Electron Microscopy of Biological Macromolecules

– An Introductory Course-

Performed from 16 April to 4 May 2012 at the

Department of Biophysical Chemistry – Rijksuniversiteit Groningen

 

Abstract

Transmission Electron Microscopes (TEM) are valuable tools for elucidating the structure of especially biological macromolecules. By making use of highly accelerated electrons and analysing their interactions with soft matter it is possible to significantly lower the diffraction limit as when compared to standard light microscopic techniques. This introductory course focused mainly on the theory behind TEM, operation in practice, and result analysis. Most important practical aspect which will be described below were finding suitable focus positions, obtaining high image qualities through accurate sample preparation, and practicing image analysis by particle overlaying.

Introduction

It is a central dogma in modern biology research that the form of biologically relevant macromolecules, such as proteins, is strongly dependent on its function. However, in order to make sense out of purely functional biochemical data it is necessary to link this data to structural biophysical information to fully grasp the structure-function relationship of a macromolecule. The observation of structures with sub-wavelengths dimensions is difficult in all microscopes, due to a diffraction limit of wave-like behaving particles like photons or electrons. This relationship was first postulated by Ernst Abbe in 1873. Form. 1 indicates how the observed spot size in an optical system depends on the wavelength λ, the diffractive index n of the medium, and the angle θ by which the particles travel through the medium. The term n sin θ is also called numerical aperture (NA) and can reach up to 1.4 in a modern microscope.

(Form. 1)

Being able to distinguish two spots from each other is called resolution and is thus mainly limited by the wavelength. For a typical light microscope the resolution limit therefore lies at around 200 nm assuming λ=550 nm and NA=1.4. Structures which are interesting for modern biological research typically have dimension at or very much below this value and can therefore not be observed by standard light microscopy.

However, by using electrons instead of photons the diffraction limit can be lowered dramatically because electrons can have a much smaller wavelength. Louis de Broglie in 1923 stated that the wavelength λ of any particle depends on its momentum p which consists out of the particles mass times its speed, where h is Planck’s constant (Form. 2).

(Form. 2)

Since the mass of an electron is constant, its wavelength therefore only depends on the acceleration speed. In modern high-voltage transmission electron microscopes (TEM) therefore resolutions of down to 0.5 Ångströms (A) (0.05 nm) have been achieved 1.

TEM microscopes are therefore a valuable tool in structural research. During this introductory course several different samples such as earthworm (Lumbricida) haemoglobin, T4 phages, and tomato mosaic virus (TMV) were examined in order to demonstrate the functionality of TEM and to gain knowledge about the operation of the microscope.

Material and Methods

An electron microscope consists out many parts which are functionally analogous to parts which are used in a light microscope. However, their construction is different and based on the electron particle properties. During the course a Philips CM12 TEM operated at 120 kV was used, which was connected to a CCD-camera. The electrons were emitted and accelerated in the top-part of the TEM from a filament (Wehnelt cylinder) by applying a high charge difference between the filament and a lower mounted anode. Electromagnetic lenses are made from iron-shielded copper coils and were used to condense, focus, and project the electron beam. A magnetic field, which is generated in the iron casing in response to the current running through the copper windings, leads to deflection and focusing of the electrons. Finally the beam is spread on the screen and can be observed through the oculars or, if the screen is lifted, through the CCD-camera system. Fig. 1 gives more details about the setup of a TEM, where (A) depicts a schematic overview of the most important components of the microscope and (B) is a photo of the outside of a Philips CM12 TEM. For clarity purposes the red dotted lines divide the TEM into three functional compartments (numbers on the right). Red arrows relate some of the functional features which are schematically presented in (A) to the real microscope in (B).

Fig. 1: Overview of a Philips CM12 TEM. (A) Electrons are extracted and accelerated from the shielded filament by the large voltage difference towards the anode. The gun alignment coils adjust the electron source (gun) with regard to the first condenser lens. Both condenser lenses concentrate the electron beam, while (fixed) apertures define the cone angle of the electrons during their way through the microscope. The objective lens focuses the beam onto the specimen, while the projector lenses magnify the transmitted electron beam on the fluorescent screen. Stigmators are applied to correct for small magnetic aberrations within the lenses which can lead to deflected beams. In addition deflector coils are used to centre the beam on the region of interest. (B) Photograph of a TEM. Some features are marked by arrows. Dotted lines relate the schematic compartments to the real ones in order to demonstrate proportions. Own creation, based on 2 and 3.

Different samples (earthworm (Lumbricida) haemoglobin, T4 phages, and tomato mosaic virus (TMV)) were obtained from the Department of Biophysical Chemsitry, Rijksuniversiteit Groningen, appropriately diluted, applied to self carbon coated and glow discharged copper 400 mesh grids, stained, and observed under the microscope. The carbon coating was obtained as follows: Rectangular mica slices were placed into a carbon evaporator which applied a thin layer of carbon to the surface. This carbon was then removed in a water bath by sliding away the mica slice from the carbon. Next, through lowering the water level, the carbon layer was applied to the copper grids which were placed on a glass slide and dried afterwards. Glow discharge was used to render the carbon layer hydrophilic in order to guarantee sufficient sample molecule adherence. Samples were diluted up to 100x in 50mM HEPES buffer and 3 µL were applied for approximately 30 seconds (haemoglobin) and 1 minute (T4 phage, TMV) on the carbon grids. Then, the grids were washed with 3 µL of buffer. Uranyl acetate was used for negative staining in one fast step for less than 5 seconds and one longer step of 30 seconds. Between sample application, washing, and staining steps excess liquid was blotted off with filter paper. The ready-to-use sample containing grids were stored in glass Petri dishes at room temperature. In order to avoid excessive damage of the sample, the low-dose system setting was used for imaging.

Recorded Lumbricida haemoglobin images were processed by 2D single-particle-analysis using the programme Groningen Image Processing (GRIP). Octopus vulgaris and Sepia officinalis hemocyanine molecules containing files were supplied by the department (recorded at 45,000x magnification with a Nikon Coolscan 8000 ED camera and scan steps of 20µm) and were also analysed.

Results

A major aim during the practical course was to learn how to operate a TEM and how to obtain good quality images of biological structures. Next to the optimal technical microscope alignments, finding the right amount of focus is a crucial step towards decent results. With the help of Fast Fourier Transformations (FFT) of the recorded TMV images (Fig. 2 (A)-(D)) it was possible to calculate whether the in-focus position chosen by the TEM operator really coincides with the physically real in-focus position. This was achieved by measuring the distance (d0) from the centre of the image derived FFT transform to the first contrast shift of this transform (first black ring, so called Thon ring). In total 11 different focal positions were chosen ranging from     -1700 nm defocus to +1700 nm overfocus (in 340 nm steps). Fig. 2 (A) and (D) display the two most diverging focus points, (B) is the defocus at which the most details are visible, and (C) is the in-focus position which was not part of the calculations. In the top left corner of every image the according FFT image is displayed. By dividing the square of d0 through the wavelength of the accelerated electrons (3.345×10-3 nm) the physically real focus position (Δf real) could be calculated.

By plotting the calculated optimal focus values Δf real against the obtained values (Δf microscope) a straight line resulted which had its y-axis intercept at approximately -137 nm (Fig. 3). This indicates that the chosen in-focus position from Fig. 2 (C) was in fact 137 nm defocused. However, in order to achieve optimal resolution with optimal contrast a certain amount of defocus, which depends on the periodicity of the sample molecule, is necessary. In order to be able to determine this value the correct starting in-focus position therefore is crucial.

Fig. 2: Results of a focal series with a TEM microscope imaging Tomato Mosaic Virus (TMV) including the Fast Fourier Transforms of the images in the top left corners. In total 11 images where taken in intervals of 340 nm. (A) displays the furthest defocus of -1700 nm, (B) has a defocus of -1020 nm and stripe patterns are clearly visible, (C) is in-focus (0 nm), and (D) is the furthest out of focus at +1700 nm. A 60K magnification was used to obtain the images.

From the results in Fig. 2 it also becomes clear how to identify the right amount of focus without making use of the calculations which led to Fig. 3. A large defocus leads to a very grainy image which has a lot of contrast, but a relatively poor resolution. Large over focus, in turn, leads to poor contrast, but good resolution. The in-focus position is marked by a strong drop of contrast. All focus positions are accompanied by typical FFT image patterns.

After having recorded high quality pictures, it was necessary to reduce the noise of the individual particles in order to be able to recognize details and separate similar but different particles from each other which might not be visible in the original dataset. Single-particle-analysis can be used to perform this task by averaging particles according to a previously defined reference consisting out of hand selected ideal particles. Averaged particles can then be split up into different classes which are defined by their bin size. During the analysis steps, the particles of the dataset are also centred and corrected for contrast differences and rotational position. For this course self-recorded worm haemoglobin (Fig. 4) and supplied Octopus vulgaris and Sepia officinalis haemocyanin molecules were analysed (Fig. 5). In the latter set it was possible to separate the molecules of the two species from each other.

 

Fig. 3: Plot of the calculated focus positions (Δf real) against the chosen focus positions (Δf microscope) resulting in a straight line which indicates that the chosen focus position was in fact approximately -137 nm off from the real focus position.

After the by-hand selection of a first reference set (not shown) the main Lumbricida haemoglobin containing files were aligned into nine different classes as shown in Fig. 4 (A). The classes are of varying qualities, but two molecular side- and  eight top-views are distinguishable. By further improving the reference set and decreasing the class size (i.e. more and individually improved classes) it became possible to generate more different top- and side-views of the molecule (Fig. 4 (B) and (C)). Based on these classes the highest quality top- and side-view was selected. Fig. 4 (D) portrays these particles in which previously blurred structural details become visible.

In addition single-particle-analysis can yield information which can lead to the separation of similar molecules and their spatial orientation from a mixture. As demonstrated by Fig. 5 the use of improving molecule references during particle analysis leads to a significant improvement of image quality. In Fig. 5 (A) the side-views are clearly distinguishable from the top-views, however, due to noise, it is not possible to separate the haemocyanin molecules of two species from this dataset. Improving the reference molecules more and more by aligning them to the cleaned dataset (reduction from 796 molecules to 754 molecules) yields strongly improved molecular details (Fig. 5 (B)).  After classification and selection of the original dataset against this improved reference set clearly distinguishable haemocyanin molecules become visible. It can be hypothesized that the two molecules are representatives of the two present species, respectively.

Fig. 4: Single-particle-analysis of Lumbricida haemoglobin. (A) Molecule classes based on a first hand-picked reference set. (B) and (C) Improved quality images by improving the reference and increasing the class size depicting top- and side-views of the molecules respectively. (D) Best quality views as selected form (B) and (C). 

 

Fig. 5: Single-particle analysis of Octopus vulgaris and Sepia officinalis haemocyanin molecules. (A) Non-analysed mix of haemocyanin molecules of both species. (B) Two distinguishable top-views and one side-view position arise from the dataset after single-particle analysis.

Discussion

During the course the focus lay on the practical application of TEM and the theory behind it in order to be able to record high-quality images and resolve simple problems which can be encountered during the procedures. Since information which is not recorded can not be seen later, it is crucial that all successive steps, starting with the sample preparation, are performed with great care and accuracy in order to yield good quality pictures which can then be analyzed. As demonstrated by Fig. 2 and Fig. 3 finding the right amount of under-focus depending on the molecular characteristics (patterns) is one of the most important steps. Balancing the needed contrast with an optimum resolution is the key to good results and requires practice. In addition by-hand selection of reference molecules from the dataset to which the whole dataset will be aligned is very important, since there is a strong selection bias. Experience with the observed molecules is an advantage because it enables the correct picking of all relevant rotations from the dataset and high accuracy classification afterwards.  An experienced electron microscopist is therefore able to extract a significant amount of structural and therefore potential functional information from a sample of biological macromolecules.

Literature

1.  Erni, R., Rossell, M. D., Kisielowski, C. & Dahmen, U. Atomic-Resolution Imaging with a Sub-50-pm Electron Probe. Phys. Rev. Lett. 102, 096101 (2009).

2.  University of Iowa Central Microscopy – Transmission Electron Microscopy. at <http://www.uiowa.edu/~cmrf/methodology/tem/tem_pg2.html&gt;

3.  Universität Regensburg Zentrum für Elektronenmikroskopie. at

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