THE HEBREW UNIVERSITY CENTER FOR NANOSCIENCE AND NANOTECHNOLOGY

Transmission Electron Microscope Tecnai T12 G2 Spirit (Cryo-TEM)


Overview of Technique

Transmission electron microscope Tecnai G2Spirit Twin T-12 allows full range of TEM analysis (imaging and diffraction) with the resolution of about 0.34 nm. The microscope is equipped with the superior high sensitive 4K FEI Eagle CCD camera which being combined with mild accelerating voltage enables TEM analysis of beam-sensitive materials like polymers, organic compounds, materials with low electron density. Also, the morphology and the crystallography of metals, ceramics, semiconductors, multi-layers, composite materials and biomaterials at the nanometer scale range could be obtained.

The T12 microscope also operates in Cryo-mode. When using the microscope in cryo-mode, specimens must be Vitrified. This means that specimens are frozen very rapidly and hence, water molecules do not form ice crystals but instead form an amorphous ice. This method has significant advantages which no other microscopy method enables; the sample is kept frozen, therefore there is no need to stain or fixate the sample. The sample remains in an aqueous solution which means that biological material such as proteins and membranes are observed in a close to natural stated and are not deformed or influenced by dehydration, staining and fixation. The same is true for the objects in aqueous dispersions and/or colloidal systems: aggregation and subsequent morphological changes could be avoided by preparation of vitrified sample and its analysis with Cryo-electron microscope.

The microscope is also equipped with the hardware for automated TEM tomography acquisition.

Basics and Tutorials

The basics of cryo-electron microscopy (Cryo-EM) are described below.

Cryo-Electron Microscopy

The cryo-EM method relates to technique that involves frozen-hydrated specimens, which must be kept around the liquid nitrogen temperature during their observation in electron microscope. This special condition is essential for biological samples involving morphological and immuno-cyto chemistry studies, but it also supports imaging of soft materials, polymers and even emulsions. The advantage of frozen-hydrated specimens is that they offer a tool to study ultrastructure without chemical fixatives, dehydration, or staining. The disadvantages are that the specimens are easily damaged by electron irradiation, and have low contrast. To solve those problems, our Tecnai G2 Spirit Twin T-12 has built-in "low dose" software to quickly access a sequence of preset conditions for searching, focusing, and recording the image. For single cryo-EM image an electron dose of 10-30e-/Å2is commonly used. For the highest resolution, the dose must be kept to the lower end of this range.

Sample Preparation for Cryo-EM

The water in frozen-hydrated specimens must be in "vitreous" (glass-like) form. If ice crystals are formed, the ultrastructure is disrupted. Fortunately, it was discovered that when an EM grid carrying a thin layer of an aqueous solution is plunged into a suitable cryogen, the water freezes rapidly enough to produce the vitreous state [1]. The maximum specimen thickness for the plunging technique is about 1 µm. The method is used to freeze small cells, organelles, and macromolecules. The cryogen of choice is liquid ethane, cooled by liquid nitrogen. The desired water layer thickness is achieved by blotting the grid after application of a drop of solution. Filter paper type, blotting time, and humidity of the air surrounding the grid, all affect the thickness of the frozen water layer. Many techniques have been designed to maintain specimen integrity during preparation, transfer and observation. For the vitrification process as such, the main parameters to control are humidity and temperature.
In our lab we use the commercially available Vitrobot Mark-IV (FEI Company) for reproducible vitrification of specimens.
[1] Dubochet, J. et al. Quart. Rev. Biophys. 1988, 21, 129.

The Vitrobot Mark IV an automated sample preparation system used to create rapidly frozen, ultra thin samples for high resolution TEM.
Cryo-EM web resources:
Electron Tomography

Automated acquisition of TEM tomography data is currently available at both our TEMs (F20 and T12) with FEI Xplore 3D software and Fischione ultra-thin tomography holder.

In electron microscopy tomography is a way to reconstruct a three-dimensional object from a series of two-dimensional images/projections which could be acquired in TEM, EFTEM and STEM modes.

The acquisition of the tilt series is normally carried out by sequential tilting of the specimen about a single axis, usually from one extreme of tilt to the other, acquiring projections either at equal angular increments. To achieve the best resolution, it is vital that the tilt is over as large a range as possible and that the number of images acquired within the series is maximized.
After the tomography data series is acquired it needs processing procedure allowing finally reconstruction of 3D structure of the tested object. The processing is performed through the obligatory alignment stage at which the individual images need to be shifted onto a common tilt axis, requiring spatial (x-y) and rotational (Ø) shift and the correction of scan or lens distortions. Accurate alignment is the key to high quality reconstructions; even very small misalignments between individual images can dramatically reduce the fidelity of the resultant reconstruction. The most widely used method for reconstruction is a real-space back-projection involving projecting each two-dimensional image into a three-dimensional reconstruction space back along the original tilt angle at which it was recorded. The superposition of these back-projected images yields a three-dimensional reconstructed object.
Tomographic reconstruction is based on the assumption that the images acquired are 'true projections of structure'; the intensity in the images must show, at least, a monotonic relationship with some function of the thickness or density of the structure. For amorphous materials, and (low atomic number) biological structures, mass-thickness contrast fulfills this 'projection requirement'. For crystalline materials possessing strong diffraction contrast STEM and EFTEM imaging could be most appropriate techniques satisfying "projection requirement". Although spatial resolution provided by the modern instruments is around a few angstroms, the overall three-dimensional resolution is dictated by the number of images (projections)
acquired in the series. As a rule of thumb, for a typical series (of ~100 images), the achieved resolution is about 1/100 of the overall object size and is therefore limited to about 1 nm^3.

For more details about the electron tomography for materials science, please, refer:

[1]. Nanoscale tomography in materials science. Günter Möbus and Beverley J. Inkson. Materials Today 2007, volume 10, 18-25.
[2] Electron tomography and holography in materials science. Paul A. Midgley and Rafal E. Dunin-Borkowski. Nature Materials 2009, volume 8, 271-280.
[3] Electron tomography. M. Weyland and P.A. Midgley. Materials Today 2004, volume 7, 32-40.

Equipment Specifications

The manufacturer is FEI Company.
W or LaB6 emitter W: I ~10A/cm2, probe size ~ 5.0 nm.
LaB6: I ~103A/cm2, probe size ~ 1.5 nm.
Point resolution 0.34 nm
Line resolution 0.2 nm
Information limit <0.20 nm
TEM magnification 22x -650kx
TWIN objective lens Maximum eucentric tilt - ±700
Selected area diffraction camera length 20-4200 mm
Maximum diffraction half-angle ±130
Cryo-TEM Sample analysis: Low Dose supported
Tomography Fully automated calibrations and image series acquisition. Reconstruction and rendering through Xplore 3D
Specimen stage Fully computer-controlled, eucentric side entry, high stability CompuStage
Specimen holders:
  • CompuStage single tilt specimen holder.
  • Philips multiple specimen holder.
  • CompuStage cryo holder (standard Gatan 60o / 70o).
  • Fischione single tilt tomography holder.
Recording systems:
  • Embedded CCD camera (FEI Eagle 4k, 16 Mega pixels).
  • Plate camera with 56 sheets of film.
Vacuum: Oil free vacuum system with turbo molecular pump, pre-pumping column, gun and specimen airlock, fully interlocked differentially pumped column