ELECTRON

MICROSCOPY
 PARTICIPANTS

• MUHAMMAD TAYYAB 209

• HAFIZA ZOHA 175
• ARFA NOOR 176
• MAIMOON AHMED 187
• MUHAMMAD AHMED 119
• ABDUL RAHEEM 169
• ALI NAVEED 172
• SULTAN SAJID 170
• ALI AKHTAR 124
• ZAIN 110
 INTRODUCTION

The word microscope is derived from greek

terms, “mikros” meaning “small” and
“skopein” meaning “to see”

A scientific instrument used to magnify and observe small objects, samples,

or tissues that are invisible or difficult to see with the naked eye
 TYPES OF MICROSCOPE

Light compound fluorescence electron

microscope microscope microscope microscope
ZACHARIAS JANSEN ROBERT HOOK ANTONIE VAN LEEUWNHOOK

a Dutch spectacle he examined the cork of is known as the father of

maker associated with tree under a microscope microbiology Made more than 250
the invention of the first and reported his findings microscopes, the most powerful of
true compound in his book “micrographia” which would magnify about 200-300
microscope which further led to the times. His descriptions of protozoa
development of cell theory using the microscopes were so
accurate that many of the described
forms are easily recognized today
ELECTRON MICROSCOPE

• A Scientific instrument that uses A

BEAM OF ELECTRONS to produce
high resolution images of tiny
structures at NANO SCALE.

• It offers resolution of 0.2NM and

magnifying power up to 1 million
times.
 Light microscope has a resolution of about 0.2 μm which greatly
compromises the usefulness for detailed studies of many
microorganisms. Since the detailed internal structure of
microorganisms cannot be effectively studied by light microscope,
electron microscope is used.

 In electron microscopy, electrons replace light as the illuminating

beam. The electron beam can be focused much as light in a light
microscope but its wavelength is about 100,000 times shorter than
that of visible light therefore electron microscope has a resolution
1000 times better than light microscopes with many electron
microscopes points closer than 0.2 nm can be distinguished and
the magnification is well over 100,000x
transmission electron microscope

Scanning electron microscope

Cryo-electron microscope
 Transmission Electron Microscope

 A Type of electron microscope that uses a beam of electrons to

produce high resolution images of the internal structures of thin
samples

 TEM consists of following components:

1. Electron gun
2. Condenser lens (electromagnet)
3. Objective lens (electromagnet)
4. Viewing screen
WORKING OF TEM
 WORKING OF TEM

1. Electron Source
• The microscope has an electron gun that emits electrons by heating a filament
or using a field emission source.

2. Beam Formation
• The accelerated electron beam is focused into a fine, coherent beam using
electromagnetic lenses (condenser lenses).
• Apertures are used to control the intensity and shape of the electron beam.

3. Specimen Interaction
• The electron beam passes through an ultra-thin specimen (thickness
typically less than 100 nm).
 WORKING OF TEM

As electrons interact with the specimen, they may:

 Transmit through (unscattered electrons).
 Scatter elastically (without energy loss) or inelastically (with energy loss),
depending on the specimen's structure and composition.

4. Image Formation
• The transmitted electrons are focused by objective lenses to form an initial
magnified image of the specimen.
• This image is further magnified by intermediate and projector lenses.
 WORKING OF TEM

5. Detection
• The magnified electron image is projected onto a fluorescent screen,
photographic film, or a digital camera.
• The resulting image shows variations in electron intensity, corresponding to
the specimen's structure, thickness, and composition.

 Applications:

 High-resolution imaging to study structures at the atomic scale.

 Studying crystal structures, defects, and grain boundaries.
 Observing cellular structures like organelles, viruses, and proteins.
 SCANNING ELECTRON
MICROSCOPE
 SEM, or Scanning Electron Microscopy, is a technique used to produce
high-resolution images of the surface of a sample by scanning it with a
focused beam of electrons.

 SEM consists of following components:

1. Electron source
2. Anode
3. Condenser lenses
4. Objective lens
5. BSE detector
6. X-ray detector
7. SE detector
8. Viewing screen
SCANNING ELECTRON
MICROSCOPE
 WORKING OF SEM

1. Electron Source
• Electron Gun: Electrons are emitted from a source (thermionic emission or
field emission) and accelerated by applying a high voltage (typically 1–30 kV).
Common electron sources:
• Tungsten filament.
• Field Emission Gun (FEG) for higher resolution.

2. Electron Beam Focusing

• The emitted electrons form a broad beam, which is narrowed and focused into
a fine, intense beam using:
• Electromagnetic lenses (condenser and objective lenses).
• Apertures to control the beam diameter and eliminate stray electrons.
 WORKING OF SEM

3. Beam Scanning
• The focused electron beam is deflected using scan coils to systematically
sweep over the sample surface in a raster pattern.
• The scanning occurs line by line, pixel by pixel.

4. Electron-Sample Interaction
• As the electron beam interacts with the sample, several types of signals are
generated due to the interaction of the high-energy electrons with the atoms in
the sample:
 Signals Generated:

 Secondary Electrons (SE):

• Ejected from the sample’s surface atoms.
• Provide high-resolution images of the surface topology.
 WORKING OF SEM

 Backscattered Electrons (BSE):

• Electrons reflected back from the sample.
• Provide compositional contrast (heavier elements reflect more electrons).

 Characteristic X-rays:
• Generated when an electron from an outer shell fills an inner-shell vacancy in
an atom, releasing energy.
• Used for elemental analysis via Energy Dispersive X-ray Spectroscopy
(EDS).

5. Signal Detection
Detectors capture these signals:
• SE Detector: Produces images with fine surface detail.
• BSE Detector: Shows atomic number contrast.
 WORKING OF SEM

• X-ray Detector (EDS): Identifies elements present.

6. Image Formation
• Detected signals are converted into electrical signals.
• The signals are synchronized with the scanning pattern to form an image on a
computer screen.
• The brightness or intensity of each pixel in the image corresponds to the
signal strength.

 Applications of SEM
 Visualizing textures and features of material surfaces.
 Examining microorganisms and tissues.
 Analyzing residues and surface evidence.
 Cryo-Electron Microscope

 The Cryo-Electron Microscope (Cryo-EM) is a powerful imaging technique

used to study biological molecules, cells, and complexes at near-atomic
resolution.

Cryo-EM consists of following components:

1. Electron gun
2. Electromagnetic lenses
3. Cryo-Stage and Sample Holder
4. Sample Grid
5. Vacuum System
6. Detectors
7. Cryogenic System
8. Imaging Software
Cryo-Electron Microscope
 WORKING OF CRYO-EM

1. Sample Preparation
• Specimen Preparation:
• A small volume of the biological sample is applied to a specialized grid with a
thin film.
• liquid is removed, leaving a thin layer of the sample.

• Vitrification:
The sample is rapidly frozen in liquid ethane at cryogenic temperatures (~-180°C).
This forms an amorphous, glass-like ice, preserving the sample in its native
hydrated state without forming damaging ice crystals.
 WORKING OF CRYO-EM
2. Sample Handling
• Cryogenic Conditions:
The vitrified grid is transferred to the microscope using a cryo-holder or cryo-stage
that maintains ultra-low temperatures to prevent sublimation or structural
changes.

• Vacuum Environment:
The microscope chamber is maintained under high vacuum conditions to minimize
electron scattering by air molecules.

3. Electron Beam Generation

• Electron Gun:
• A high-energy electron source generates a coherent electron beam (100–300
kV).
• Electrons are focused and directed towards the sample using electromagnetic
lenses.
 WORKING OF CRYO-EM

4. Electron-Sample Interaction
• Transmission and Scattering:
• The electron beam passes through the thin vitrified sample.
• Some electrons pass through unscattered, while others scatter elastically or
inelastically, interacting with the sample's atomic structures.

5. Image Formation
• Direct Electron Detector (DED):
• The scattered and transmitted electrons form an image on a direct electron
detector, which captures high-resolution 2D images.
• Unlike traditional detectors, DEDs are highly sensitive and capable of
detecting fine structural details.
 WORKING OF CRYO-EM

6. Data Acquisition
• Multiple Images:
• Thousands of 2D images are taken from different angles or particles to ensure
a comprehensive dataset.
• Cryo-Tomography (if applicable):
• For larger or heterogeneous samples, images are collected at multiple tilt
angles. This data is used to reconstruct a 3D tomogram.

7. Image Processing
• Alignment and Averaging:
• In single-particle Cryo-EM, individual images of identical particles are aligned
and averaged to enhance signal-to-noise ratio.
• Noise Reduction:
• Advanced algorithms reduce noise to enhance image clarity.
 WORKING OF CRYO-EM

• 3D Reconstruction:
• The processed images are combined using computational methods to produce
a detailed 3D structure of the sample.

8. Structural Analysis
• Atomic Modeling:
• The reconstructed 3D structure is analyzed to determine atomic positions,
molecular conformations, and interactions.
• Functional Insights:
• Researchers interpret the structural data to understand biological mechanisms
or design drugs.
 WORKING OF CRYO-EM

 Applications:
 Protein structure determination (e.g., enzymes, ribosomes).
 Drug design and discovery.
 Virus and cellular imaging (e.g., SARS-CoV-2 spike protein).
 Molecular and cellular biology research.
THANK YOU SO MUCH