UP MS Department of Medical Biology
Beáta Bugyi
Molecular cell biology 1.
Morphological techniques 2.
Electron microscopy
Keywords
Resolution limit. Electron microscopy. Electron microscope. Principles of transmission, scanning and
cryo-electron microscopy. Sample preparation, contrasting methods.
1 / 17
Introduction
light microscopy phase-contrast microscopy fluorescence microscopy
scanning EM - SEM transmission EM - TEM cryo-EM
1 1
https://www.sciencephoto.com/media/471609/view/thale-cress-cell-tem
2 / 17
Reminder
Resolution limit, resolving power
Resolution limit: the shortest distance between two points of the object that can be distinguished as separate entities
on the image (d)
Resolving power: inverse of the resolution limit (1/d)
resolution limit ↓ resolving power ↑
The resolution limit is this red bar. In the first example, two features of the object are separated at a distance
larger than the resolution limit (larger than the red bar). Therefore, they appear as separate entities in the image.
In the second example, the two features of the object are located closer than the resolution limit, in this case, they
appear as a single entity on the image. They cannot be distinguished or resolved; this information is lost.
3 / 17
Problem
The resolution of the human eye is limited: optimal distance of clear vision (250 mm); d ~ 0.1 mm (1 arc minute)
The resolution of the light microscope is limited: d ~ 200 nm
𝜆
𝑑 = 0.5
𝑁𝐴
wavelength (λ) ↓ resolution limit (d) ↓ resolving power ↑
? Can we use “illumination” with shorter wavelengths?
Using electromagnetic radiation (X-rays, gamma) with wavelengths shorter than visible light in imaging following
the principles of microscopy is difficult.
! Solution → electron beam, de Broglie matter-wave theory
A particle having 𝐸 energy and 𝑝 = 𝑚𝑣 momentum can be considered a wave with wavelength:
ℎ
𝜆=
𝑚𝑣
velocity (v) ↑ wavelength (λ) ↓
At sufficiently high velocities, the wavelength of electrons ~ nano (10-9), piko (10-12) meter
1929 Nobel Prize in Physics
Prince Louis-Victor Pierre Raymond de Broglie
"for his discovery of the wave nature of electrons"
4 / 17
Electron microscopy (EM)
1974 Nobel Prize in Medicine
Albert Claude, Christian de Duve, George E. Palade
"for their discoveries concerning the structural and functional organization of the cell"
1986 Nobel Prize in Physics
Ernst Ruska
"for his fundamental work in electron optics, and for the design of the first electron
microscope"
5 / 17
Electron microscope
video: Hajnalka Ábrahám, Gergely Berta
2
https://www.sciencedirect.com/science/article/pii/S254252932030081X
6 / 17
Electron source/gun, accelerating electrons
Electrons escape from the cathode due to heating.
Electrons are accelerated (v, velocity) by an electric
field (U).
1
The kinetic energy of electrons: 𝐸 = 𝑚𝑣 2
2
The work done by the electric field: 𝑊 = 𝑒𝑈
The work done by the electric field increases the
kinetic energy, and thus the velocity of the electron:
1 𝑒
𝑒𝑈 = 𝑚𝑣 2 → 𝑣 = √2 𝑈
2 𝑚
The wavelength of the electron:
ℎ ℎ 1
𝜆= = = 1.225 (𝑛𝑚)3
𝑚𝑣 √2𝑒𝑚𝑈 √𝑈
Calculation
What is the speed and wavelength of an electron accelerated with an accelerating voltage U = 200 000 V?
𝑚2 𝑘𝑔
𝑒 = 1.6 × 10−19 𝐶, 𝑚 = 9.109 × 10−31 𝑘𝑔, ℎ = 6.626 × 10−34
𝑠
𝑒 8
𝑚
𝑣 = √2 𝑈 = 2.65 × 10
𝑚 𝑠
1
𝝀 = 1.225 = 𝟎. 𝟎𝟎𝟐𝟕 𝒏𝒎 = 2.7 × 10−12 𝑚 ~ 100 000 × smaller than the wavelength of visible light
√𝑈
3
Does not consider relativistic mass.
7 / 17
TEM & SEM
Transmission EM (TEM)
▪ illuminates through the sample
▪ 2D image
▪ analogous to light microscopy
Scanning EM (SEM)
▪ scans the sample
▪ 3D image
▪ principle of surface scanning: it collects data from the
surface of the object from point to point, assigning
pixels (location, value of the physical parameter) to
each point, the data is displayed as an image
8 / 17
What and why do we see in a TEM image? - interaction of the electron beam with the sample
4 5
The scattering of electrons is proportional to the atomic number:
▪ low atomic number elements (e.g. H, O, C, N) scatter less
biological samples are transparent to the electron beam, most of the electrons pass through them without
interaction (electrons are not scattered)
▪ high atomic number elements (e.g. heavy metals, uranium, lead) scatter more
4
Medical Biophysics
5
https://en.wikipedia.org/wiki/Electron_scattering
9 / 17
What and why do we see in a TEM image? – contrast based on atomic number
▪ preparation of ultra-thin samples (~ 50 - 100 nm) that can pass through the electron beam
▪ creating a contrast with heavy metal staining (heavy metal salts, e.g. uranyl-acetate, lead-citrate
phosphotungstic acid,…)
TEM image is a pattern of light and dark points
The transmitted (not scattered) electrons reach the detector and cause flashes on the fluorescent screen located at
the bottom of the observation chamber (bright points of the image). Electrons elastically scattered by heavy metal
atoms are excluded from the beam path, they are not detected. The pixels of the object points with which the
electrons interacted (electron scattering points) will be missing from the image (dark points of the image).
scattering of electrons: yes/no appearance on the image: bright/dark
low atomic number elements (e.g. elements frequently bright
occurring in biological samples: H, O, C, N)
NO
high atomic number elements (e.g. heavy metals, dark
uranium, osmium, lead)
YES
6
6
https://www.sciencephoto.com/media/471609/view/thale-cress-cell-tem
10 / 17
Sample preparation, labeling techniques, contrasting methods
1. chemical fixation – double fixation
aldehyde (e.g. formaldehyde, glutaraldehyde): cross-linking of proteins
osmium tetroxide (heavy metal): unsaturated fatty acids, membrane
2. dehydration – vacuum
acetone, ethanol
3. embedding – strength
synthetic resin (e.g. epoxy resin)
4. sectioning
ultramicrotome (~ 50 - 100 nm thick slices)
5. transfer to the support surface (microgrid, grid)
heavy metal salts, e.g. uranyl-acetate, lead-citrate phosphotungstic acid,…
immunogold
antibody + gold colloidal particle (Ø ~ 5 - 30 nm)
rotary shadowing, angular shadowing; metal vapor
freeze-fracture, freeze etch; metal vapor
11 / 17
Contrast – positive staining, negative staining
▪ positive staining ▪ negative staining
stains the cellular component (makes it electron- stains the environment (makes it electron-scattering)
scattering) cells: bright, background: dark
cells: dark, background: bright
(A) positive and (B) negative contrast:
(A) the heavy metal salts bind to individual components of the biological sample
(B) the heavy metal salts fill the space individual components of the biological sample
12 / 17
7
Actin filaments (left), actomyosin (right), negative staining, TEM
https://www.umassmed.edu/cemf/Negative-Staining/
7
https://www.protocols.io/view/Positive-and-Negative-Staining-of-Viruses-on-TEM-G-day2fv.html
13 / 17
Cryo-electron microscopy (cryo-EM)
▪ transmission EM
▪ allows samples to be observed without staining, in their
natural aqueous environment, resulting in a 3D structural
image with high (atomic) resolution
▪ cryogenics: deals with the behavior of materials at very low
temperatures
▪ cryofixation/vitrification: the sample is frozen very quickly
(106 K/s) in its hydrated state to prevent the formation of ice
crystals
liquid N2 (77 K = - 196 oC): protein samples voltage-dependent potassium channel
liquid ethane (189 K = - 89 oC): thin biological samples https://doi.org/10.7554/eLife.37558
1 Å = 0.1 nm
2017 Nobel Prize in Chemistry
Jacques Dubochet, Joachim Frank, Richard Henderson
"for developing cryo-electron microscopy for the high-resolution structure determination of
biomolecules in solution"
14 / 17
https://www.youtube.com/watch?v=qc2PbmI5qMw
15 / 17
Cryo-EM workflow
cryofixation imaging using the TEM mode
identify molecules with a similar orientation, 2D reconstruction of the 3D model of the molecule
16 / 17
17 / 17
