Electron Microscope: Understanding its Types and Components in Biology

Electron Microscope: An Introduction

Dr. Saif Alharthy
Assistant Professor of Toxicology and Clinical Chemistry

What is Electron Microscopy (EM)?

• A technique to:
• Obtain high resolution images of biological and non-biological specimens.
• Investigate the detailed structure of tissues, cells, organelles and macromolecular complexes.
• Two main types of electron microscope:
• Transmission EM (TEM)
• Scanning EM (SEM)

Transmission Electron Microscope (TEM)

. Transmission electron microscope (TEM) is a technique used to observe very tiny details of very small specimens by using a beam of electrons that pass through a very thin section (sample).
. First developed was by German scientists Max Knoll and Ernst Ruska in 1931.

TEM Capabilities and Requirements

• TEM provides:
• Higher magnification and resolution more than in a light microscope
• This because of wavelength of an electron is much shorter than that of a photon
• TEM requires:
• Ultrathin specimens (typically thinner than 100 nm) so that the electrons pass.

Fundamental Principle of TEM

. The fundamental principle:
· A series of electromagnetic lenses and apertures are placed throughout the microscope's column to focus the electron beam on the sample, minimize distortions, and magnify the resulting image onto a phosphor screen or a specialized camera.

Components of the Transmission Electron Microscope

• TEMs are composed of five key components:
• High voltage source
• Vacuum system
• Microscope column
• Detectors (e.g., imaging cameras, spectrometers)
• Control computers and software

electron source
electron beam
Illumination system
condenser lenses
condenser aperture
objective lens
sample
objective lens
Image (or diffraction
pattern) formation
objective aperture
selected area aperture
intermediate lenses
Magnify image
(or diffraction
pattern) onto
screen/camera
projector lens
fluorescent screen

TEM Column and Electron Gun

• At the top of the column is the electron gun which couples to a high voltage source used to set the kinetic energy of the electron beam.
• Typical accelerating voltages range from 80 kV up to 300 kV.
• The microscope column consists of a series of electromagnetic lenses and apertures to focus the electron beam onto the sample and magnify the TEM image onto the viewing screen (or detectors).
• A vacuum system is used to maintain the required vacuum levels throughout the column.

Electron Gun Types

. It is also called the electron source.
· Two main types of electron guns for TEM:
· Thermionic
· Field emission.
· The characteristics of the emitter will determine the resolution limit of the microscope

Type of Electron Sources

ANODE
ANODE 2
ANODE 1
WEHNELT
CYLINDER
LAB6 OR CEB6
CRYSTAL
TUNGSTEN
FILAMENT
TUNGSTEN
TIP
THERMIONIC EMISSION SOURCE
FIELD EMISSION SOURCE

Comparison of Electron Gun Characteristics

Illumination System

• This system takes the beam from the electron gun and guides it to the sample.
• To do this, a combination of two condenser lenses and at least one aperture at the exit plane of the second condenser lens (in combination with tilt and deflection coils) are needed.
• The condenser lenses are adjusted to form a broad beam at the sample that is several micrometers in diameter
• The upper pole piece of the objective lens can act as a third condenser lens to create an even more parallel beam

electron source
electron beam
Illumination system
condenser lenses
condenser aperture
objective lens
sample
objective lens
Image (or diffraction
pattern) formation
condenser aperture
selected area aperture
intermediate lenses
Magnify image
(or diffraction
pattern) onto
screen/camera
projector lens
fluorescent screen

Electromagnetic Lenses

• Lenses are used to control the path of the electrons. Because electrons cannot pass through glass, the lenses that are used are electromagnetic.
• They simply consist of coils of wires inside metal pole pieces. When current passes through the coils, a magnetic field is generated. As electrons are very sensitive to magnetic fields, their path inside the microscope column can be controlled by these electromagnetic lenses simply by adjusting the current that is applied to them.
• Two types of electromagnetic lenses are used:
• The condenser lens is the first lens that electrons meet as they travel towards the sample. This lens converges the beam before the electron beam cone opens again and is converged once more by the objective lens before hitting the sample. The condenser lens defines the size of the electron beam (which defines the resolution), while the main role of the objective lens is to focus the beam onto the sample.

1
e-
a)
b)
Source of
electrons
Magnetic lens
Axk
Focal point
Electron
trajectory
Copper
windings
Iron shroud
electron beam
magnetic pole
(north pole/south pole)
flux path< />sample location
for In-Lens
coil
lens magnetic
field area
sample location
for Out-Lens

Objective Lens Function

. It forms the images and diffraction patterns (DPs) that are magnified onto the viewing screen or camera.
· The object is the transmitted electrons just at the exit surface of the specimen. The objective lens disperses the electrons emerging from the exit-side of the specimen in the back focal plane (BFP), where the diffraction pattern is found, and recombines them in the image plane. To generate the high-resolution images of atomic columns, the focal length of the objective lens must be minimized to maximize magnification. Thus, the object distance, or distance between the specimen and the objective lens must be very small.
PARALLEL ELECTRON BEAMS
1
1
1
M
f
do di
THIN SPECIMEN
OBJECT PLANE
A
OBJECT DISTANCE , do
OBJECTIVE LENS
FOCAL LENGTH, f
BACK FOCAL PLANE
1
DIFFRACTION PATTERN
OBJECTIVE APERATURE
IMAGE DISTANCE, d;
IMAGE PLANE
IMAGE
OPTICAL AXIS

Intermediate and Projector Lenses

• The intermediate and projector lenses magnify the image or diffraction pattern onto the viewing screen.
• The projector lens is the final lens in the column that projects the image/DP onto the viewing screen or camera.
• Multiple intermediate lenses are used to adjust the degree of magnification of the image or DP produced by the objective lens.

electron source
electron beam
Illumination system
condenser lenses
condenser aperture
objective lens
sample
objective lens
Image (or diffraction
pattern) formation
-
objective aperture
selected area aperture
Magnify image
(or diffraction
pattern) onto
screen/camera
intermediate lenses
projector lens
fluorescent screen

Strengths and Limitations of Transmission Electron Microscopes

Advantages of TEM

• Offers the highest and most powerful magnification of any microscopy technique.
• Enables nano-analysis: the ability to collect local information about composition and bonding which can be correlated to high resolution images.
• The ability to visualize very tiny details of the samples.
• High quality micrographs (images)

Disadvantages of TEM

• Limited sampling
• Complex image interpretation:
• Possibility of electron beam damage, especially for light elements, biological samples, and soft materials.
• Difficult sample preparation to create extremely thin specimens; thin samples often result in imaging artifacts.
• Very expensive
• Using toxic materials while preparing the samples
• TEM requires big place to install because it is very huge microscope

Sample Preparation for TEM

Sample Preparation Steps

• Fixation
• Secondary Fixation
• Dehydration
• Infiltration
• Trimming
• Cutting/Microtome
• Staining

Fixation Process

• Primary Fixation
• This method is used for stabilizing biological samples. Chemical substances are used to cross link protein molecules with nearby molecules. The most frequently used chemical in this method is glutaraldehyde.
• Secondary Fixation
• To increase the contrast of the minute structures inside the specimen and give more stability by using osmium tetroxide (OsO4).

Dehydration in TEM Sample Prep

. It is the process by which the water content in the specimen is gradually replaced with a graded series of dehydration agent: an organic solvent, such as Ethanol and acetone. (50, 60, 90, 100% of acetone)
· Dehydration is important as the epoxy resin used in further steps does not mix with water.

Infiltration and Embedding

. In infiltration, epoxy resin is used to penetrate the cell, which will then occupy the space and make the sample hard enough to bear the pressure of sectioning or cutting. This process is also called embedding. The resin is then kept in an oven at 60° overnight to allow for setting. This process is called polymerization.
EPON resin
Skin
EPON resin
Mold
B
C
D
Resin
E
Skin
BA
block
CA
VS
BS
OS
TA
Chuck
F
0
19/4KO2ac
19/4-WTCd
Steps to trim block face
¥ Razor
IRL
19/4KO2tb

Trimming, Cutting, and Staining

. The sample should be semi-transparent to allow the passage of electron beams through it by sectioning (cutting) the sample into fine sections using a glass or diamond knife.
. The sections (30 nm - 60 nm) moved to a copper grid to be viewed under the microscope.
· Staining in this process, uranyl acetate (UA), followed by lead citrate are used to increase the contrast between different structures in the specimen, and also to scatter the electron beams.
ultra 35°
0 30C. elegans
Specimen fixed and
embedded in plastic
Specimen sliced into ~50 nm
sections with a diamond knife
Sections picked up on
a TEM grid
A cross-section of C. elegans
TEM imaging
A grid mounted on the
sample holder for TEM

Ultramicrotome Section Color Reference Chart

A
C
B
Sample Slices
TEM Cu grid
C Film
Flat Sample Slices
1
TEM Cu grid
240 -GA International
8
0039
XST XARA
B
C
B
G
H
anna
J
K
L
M
..
.
O
O
21-3
P
Q
Z
O
E
FS. No

Light Microscope vs. Electron Microscope Comparison