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Introduction to electron microscopy Andres Kaech April 2012

1

Table of content

1. Introduction ................................................................................................................................................ 2

2. Principle of SEM and TEM ........................................................................................................................ 4

The electron source .......................................................................................................................................... 5

Electromagnetic lenses .................................................................................................................................... 6

Specimen holders and stages ........................................................................................................................... 8

3. Electron specimen interactions ................................................................................................................. 10

4. Contrast formation and imaging in TEM ................................................................................................. 12

5. Contrast formation and imaging in SEM .................................................................................................. 15

Imaging using secondary electrons ................................................................................................................ 15

Imaging using backscattered electrons .......................................................................................................... 17

Elemental analysis using x-ray ...................................................................................................................... 18

Artefacts ........................................................................................................................................................ 19

6. Sample preparation ................................................................................................................................... 21

Preparation of cells and tissues for transmission electron microscopy .......................................................... 21

Preparation of cells and tissues for scanning electron microscopy ................................................................ 24

7. Literature .................................................................................................................................................. 27


2

1. Introduction

Microscopy enables a “direct” imaging of organisms, tissues, cells, organelles, molecular assemblies and even individual proteins. Analytical techniques as used in molecular biology provide a data set which represents cellu-lar processes in an abstract way. Therefore, microscopy was and still is an important, complementary technique to visualize the macro and/or microscopic structure and to assign structure to function and vice versa.

Microscopy techniques, their resolution limit, scales and corresponding objects are depicted in figure 1.1. The typical size of eukaryotic cells is 10-20 micrometer and their subcellular components can not be seen by naked eyes. Additionally, cells are colorless and transparent. The organelles or other constituents have to be stained to make them visible in the light microscope. A variety of histological stains and fluorescent markers have been developed.

Imaging the ultrastructure of cellular components requires electron microscopes which have a much higher re-solving power than light microscopes. The physical properties of electron microscopes (e.g. high-vacuum) de-mand specific preparation and staining techniques to reveal the ultrastructure of cells and tissues.

Fig. 1.1: Resolution limit of different imaging techniques, involved radiation and size of biological objects.

The limit of resolution of an optical system depends on the numerical aperture and the wavelength of light (Law of Ernst Abbé). This law holds as well for electrons, of which the speed determines their wavelength: The higher the speed of the electrons, the smaller the wavelength and the better the resolution. The practical resolution of a transmission electron microscope (TEM) run at 100 kV (acceleration voltage that determines the speed) is ap-proximately 0.5 nm and exceeds the resolution of light microscopes by far. In scanning electron microscopes (SEM), the effective resolution is around 1 nm. The resolution of modern electron microscopes is much better than the resolution that a prepared biological specimen can provide due to preparation.

The most important and challenging part of electron microscopy of biological material is the preparation proce-dure. With every preparation step artifacts are introduced. Electron microscopists have put a lot of effort in the preparation of the specimens to reduce the artifacts to a minimum and to image the specimens in a more lifelike state. The image of the specimen can never be better than the entity of the introduced artifacts during prepara-tion.

In combination with immunocytochemical methods, electron microscopy is powerful method to label and high-light single, specific proteins enabling a correlation of the ultra structure and its function.

Note: Preparation of biological specimens for electron microscopy is often a very time-consuming process.

Atoms

1 mm

100 m

10 m

1 m

100 nm

10 nm

1 nm

0.1 nm

Cells

Red blood cells

Bacteria

Mycoplasma

Viruses

Proteins

Amino acids

Radio

Infrared

Visible

Ultraviolet

x,-rays

Human eye

Electron microscope

Light microscope

Resolution Limit Wavelength/Size Object

MRI, CT

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April 2012

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Introduction to e

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10

3. Electron specimen interactions

The electron beam (TEM and SEM) hits the sample and either passes the sample unaffected or interacts with it. Three different interactions of electrons with an atom can be generally discerned (Fig 3.1):

i) An electron of the primary beam is scattered by the electrostatic interaction with the positively charged nucleus of an atom at an angle of more than 90°, yielding backscattered electrons. This type of electrons has practically the same energy as the ones of the primary beam.

ii) An electron of the primary beam is scattered by the electrostatic interaction with the positively charged nucleus of an atom at an angle of less than 90°, yielding elastically scatterd electrons. Al-so these electrons do not loose energy and therefore are referred to as elastically scattered electrons.

iii) Electrons can also loose energy while interacting with the “electron cloud” of the atom. These are called inelastically scattered electrons. This interaction can lead to the following processes (see also http://www.matter.org.uk/tem/default.htm):

Inner-shell ionisation An electron is pushed out of the electron cloud. The electron „hole“ is filled by an electron of an outer shell: Surplus energy is either emitted as characteristic x-ray or transferred to another elec-tron, which is emitted (Auger electron).

Bremsstrahlung (continuum x-rays) Decceleration of electrons in the Coulomb field of the nucleus ⇒ Emission of x-ray carrying the surplus energy ΔE ⇒ Uncharacteristic x-rays

Secondary electrons (SE) Loosely bound electrons (e.g., in the conduction band) can easily be ejected ⇒ low energy (< 50 eV)

Phonons Lattice vibrations (heat) ⇒ beam damage

Plasmons Oscillations of loosely bound electrons in metals

Cathode luminescence

Fig. 3.1: Interaction of electrons with an atom. Note: Inelastically scattered electrons lead to a variety of processes like heating, radiation and electron emission as described in the text.

Inelastic(low angle, E=E0-∆E)

Unscattered(E=E0)

Primary electrons (E0)Backscattered electrons (E=E0)

Elastic(higher angle, E=E0)


11

Almost all types of electron interactions can be used to retrieve information about the specimen. Depending on the kind of radiation or emitted electrons which are used for detection, different properties of the specimen such as topography, elemental composition can be deduced.

The follwing figure 3.2 shows the interaction of the electron beam with the specimen. In biological TEM analy-sis, the elastically scattered electrons are mainly used for imaging. The inelastically scattered electrons will be recorded as well with the imaging device (e.g. CCD camera). They have a slightly lower energy (a longer wave-length) than the primary electrons. In conclusion, they are deviated less strongly in the electron lenses and do not merge at the same spot as the elastically scattered electrons (chromatic aberration). They contribute to unsharp-ness of the image. In high-end instruments, the inelastically scattered electrons can be filtered out with an energy filter.

In SEM, mainly secondary electrons are used for imaging of biological specimens. These electrons have a very low energy (around 50 eV) compared to the energy of the primary electrons (up to 30 keV). Due to the low ener-gy, these electrons can escape only from the surface area of the specimen (for details see chapter 5) and therefore provide information about the surface topography. Also backscattered electrons are frequently used for imaging with an according backscatter detector. The number of electrons which are backscattered from a certain spot of the specimen depends on the local elemental density of the specimen. Hence, backscattered electrons provide a “density image” and information about the elemental composition, respectively. Backscattered electrons are also more resistant to charging due to their high energy.

X-rays are always produced when matter interacts with an electron beam. The energy of a part of the emitted x-rays is specific for different atoms and can be used for elemental analysis in TEM and SEM using special detec-tors. However, the analysis of light elements is rather difficult and the high corrugation of biological specimen surfaces makes the interpretation of elemental maps very demanding. A lot of experience is necessary to prevent pit falls.

Fig 3.2: Interaction of electrons with specimen/matter, induced radiation and emission. Transmitted electrons like elastically scattered electrons are typically used for TEM imaging of biological specimens. Secondary electrons and backscattered electrons are mainly used for imaging in the SEM.

PrimaryPrimary electronselectrons

UnscatteredUnscattered electronselectrons

ElasticallyElastically scatteredscattered electronselectrons

Inelastically scattered electrons

SecondarySecondary electronselectronsBackscatteredBackscattered electronselectrons

AugerAuger electronselectronsHeat

Cathode luminescenseX-rays

Specimen Interaction volume

SEM analysis

TEM analysis

Introduction to e

12

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Introduction to e

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15

5. Contrast formation and imaging in SEM

In SEM, an electron beam with a tiny spot is guided pixel by pixel over the surface of a specimen, e.g. 1024 x 768 pixels. At every pixel, the beam stays for a defined time and generates a signal (e.g. secondary electrons) which are detected, amplified and displayed on a computer screen. The scanning of the beam is synchronized with the display of the computer screen. The signal deriving from every single pixel on the sample is simultane-ously displayed on the corresponding pixel of the computer screen and finally forms the image. Note: There is no CCD camera for image acquisition in SEM! The image consists of displayed grey levels, e.g. 256 gray levels in an 8 bit image. The magnification is changed by scanning a smaller area with the same number of pixels. The pixel size therefore gets smaller and the resolution is increased.

The beam of electrons wich hits a spot of the surface of the specimen interacts with a whole volume of the spec-imen. The interaction volume looks like a pear (figure 5.1). Secondary electrons are produced in the whole vo-lume but can escape only from a small volume close to the surface of the specimen due to their low energy (around 50 eV). Backscattered electrons manage to escape from a larger depth since their energy is the same as the primary electrons. On their way through the sample, backscattered electrons can again interact with atoms and produce secondary electrons (called SE 2 electrons). X-rays can escape from the whole interaction volume.

The size of the interaction volume is dependent on the acceleration voltage of the primary electrons and the atomic number of the atoms close to the surface. Heavy atoms decelerate the beam more than light elements and therefore reduce the interaction volume.

Fig. 5.1: Interaction volume of the electron beam with a bulk sample. Escape depths of SE, BSE, and x-rays are indicated. Note: The SE escape depth is independent on the energy of the primary electrons (PE).

Imaging using secondary electrons

Secondary electrons (SE) are mainly used in SEM for imaging the surface topography of biological specimens. They are detected with an Everhart Thornley detector. The principle of detection is shown in figure 5.2. The SE are collected by a collector grid. A voltage of + 200 to 500 V is applied to the collector grid which attracts the low energy electrons. The SE then hit a scintillator which converts the electrons to photons. The photons are guided by a light conducting tube on the photomultiplier tube (figure 5.3), where the photons again are converted to electrons that are amplified finally leading to an electrical signal. The current is translated into a gray value and displayed on the screen of the monitor.

…interaction volume RPE

SE escape depth (E < 50 eV)

BSE escape depth(E = E0)

X-ray escape depth

“high voltage” “low voltage”

Introduction to e

16

Fig 5.2: Princ

Fig. 5.3: Work

The SE escaptrons. Howevaction volumuppermost lavoltages is hitrate deeply i(atomic numbnum. Since bSE escaping volume. The surface, biolothe signal is llower areas cfigure 5.4 an

electron microsco

ciple of SE detec

king principle of

pe depth (λ) aver, the percen

me R is larger aayer. In concluigher and provinto the specim

mber) of the spebiological specthe specimen result is a fain

ogical specimlocalized to thcan not escape

nd figure 5.5.

Primary ele

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opy

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of a photomultip

as depicted in fntage of the Sat lower accelusion, the numvides a strongmen and the pecimen. It is 1cimen consist is very low. Mnt, unsharp sig

mens need to behe surface, mae through the h

+200

Electron

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verhardt Thorn

plier tube. The a

figure 5.1. is iE produced inleration voltag

mber of the emger signal. Theproduced SE c10 – 100 nm fo

of light elemeMoreover, thegnal with verye coated with any more SE aheavy metal la

+7-1

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Andres Kaech

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pril 2012

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SE

microscopy

g of the specimeprimary electroast. The contrasvoltage is select

represent a vieted and therefornizable. Right. t a high signal ns).

ckscattered el

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E

R

PE

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PE

SE

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surface of the SE, a differentme energy as trected back to

ure 5.6).

P

the surface. Thef SE escaping try beam and th

membrane of freures are obscurm localizing thenal protein pat

specimens but arrangementthe primary elowards the obj

SE esca

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Primary electSecondary elExcited volumSE escape de

April 2012

17

eriving directlyt different spotsrface when the

yeast. Left: Theginations of thesurface provid-le (bright spots

tection of dif-tor is applied.(much higher

Therefore, the

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2

7

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18

Fig. 5.6: Arrangement and working principle of a BSE detector. The scintillator is arranged circular around the primary beam just beneath the objective lens. BSE hit the scintillator and produce photons, which are guided to the photomultiplier tube. As for the SE detector, the signal is amplified, converted to a gray level and dispayed on the computer screen.

Figure 5.7 is an example, how the BSE detector can be used to get additional information of the specimen, e.g. detection of gold particles on the surface of red blood cells.

Fig. 5.7. Red blood cells labelled against surface proteins. The antibody is connected to a gold particle (heavy metal aggre-gation). Whereas the SE signal provides the surface topography, the BSE image distinctly shows the gold particles. The images were recorded simultaneously at 25 kV acceleration voltage. The whole sample is coated with a thin layer of heavy metal (ca. 2 nm). SE derive mainly from this layer and therefore do not clearly accentuate the gold particles, which have a size of around 20 nm. On the contrary, a lot of elctrons are backscattered where the gold particles are, but only very few are backscattered from the thin metal coat.

Elemental analysis using x-ray

X-rays produced in the specimen can be used for elemental analysis of the specimen (Fig 5.8). The x-ray energy is specific for different elements. A dedicated x-ray detector detects the x-rays deriving from every spot of the sample and assigns the according element. Elemental mapping is possible for any area of interest. In particular the interpretation of x-ray spectra is very challenging and needs a lot of experience. A highly corrugated surface of a specimen – typical biological specimen - can lead to misinterpretation. Quantitative analysis with biological specimens is usually not possible.

Scintillator Light pipe Photomultiplier

Primary electrons

BSE

SE BSE

Introdu

Fig 5.8

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Beamdestro

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8: Elemental an

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9: Charging arleaf (right)

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microscopy

nalysis using x-

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with a thin hconducting sp

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beam penetrat charging. A theavy metal lpecimens.

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April 2012

19

ates the speci-typical charg-ayer not only

xy surface of a

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Introduction to e

20

Fig. 5.10: Bea

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Andres Kaech

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can. Scale bar 5

pril 2012

500 nm.


21

6. Sample preparation

As we have discussed in the earlier sections, electron microscopes are run under high vacuum conditions. A high vacuum, however, is a very unfriendly environment for biological specimens like cells and tissues (or condensed organic hydrated matter in general). Electron microscopes pose the following prerequisites to the specimen for investigation.

Resistant in the vacuum

Electron beam resistant

Providing contrast

Penetrable for electrons (transmission electron microscope)

Large biological specimens (cells, tissues) do not fulfill any of these requirements and have to be treated in order to be investigated in the electron microscope. Typically, the specimen is turned into a solid state to make it suita-ble for vacuum and resistant in the electron beam. Moreover, it has to be cut to very thin sections of ca. 100 nm for transmission electron microscopy. Such a specimen still does not provide contrast. Biological material is basically composed of light elements like C, O, H, N, S, P, which do not interact with electrons. Selective stain-ing or contrast enhancement with heavy metals is required to form an image as already pointed out in chapters 4 and 5 (contrast formation and imaging in TEM and SEM).

Preparation of cells and tissues for transmission electron microscopy

Typical preparation procedures for cells or tissues for transmission electron microscopy include the steps depict-ed in figure 6.1.

Figure 6.1: Left: Classical preparation pathway for transmission electron microscopy. The whole process is performed at room temperature. Right: Cryo-preparation pathway for transmission electron microscopy. The specimen is frozen and slowly warmed up during freeze-substitution (low temperature dehydration).

The classical preparation is performed at room temperature. During fixation, the native biological specimen is chemically stabilized with chemical fixatives like glutaraldehyde and osmium tetroxide. All biological processes are arrested during fixation. The fixed specimen is ready for dehydration with a sequence of different ethanol concentrations until it is completely dehydrated. Subsequently, the ethanol is exchanged with a monomer solu-tion of a plastic formulation (e.g. Epon/Araldite) in a sequence of rising concentrations of plastic components dissolved in ethanol or acetone. The monomers are polymerized by heat or UV light and provide a hard plastic block containing the embedded specimen. This enables cutting of thin sections (100 nm) in an ultramicrotome. The sections are stained with uranyl-acetate and lead citrate. Uranium and lead ions selectively bind to lipids, proteins and nucleic acid in the specimen and provide a distribution of electron dense material and finally the image in the electron microscope (Figure 6.2).

Embedding

Fixation

Dehydration

Staining (uranyl-acetate, lead-citrate)

Thin sectioning

TEM

Classical preparation(chemical fixation at RT)

Cryo-preparation(cryo-fixation)

Introduction to e

22

Figure 6.2: TE

A lot of artifto minutes) apreparation pdehydration a

Cryo preparastep, the specing is very fcause of the ultra structurproperties of 6.3) is achiev

Once the speperatures (e.gplastic, sectio

electron microsco

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Andres Kaech

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Figureformat

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e particles likees by a procesng the particlea short incuban layer of theis added on thconds or minuoscope. The hey and provide can be omitted

e 6.4: TEM miction is shown in

microscopy

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The principle of

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April 2012

23

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s attach to thesten, uranium

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24

Preparation of cells and tissues for scanning electron microscopy

Typical preparation procedures for cells or tissues for scanning electron microscopy include the steps depicted in figure 6.5.

Figure 6.5: Left: Classical preparation pathway for scanning electron microscopy. The specimen is chemically fixed and dehydrated as described for classical preparation for TEM, followed by critical point drying and coating. Right: Cryo prepa-ration pathway. The specimen is frozen, freeze-fractured, partially freeze-dried, coated and imaged in the cold state in the SEM (Cryo-SEM). The specimen always stays at low temperature and undergoes physical treatment only.

The classical preparation procedure for scanning electron microscopy involves critical point drying (left path in figure 6.5). Air drying of specimens at room temperature leads to a collapse of the sample structure caused by the high surface tension of water. Critical point drying was developed in order to prevent the destroying forces of the surface tension. The first steps including fixation and dehydration are performed the same way as described for the classical preparation for TEM. After dehydration, the ethanol is exchanged with acetone. In a dedicated device (critical point dryer) the acetone is exchanged with liquid pressurized carbon dioxide. Subsequently, the temperature and the pressure in the chamber of the critical point dryer are increased until they reach the critical point of CO2 (31°C, 74 bar). The CO2 in the critical state (neither gas nor liquid) is released from the chamber very slowly providing a gently dried specimen. The process is shown in figure 6.6.

Fixation

Exchange of acetone with pressurized liquid

CO2

Dehydration(ethanol, acetone)

Critical point drying

Chem. fixationChem. fixation Cryo-fixationCryo-fixation

Coating (Contrast)

SEM

Dry specimen

Partial freeze-drying

Freeze-fracturing

Frozen hydrated specimen

Cryo-SEM

No ch

emica

l treatm

ent!

Introdu

Figure

The celectrthe scing (F

Note: aqueo

Figure

Similachemicryo tfor cr120°Cprovid

uction to electron

e 6.6: Phase dia

critical point don beam evap

canning electrFigure 6.7).

The critical pous specimens

e 6.7: Critical p

ar artifacts as ical fixation atechniques forryo preprationC) in a dedicding cross-fra

microscopy

agram of carbo

dried specimeporation. The on microscop

point of waters since it woul

point dried liver

during classiand dehydratior SEM (right p

n for TEM. Onated freeze-fr

acture planes a

Pres

on dioxide. The

en is coated wheavy metal

pe to the surfa

r is at 374°C ld be destroye

r tissue, coated

cal TEM prepon at room tempath in figure nce the specimracturing macas well as fract

solid

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Andres

red line in the p

with a thin laylayer renders

ace providing

and 221 bar. ed at the critica

d with platinum.

paration are inmperature. Ma6.5). The free

men is adequachine. The frature planes th

liquid

gas

SS

EE

Phase diagram o

Kaech

panel shows the

yer of heavy s the specimenthe required y

Critical pointal point condi

Blood vessel w

ntroduced duriany of these aezing or cryo ately frozen, iacturing throu

hrough biologi

SS StartingEE End poinCC Critical p

CC

of CO2

e process of cri

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t drying is nottions required

with erythrocyte

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pointntpoint

Temperature

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nm) by sputtand localizes

rons and contr

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es and leukocyte

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April 2012

25

ing.

ter coating ors the signal inrast for imag-

ectly with the

e.

volves as wellagain by usinge as describederatures (e.g. -ndom process).

2

5

r n -

e

l g d -s

Introduction to e

26

Figure 6.8: Frfracture face o

After fracturzen water (palow temperatcedure excluperatures thro

Figure 6.9: Crwith platinum.

electron microsco

Freeze-fracturingof the lipid bilay

ring, the visibiartial freeze dture for imagi

usively involvough the who

ryo-SEM image.

opy

g of biologicalyer. Cyt…Cytos

ility of the ultdrying). The fring in the cry

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trastructure cafracture face oyo scanning elprocesses. Thepreparation an

ure frozen mou

Andres Kaech

F…plasmatic fris cross-fractur

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use brain tissue

racture face of red in the cell o

ed by sublimaen finally is coscope (figure

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This preparationd stays at low

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pril 2012

asmatic

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d coated


27

7. Literature

Physical Principles of Electron Microscopy, Ray F. Egerton, Springer Verlag, 2007.

Griffith G. (1993). Fine Structure Immunocytochemistry. New York, Berlin, Heidelberg. Springer Verlag. ISBN 0-387-54805-X.

Electron microscopy methods and protocols / ed. by M.A. Nasser Hajibagheri. - Totowa, N.J. : Humana Press, cop. 1999. (Methods in molecular biology ; vol. 117)

Electron microscopy : methods and protocols. - 2nd ed. / ed. by John Kuo - Totowa, N.J. : Humana Press, 2007. (Methods in molecular biology ; 369)

Electron microscopy : principles and techniques for biologists / John J. Bozzola, Lonnie D. Russell. - Boston : Jones and Bartlett, 1991. (The Jones and Bartlett series in biology)

Funktionelle Ultrastruktur : Atlas der Biologie und Pathologie von Geweben / Margit Pavelka, Jürgen Roth. - Wien : Springer, 2005.

Cavalier A., Spehner D., Humbel B. M. (2008). Handbook of Cryo-Preparation Methods for Electron Microsco-py, CRC Press, Boca Raton

Harris, J. R (1997). Negative Staining and Cryoelectron Microscopy. Oxford, BIOS Scientific Publishers Lim-ited

Echlin P. (1992). Low-Temperature Microscopy and Analysis. New York, Plenum Press. ISBN 0-306-43984-0.

Robards A. W., Sleytr U. B (1985). Low Temperature Methods in Biological Electron Microscopy. Practical Methods in Electron Microscopy, Volume 10. Edited by Glauert A. M. Amsterdam, Elsevier Science Publishers B. V. (Biomedical Division)

Steinbrecht R. A. and Zierold K. Cryotechniques in Biological Electron Microscopy (1987). New York, Berlin, Heidelberg. Springer-Verlag. ISBN 0-387-18046-X

http://www.matter.org.uk/tem/default.htm

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