3D Reconstruction of magnetosomesby Cryo-Electron tomography

Course Manual

Max Planck Institute of Biochemistry (MPIB)Department of Molecular Structural Biology

Supervisors:Shoh Asano tel: 089/85782034 room: C2Zdravko Kochovski tel: 089/85782639 room: C134/135

Advanced Lab Course - SS 2012

Technical University of Munich

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Course location:Max-Planck-Institut of BiochemistryAm Klopferspitz 18D-82152 Martinsried

How to get there:Take Bus 266 (direction Planegg) from GroßhadernExit at Martinsried, Max-Planck-InstituteWait at the main entrance of the institute to be picked up

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Contents

1 Aim of Experiment 4

2 Background 52.1 Theoretical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.1.1 Light microscopy and the resolution limit of visible light . . . . . 52.1.2 Transmission electron microscopy - TEM . . . . . . . . . . . . . . 62.1.3 Electron tomography . . . . . . . . . . . . . . . . . . . . . . . . . 72.1.4 Biological Sample - Magnetosomes . . . . . . . . . . . . . . . . . 8

2.2 Practical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92.2.1 The electron microscope . . . . . . . . . . . . . . . . . . . . . . . 92.2.2 Tomographic acquisition . . . . . . . . . . . . . . . . . . . . . . . 122.2.3 Tomographic reconstruction . . . . . . . . . . . . . . . . . . . . . 132.2.4 Specimen preparation . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.3 Further reading/Literature . . . . . . . . . . . . . . . . . . . . . . . . . . 13

3 Proceeding of the experiment 15

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1 Aim of Experiment

To get an overview of cryo-electron tomography (Cryo-ET) as a method allowing three-dimensional visualization of frozen-hydrated, vitrified biological material at molecularresolution. The experimental procedure will step through the preparation of magneto-somes for cryo-electron microscopy by plunge freezing in liquid ethane, data acquisitionfor tomography and the subsequent computational reconstruction and interpretation ofthe tomographic data.

Figure 1: Cryo-ET workflow (modified from VanHecke et al. [1])

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2 Background

2.1 Theoretical

2.1.1 Light microscopy and the resolution limit of visible light

Light microscopy and its derivative fluorescent microscopy are commonly used for bio-physical measurements. One of the downsides is the lack of resolution. A simple cal-culation reveals that classical microscopy with visible light can only attain a resolutionof roughly 200 nm. If we take a look at the diffraction image of a single illuminationpoint through a small lens we will see the Airy pattern with the Airy disc located in themiddle (defined by the distance center to first zero (black) of the radial pattern). TheRayleigh criterion defines something ”resolvable” if the Airy maximum of one objectfalls at least in the first Airy minimum of the other (Figure 2). If we calculate thefirst zero of the diffraction image with the help of the first Bessel function we get thewell-known formula for the angular resolution. By converting it to spatial resolution,we can approximate the spatial resolution by:

R = 1.22λ

2NA≈ λ

2NA(1)

Numerical aperture (NA) is defined as NA = η sinα, where η is the refractive index ofmedium the lens is working in (ηwater:1.333, ηoil:1.515) and α is half the angle of cone ofmaximum possible light entering a lens. With violet (400nm) as the visible light withthe shortest wavelength we get roughly 200 nm, assuming NA = 1.

Figure 2: Airy patterns from a couple of illumination points. Rayleigh resolvabilitycriterion used (first zero Airy pattern = maximum second Airy pattern). (Modifiedimage from Nikon, http://www.microscopyu.com/)

Today, ”superresolution” techniques like STEM, STED, STORM, 4PI, etc. provide abetter resolution of up to tens of nanometers with visible light by circumventing Abbe’slaw (resolution=half wavelength). This can be done for example by modifying the

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excitation light and using the nonlinear nature of fluorophores. All mentioned light mi-croscopy techniques have in common that they can be used with live specimen. At thehighest end of resolution we have X-ray crystallography. With a wavelength of around1 Angstrom (thus roughly the same scale as chemical bonds) it is possible to detectdiffraction patterns of highly ordered crystals. Atomic resolution is routinely possibleto attain, but only with given coherent beam and a crystalline specimen. Furthermorethe need of a synchrotron in order to get coherent X-ray light source for high qualitymeasurements poses a limitation.Electron microscopy fills the ”resolution gap” between light microscopy and x-ray diffrac-tion. The resolution can be up to a few Angstroms, but in most biological applications itis around a few nanometers. The big advantage is that it is not necessary to crystallizeor purify (as in the case of NMR) the sample. Unlike light microscopy, observing livespecimen is generally not possible in electron microscopy (at least not at sufficientlyhigh resolution).

2.1.2 Transmission electron microscopy - TEM

This ”original” form of electron microscopy needs highly accelerated electrons emittedfrom a cathode to be able to penetrate and image thin samples successfully. In orderto record the sample properly, a slim, fully focused and monochromatic electron beamis needed. Shaping the beam and focusing is done by several (metal) apertures andelectro-magnetic coils (equivalent to the lenses in light microscopy). The transmittedbeam is then magnified and projected on a screen/detector. Contrast is being createdby two distinct mechanisms: Absorption and Phase-Contrast. The first can be alsonamed amplitude contrast, where thicker spots and areas with high atomic number Zabsorb all electrons and thus appear as black spots on the final image. This effect canbe described as an exponential decay of the incoming electron’s number (atomic massnumber A, mass density ρ, thickness t and the elastic scattering cross section σ of thesample):

n = n0e−NA

Aσρ t (2)

This formula is rather intuitive and is comparable to Beer’s law for absorption of lightin material. Phase contrast on the other hand is due to electron wave interference inthe image plane, where the phase of electrons is shifted during sample penetration.Assuming a weakly scattering sample, the scattered wave function Ψsc can be describedlike this:

Ψsc = Ψ0eiφ ≈ Ψ0(1 + iφ) (3)

Combining two more effects (contrast transfer function and aperture limitations), a goodapproximation of our final wave function is as follows:

Ψfinal = F−1(A(k)CTF (k)F (Ψsc)) (4)

where A(k) is the term introduced by aperture limitations, CTF (k) is the contrast trans-fer function and F/F−1 the Fourier transform and the inverse Fourier transform of a

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given function respectively. The CTF includes effects of changing focus (over/underfocus)as well as of spherical aberration. The final image is calculated as usual:

Imagefinal = Ψfinal ∗Ψ∗final (5)

2.1.3 Electron tomography

Electron tomography is performed by tilting an object in the EM and computationallymerging the two-dimensional projections obtained at the different tilt angles. The resultis a three-dimensional image (volume), called ”tomogram”. The resolution of such atomogram is non-isotropic. X/Y (in-plane) resolution is the same but the resolution indepth (Z) is highly dependent on tilting (range, amount and geometry) and is usuallymuch worse. The mathematical principle behind tomography goes back to JohannRadon in 1917. The ”Radon transform” describes the integral transform of a knownfunction over straight lines. The inverse function is needed in our case where we canapproximate an unknown 3D-volume via its 2D-projections (Radon transforms). Thismeans that we need to tilt the sample to the highest possible angle and with the lowestpossible angular increment to get the best possible sampling.

Figure 3: Diagram showing steps from object to projection to reconstruction. Bothelectron optics and projections are done in the microscope, while the reconstruction isdone in the computer. (Taken from VanHecke et al. [1])

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Additional tilting geometries like dual tilt (another tilt in perpendicular plane) or conicaltilts make the resolution more isotropic.In Fourier-space, we can visualize the tomographic principle by using the projection slicetheorem. It states that the 2D Fouriertransform (F2) of a 2D projection (P2) of a 3Dvolume (V) is exactly the same as a 2D slice (S2) out of the 3D Fouriertransform (F3)of the same 3D volume (V):

F2P2V = S2F3V (6)

As we can see, with each additional projection, we get another slice in the Fourierspaceand thus the approximation of the 3D volume is more exact. In the case of biologicalsamples, one has to think about radiation damage (e.g. forming small gas pockets) andthus a trade-off of having a lot of different projections and not damaging the sample isnecessary. Usually the dose is limited to about 120 electrons per square Angstrom in acomplete series.

2.1.4 Biological Sample - Magnetosomes

The samples we want to take a look at are so called ”magnetosomes”. They are definedas bacterial organelles which can be found in magnetotactic bacteria. Magnetic particles(magnetite crystals Fe3O4) connected in a chain ensure the magnetotaxis, the ability ofcombining to sense the surrounding magnetic field and coordinate movement in searchfor food.The most famous example is the bacterium Magnetospirillum, which was isolated frompond water in 1975 by Robert Blakemore. The bacterium lives in microaerophilic (lowoxygen) environment. Due to the presence of the magnetosomes, Magnetospirillum canalign itself according to the Earth’s magnetic field. This facilitates the movement alongthe oxygen gradient.

Figure 4: A magnetosome with magnetite crystals (red arrow) showing magnetization( c©Andre Scheffel)

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Figure 5: Different morphology of the magnetite chain ( c©Andre Scheffel)

The Magnetosomes are small enough in size to be recorded with electron microscopy.The characteristic magnetic particles can be easily identified due to the high atomicnumber Z of Fe, thus making it an attractive specimen for transmission electron mi-croscopy.

2.2 Practical

2.2.1 The electron microscope

The electron microscope which we will use in this course is a FEI Tecnai G2 Polara(Figure 6). It can be cooled down to liquid Nitrogen temperature and is equipped witha FEG for electron generation. The FEG is a [Schottky type] Field Emitter Gun, whichuses a sharp tungsten crystal tip from which electrons can be extracted by field emissionwith a high coherence in space (”brighter”) and time (smaller energy spread).

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Figure 6: FEI Tecnai G2 Polara - the microscope used during this practical course

Figure 7: Images of bacterium Magnetospirillum embedded in vitreous ice taken withFEI Tecnai G2 Polara at Martinsried. (a) low mag. image (ca. 4000x). (b) high mag.image (ca. 37000x) of the highlighted area from (a).

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Figure 8: A rough scheme of an electron microscope column. The beam travels fromtop to bottom. ( c©G. Schweikert, modified by S. Asano)

The illumination system consists of several electro-magnetic lenses and can be furtheradjusted by three apertures. The sample stage can be tilted up to 70 ◦ and is completelycomputer-controlled. Furthermore a GIF, Gatan Image Filter is installed, which can”filter out”/remove inelastically scattered electrons before they hit the detector. Amagnetic prism is used to separate slower electrons [with less energy] around a 90 ◦

angle and a slit ”selects” the desired energy range. The filtered electron beam thenhits the scintillator and converts the electron signal into light, which gets captured viaa separately cooled CCD. Up to six microscopy samples can be loaded via the MSR(Multi Specimen Rod) and inserted into the column with another rod, the InsertionRod (IR). A dewar on the right part of the column is used as a reservoir for liquidnitrogen to ensure cryo (< −160 ◦C) conditions within the microscope for long timeperiods (∼24h). The acquisition of the tomographic series is done by software suppliedwith the microscope and is done in an automated fashion.

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2.2.2 Tomographic acquisition

As mentioned earlier, it is necessary to work under extremely low electron exposure(”low-dose”) conditions. As we work in medium-high magnification, we have to be care-ful that the sample does not drift away and the intended defocus stays the same forall tilts. For that purpose the software automatically takes images before the sampleacquisition on a nearby spot along the tilt axis and correct for defocus changes. Further-more, by comparing that image to the image taken with the previous tilt, it is possibleto account for drifts and is used for tracking the original position [See in problems howthis is done mathematically.] In the software, the following steps are done in sequence:tilting, correcting for image shifts, correcting for defocus, acquisition. We are radiatingthe sample position only in the Acquisition step to keep the electron dose low. Thissequence is aptly named: ”Low-dose acquisition scheme” Figure 9 To get a higher con-trast, we use energy filtering and get rid of all inelastically scattered electrons. The2048 x 2048 pixel CCD then records the image and sends it to the attached workstationcomputer for storage.

Figure 9: Low-dose acquisition scheme

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2.2.3 Tomographic reconstruction

Once a set of projection is acquired, the single images need to be computationallymerged. Simple crosscorrelation of images is not sufficiently accurate to align the imagesproperly due to the low signal to noise ratio. Thus user input is needed to click on fiducialgold markers, which absorb electrons very well and thus will show as ”black dots” onthe micrographs. This has to be done with multiple markers for all images in orderto accurately describe image shifts or even rotations between single images. With asufficient set of markers the reconstruction can be then done via specialized software.(In this case via the TOM toolbox software from our department [2].)

2.2.4 Specimen preparation

There are different choices of how one prepares (and fix) samples for electron microscopy.Fixing samples with chemicals (e.g. Aldehyde) is the most common procedure. Instaining protocols, heavy metal solutions (e.g. Uranylacetat) are applied on proteins orother objects. The technique that preserves biological samples closest to their nativestate (which we use in this course) is cryo-fixation. A sample in native environment(e.g. buffer) is rapidly transferred to a very cold (∼ −160 ◦C) reservoir, where the waterimmediately freeze without forming the hexagonal lattice (plunge freezing: Figure 1). This rapid freezing process is called ”vitrification”. Depending on the dimensionsof the sample, sectioning is required to cut thicker samples into thin sections via avery sharp knife. Transmission electron microscopy needs specimen of 1µm or less inthickness, otherwise absorption and the higher fraction of inelastically and multiplescattered electrons will deteriorate the image quality.

2.3 Further reading/Literature

For a quick overview of Cryo-Electron Tomography:

D. Vanhecke, S. Asano, Z. Kochovski, R. Fernandez-Busnadiego, N. Schrod, W. Baumeis-ter, and V. Lucic. Cryo-electron tomography: methodology, developments and biologicalapplications. Journal of Microscopy, 242(3):221227, 2011.

AJ. Koster, R. Grimm, D. Typke, R. Hegerl, A. Stoschek, J. Walz, and W. Baumeis-ter. 1997. ”Perspectives of Molecular and Cellular Electron Tomography.” Journal ofStructural Biology 120 (3) (December): 276-308.doi:10.1006/jsbi.1997.3933.

For deeper theoretical insights on tomography in general and all aspects relating to it:Joachim Frank, ”Electron tomography”, Springer

A very broad review of ”magnetosomes”: Faivre and Schuler, ”Magnetotactic bacteria

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and magnetosomes”, Chemical Review Volume 108, Issue 11, pages 4875-4898, 2008

3 Proceeding of the experiment

• Introduction to the experiment and a guided tour through the department (1:30h)

• Collecting sample and observing it under the light microscope (1:30h)

• Freezing of sample in liquid ethane (1h)

• Lunch

• Electron microscopy, tomographic data acquisition (2h)

• Computational work and reconstruction (2h)

• Debriefing (0:30h)

References

[1] D. Vanhecke, S. Asano, Z. Kochovski, R. Fernandez-Busnadiego, N. Schrod,W. Baumeister, and V. Lucic. Cryo-electron tomography: methodology, develop-ments and biological applications. Journal of Microscopy, 242(3):221–227, 2011.

[2] S. Nickell, F. Forster, A Linaroudis, W. Del Net, F Beck, R Hegerl, W Baumeister,and J. M. Plitzko. Tom software toolbox: acquisition and analysis for electrontomography. Journal of Structural Biology, 149(3):227–234, 2005.

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