordinator provided text and pictures for the factsheet and his copyright is acknowledged
http://ec.europa.eu/research