Introduction of electron microscopy

Electron microscope (EM), if not specified, usually refers to transmission electron microscope (TEM). There is another type of electron microscope: scanning electron microscope (SEM). As SEM works in a totally different mechanism, it is more like scanning probe microscope SPM than EM. Traditionally, SEM should be referred as SEM, not as EM. This lecture deals with TEM only, SEM will be discussed later. Electron microscopy is a microscopic method of 80 years old. It is still the corner stone of ultra-high resolution microscopy method. Even the newest super resolution microscopy method use electron microcopy to compare or verify its resolution obtained: i.e. EM is the golden standard of ultra-high resolution in nanometer, sub-nanometer and pico-meter range. It is still the most reliable way to approach atomic resolution. One can reach 1-2 nm resolution effortlessly on routine TEM, and resolution with a little bit tweak.

Brief History of Electron microscope 1931: Ernst Ruska: a student at the Technical College in Berlin, together with Max Knoll, built-up a prototype of TEM, capable of 400 x magnification. 1933: Ruskas improved TEM exceeded LM resolution. 1939: first commercial TEM was built by Siemens, resolution 10 nm. 1944: resolution of TEM reached 2 nm 1986: Ruska received Nobel Prize in Physics for his contribution to EM.
Fig. 7- 1

Principle of electron microscope

1897, Joseph John Thomson discovered electron while studying cathode ray. Electron mass: m = 9 x kg, of proton mass; Its calculated classical radius: 2.8 x nm, very small. (Just as a comparison: now the highest resolution of EM: 50 pm: 5 x Electron as wave In 1924, Louis Victor de Broglie proposed a wave is connected to electron

nm)

= ;

P (momentum) =

so: =

h is Plancks constant: 6,626 x This wave is therefor called de Broglie wave, or matter wave. Matter wave exists universally in any matter. Matter wave is easy to be confirmed in elementary particles like proton and neutron, and even in large molecules. (Predicted Matter wave for human body = nm, too short to be detected.

Voltage and electron wavelength

Wavelength of electron in electron microscope depends on accelerate voltage applied

(nm) V is the acceleration voltage used.

nm; or roughly: =

nm

is inversely related to the accelerate voltage, higher V, shorter . Table 7-1 Wavelength of electron under different accelerate voltage Acc V (KV) Non-relative (pm) *Relative 10 12.3 50 5.5 60 5 100 3.86 3.70 200 2.73 2.51 300 2.25 1.97 1000 1.22 0.87

* Relative wavelength: when speed close to light speed, the relative effects have to be considered.

Electron wavelength and EM resolution Resolution of light microscope d = 0.61 : (when =550 nm, NA=1.4, d=240 nm). Objective lens aperture angle 67 -72 (maximum) Currently, with a carefully designed, manufactured light microscope, all aberrations are corrected, the resolution is only limited by diffraction of light: diffraction limited resolution. Look at 1-2 pm electron wavelength at 300 KV to 1000 KV, the resolution should reach 0.5 pm if we follow calculation for light microscope. But EM resolution even with big efforts now reaches only 50 pm, there are still 1000 times less than the limit. (Resolution of high-end TEM is in the range of 100-200 pm, only extra high resolution EM can reach 50 pm, resolution of TEM for most biological sectioned material is about 2 nm) Diffraction is not yet the limiting factor of EM resolution. EM has not yet reached diffraction limited resolution. EM resolution can be estimated as

; or by

formal formula: dmin = 0.43

; (a is half aperture angle in radiance, v is accelerate voltage, Cs is coefficient of spherical aberration) 2

Structure of TEM
Basic structures: Unlike light microscope we learnt before, which consists of several optical lenses made of glass and movable mechanism to adjust height of lens for focusing, electron microscope is a stiff metal column consists several heavy blocks of metal. The metal blocks are holder for coils around soft iron core: it is called electromagnetic lens, no moving mechanism for focusing, focusing and defocusing are all performed by current flowing in the coil. Electromagnetic lens can converge electron beam like convex glass lens converge light, but it cannot diverge electron beam. There is no concave lens equivalent of electromagnetic lens. Fig. 7- 2 Electromagnetic lenses are always working in pair to guarantee electron beam is shifted in parallel rather than tilt away. Electron magnetic lens are notorious bad lens, full of aberrations, but not easy to correct. That is the reason why resolution of electron microscope is far from reaching diffraction limit. Main parts of electron microscope Illumination system Image formation system Image recording system Electron source for illumination (Electron emitter): Thermionic electron gun with tungsten hair pin filament: Most common in routine EM for biological, medical sciences, cheap LaB6 crystal emitter: More brightness, commonly used in EM for material science, cost more Field emission gun (FEG): Used in high resolution EM, most expensive The main differences among three electron emitter are in four aspects: 1) Electron beam energy spread (Delta E): The smaller, the better: Electron emitted has better coherence 2) Probe size: resolution of electron microscope is related to probe size: smaller is better. 3) Brightness: high resolution, high magnification and thick specimen require more brightness 4) Life time: money-wise and efficiency-wise, uninterrupted running time. 5) Price: high purchase and maintenance price for FEG

Fig. 7- 3

Table 7-2 comparison of EM electron emitter

Emitter W-filament LaB6 FEG

E 1-3 eV 1-2 eV < 1 eV

Probe size 30-100 m 5-50 m < 5 nm

Brightness A / cm2 105 10 10

6 9

Life time hour 50-150 200-1000 > 1000

price < 100 1000 ---

Electron Gun: Electron emitter (tungsten filament, LaB6, or FEG) Cathode and anode Accelerate voltage 40-120 KV: conventional voltage EM 200-400 KV: high voltage EM > 1000 KV: ultra-high voltage EM (Highest voltage 3200 KV)

Condenser
Double condenser system First condenser C1: converge beam from gun control spot size: smaller spot size, better image quality Second condenser C2: Control illumination field on specimen Brightness control Condenser aperture: (on second condenser) 3-4 size: diameter 100-400 m, selectable Determine illumination area Cut off unnecessary illumination Reduce beam irradiation Reduce specimen damage
Fig. 7- 5 Fig. 7- 4

Objective lens and image formation in EM

Objective lens is the most important part in EM. In TEM, specimen very close to objective lens to guarantee a Large collecting angle and produce a reasonable Magnification at first image plane. The magnification from objective is not high, it is usually < 50x. Objective lens has a rather large depth of field, up to 1 m at 10 000 x magnification. This means, all specimen details, up and down through the whole thickness of the specimen, are all in sharp focus as long as it is properly focused (as the specimen thickness is usually < 100 nm, maximum 200 nm). Good or bad? Good point is: it is easier for user to operate microscope.
Fig. 7- 6

Bad point is: the z-overlapping is inevitable; all structures in the real 3D dimension (though only 100 nm range) are added together as a 2D image. Users must use other strategies to get 3D view if it is desirable. Objective lens is vulnerable to spherical aberration and chromatic aberration; correct these aberrations is very difficult, if not impossible (discussed later in detail) The objective aperture is rather small in EM objective lens. There are 3-4 exchangeable aperture at size of 20, 40, 60, 100 m diameters. In light microscope, NA is the resolution limiting factor: d = 0.61 : (when =550 nm, NA=1.4, d=240 nm). The objective lens aperture angle: 67 -72 at this resolution limit. Since electron wave length is much smaller than EM resolution, the diffraction angle caused by specimen details is smaller than aperture angle determined by objective lens aperture size. Unlike in light microscope, NA is the ultimate factor for resolution, the EM objective lens aperture size has no influence on resolution; its main function is to limit highly scattered stray electrons to increase contrast. EM objective lens aperture angle is very small: 0.030-0.050 rad (1.71 -2.86) But diffraction angle caused by specimen detail under electron wave is even smaller When resolution of TEM is in the range of 100-200 pm (0.1-0.2 nm): For 100 kV: = 3.9 pm, if the details are at 200 pm level Diffraction angle = arcsin = 0.0195 rad Diffraction is not yet the limiting factor of EM resolution. EM resolution is still 100 times worse than diffraction limited level (EM resolution formula dmin = 0.43 no aperture angle included). Like image formation in light microscope, the structural details in the specimen diffract electron ray to different extent, depending on size of the details and wavelength of the electron. As long as the diffraction angle is still within the collecting angle of the objective lens, it will be resolved. The resolved details are separated to different parts on the back focal plane of the objective lens. Then, depends on imaging mode, it goes in two ways: Image mode: the electron ray is projected to the first image plane, interference constructively or destructively to produce an image with different electron density. The first intermediate lens is focused on the first image plane and it passes rays through two to three stages of intermediate lenses and one projector lens, final image is formed on the viewing screen. The final magnification achievable is up to 1 million times. Diffraction mode: the first intermediate lens (also called diffraction lens in this case), is focused on back focal plane of objective lens instead of first image plane as in image mode. The lower intermediate lenses do not participate in the image formation. The diffraction pattern formed on BFP is mapped on the viewing screen via projector lens, interference to form diffraction image.

Fig. 7- 7

After projector lens, the signal is still electron wave with different density. Human eyes are totally blind to them, although photographic film camera can record them directly (CCD camera need a scintillator to covert electron to photon, a new type of Direct Electron digital camera using CMOS as sensor can capture electron without scintillator) To enable user view the image before taking image, the image-carrying electron beam from projector lens is projected onto a special image display device: phosphor viewing screen, where the electron is converted into photon by the phosphor coating on the screen. The phosphor coating thickness and grain size affect image quality and brightness, but only on the image viewing screen. Fine grain has high resolution but very dim brightness. There is no need for high resolution on the viewing screen, as final image is not taken from there, the brightness is more important for human eyes to find region of interesting. The grain size is in range of a few m.

Limiting factors of TEM resolution

As electron wavelength predicts much better than EM actually achieved EM objective aperture is not a limiting factor for EM resolution Diffraction is not yet a limiting factor of EM resolution What are the main factors which restrict EM resolution substantially? Aberration is ultimate limiting factor for TEM resolution.

Aberrations in TEM
Chromatic aberration: (Cc: coefficient of chromatic aberration) Cannot be corrected completely, but be minimized by using: 1) more coherent electron beam 2) smaller spot size 3) electron source with smaller energy spread Currently, chromatic aberration is not a big problem, as spherical aberration is a greater obstacle for achieving high resolution in TEM. Spherical aberration ( coefficient of spherical aberration) It is the biggest limiting factor for TEM resolution, and very difficult to control. Resolution of TEM is determined by wavelength and spherical aberration: As the TEM resolution formula shows: dmin = 0.43 ; From built-up of the first TEM 1933, it took 62 years for the first practical spherical aberration corrector Cs being developed in 1995. Another eight years for the first commercial Cs corrected TEM: in 2003 by JEOL Now Cs corrected TEM reaches 50 pm resolution.

Fig. 7- 8 Cs corrected TEM: Jeol JEM 2100

Other practical limiting factors of TEM resolution (Can be controlled if doing properly) 1) Astigmatism: Exists in both condenser and objective lens, intermediate lens Correction: Stigmator built-in under every lens group. Condenser stigmator Objective lens stigmator Projector lens stigmator It should be corrected by user regularly

Fig. 7- 9 astigmatism

Objective lens astigmatism

Obj. lens astigmatism corrected

Fig. 7- 11

Obj. lens astigmatism

Fig. 7- 10

2) Proper focusing and optimal underfocus (OUF): Optimal underfocus increase image contrast. Optimal underfocus can be judged by using Fresnel fringe that comes as the result of near field diffraction: Fresnel diffraction. Different magnification needs different optimal focus. With a little bit practice, your eyes can be trained to get optimal focus of a given magnification.

Fig. 7- 12

3) Specimen thickness, contrast Specimen thickness is inversely related to TEM resolution; contrast increase the visibility of resolved fine details.

Image contrast and its enhancement

Image formation and contrast method in TEM Contrast mechanism Scattering contrast: It is the most common contrast mechanism in TEM image. When electrons pass through specimen, they are scattered away by interaction with nucleus in the specimen: change direction without energy loss: elastic scattering (Rutherford scattering). Scattering contrast is directly related to the atomic number Z in the specimen. The higher the Z-number, it scatters electron more strongly, thus has higher contrast. The biological specimen, unfortunately, has dominant light atoms like C, H, O, N, very little scattering contrast. They need electron staining with heavy metal like uranium and Lead to enhance contrast. It is also related to specimen thickness: thicker specimen has higher contrast, but less resolution. Diffraction contrast: used in electron diffraction imaging only. Darkfield contrast: possible, mainly for crystalline material, rarely used in biological specimen. The resolution is poor in darkfield like in light microscope. Because the high quality central, axial electron beam is blocked, only peripheral beam is used, which has poor quality, higher aberration and astigmatism thus has poor resolution. Phase contrast: insert an in-column phase plate within TEM at objective lens back focal plane to introduce phase shift, which convert the phase difference of the electron beam exiting the specimen into intensity difference. This is a rather new approach used in high resolution TEM, mainly for crystalline material with lattice structure. It can also be used for unstained biological specimen. Absorption contrast and color contrast do not have any roles in EM image formation.

TEM sample preparation

Fundamental problems for biological specimen in TEM Consists of very content of light elements: C, H, O, N, interacts with electron weakly, no contrast Very sensitive to electron beam radiation damage Thick: the thinnest cell of 5 m is too thick for electron to penetrate: (Max 200-300 nm thick even at 300 KV) Full of water: ionize and discharge in high voltage field. Preparation of biological specimen for EM From 1933, it took more than 30 years to establish proper ways to prepare biological specimen for TEM with decent image quality Essential specimen requirement for TEM It must transmit sufficient electron to form image It must scatter electron enough to produce contrast It must stable under electron beam bombardment It must be at a size to fit small TEM specimen holder It took long time and step by step to meet all these requirement. Milestones of TEM specimen preparation In early 1950 Osmium fixation and methacrylate embedding 1950-60 improvement on microtome (thermal expansion knife advance) and knifes making (Glass knife making or commercial diamond knife) In 1960s epoxy resin replaced methacrylate for embedding In 1963 Glutaraldehyde was used as primary fixation, a revolutionary step for improving fine structure of biological specimen. Now Glutaraldehyde plus Osmium fixation is the golden standard for TEM. 8

Methods of preparing biological specimen for TEM

First step: specimen fixation Purpose of fixation: 1) Stop all biological activity at the time of fixation. 2) Prevent anoxia induced morphologic changes at ultrastructural level: (most import for electron microscopy, as 2-3 minutes lack of oxygen supply to cells will produce EM-detectable damages) 3) Prevent enzyme induced structural destruction (autolysis) (minimum requirement for all microscopic method) 4) Protect specimen against subsequent rigorous treatment like chemical dehydration, embedding etc. (extraction of cellular components by organic reagent is a main reason of bad structural preservation) Methods of fixation Chemical fixation: Glutaraldehyde GA: (1-5% ) ultimate important for preserving fine structure . GA forms extensive, Irreversible crosslink among protein molecules Disadvantage: Slow penetrating, auto-fluorescence. Destroys antigenicity of many antigens, not good for immuno-EM. Paraformaldehyde PFA: (4%) Weak, reversible fixation, not enough to preserve fine structure for EM if used alone Advantages: 10 times faster penetrating than GA. Preserve antigenicity well. Combination of GA and PFA for bigger tissue bloc Perfusion fixation with PFA_GA for animal before demising Other ways of fixation Freezing: Quick freeze specimen for frozen section: IEM Cyo-fixation and cyo-EM observation Combination of freezing and chemical fixation Quick freeze fixation + freeze substitute with chemical fixation Good results but time consuming, need special device High pressure freezing Post-fixation with Osmium tetroxide Dehydration: required both for TEM operation and embedding procedure Use ethanol: better contrast, less brittle, but not miscible with resin. Need propylene oxide as intermediate agent to go to resin. Use acetone: dehydrate more thoroughly, make specimen more brittle. Miscible with resin, can go into resin directly. Embedding: embedding into resin to get increased, homogeneous hardness for ultrathin sectioning. Epoxy resin is commonly used: Resin harden tissue to get good sectioning property Resin protect specimen from electron beam radiation damage. Semi-thin and thin sectioning with ultra-microtome Semi-thin section at 1 m, toluidine blue staining for localization position of final sectioning Ultra-thin sectioning at 50-100 nm

Electron density staining of section: Uranyl acetate and Lead citrate double staining Use heavy metal salt bound to specimen to increase specimens electron density: electron staining.

Positive staining:
Uranyl acetate (UAc), Uranyl ion (Z=92, A=238) Uranium staining on section: Negatively charged phosphate groups: NTP in nucleic acid, DNA, RNA, ribonucleoprotein Phosphate groups in phospholipid of lipid bilayer, membrane staining Free amino group in protein, glycogen Any osmicated reactive groups Uranium en bloc staining (stain tissue bloc after GA fixation, before sectioning): Act as both fixative and stain, Enhance contrast for tissue with APUD Enhance contrast especially for cytology, cultured cells. Selective staining: Differential staining of RNA with EDTA (no DNA stained) Lead Citrate Pb ion (Z= 82, A=207) Lead general staining: Affinity to cellular components, increase general contrast Stain selectively for RNA-containing structure and glycogen in non-osmicated specimen. Lead differential staining for ribosome and glycogen: De-osmium with periodic acid (or H2O2): Lead Enhance glycogen only.

Whole mount cell TEM, no sectioning

Cell thickness 6-15 um is too thick at central area for electron to penetrate Only periphery region of the cell: leading edge, filopodia or lamellapodia can be viewed. Especially good for cortical actin cytoskeleton

Fig. 7- 13

Negative staining
Used for non-sectioned materials: Bacterial Virus particle Macromolecules: protein and polysaccharide Commonly used negative stains (metal ion with Z > 40) Uranyl acetate UAc Potassium phosphotungstate KPT (Tungsten, W: Z= 74 A=184) Molybdenum Z=42, A=96

Fig. 7- 14

10

Phase contrast for unstained biological specimen Phase contrast cannot be routinely appreciated unless a special modification is done to the TEM. Phase contrast requires a in-column phase plate to shift the small phase difference produced by specimen to a larger scale to generate amplitude difference for yielding electron density difference.

Fig. 7- 15

By Dr. Fang Zhao in October 2011

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