Transmission electron microscope Dr Mounika

1. INTRODUCTION TO ELECTRON MICROSCOPY,
TRANSMISSION ELECTRON MICROSCOPY IN DETAIL-
INCLUDING FIXATION, PROCESSING & SECTIONING
OF SPECIMENS FOR TEM STUDY
DR. MOUNIKA. S
Post graduate student
Department of Oral Pathology & Microbiology
SRM Dental College,
Ramapuram, Chennai, India

2. CONTENTS
 APPLICATIONS OF TEM
ADVANTAGES &
DISADVANTAGES OF TEM
 SAMPLE PREPARATION
 FIXATION
 PROCESSING
 SECTIONING
 STAINING
 INTRODUCTION
 COMPONENTS OF ELECTRON
MICROSCOPE
 TRANSMISSION ELECTRON
MICROSCOPY
 PRINCIPLES OF TEM
 ELECTRON INTERACTION IN
TEM
INSTRUMENTATION- VACCUM,
ELECTRICAL, MICROSCOPE
COLUMN& ELECTRON OPTICS 2

3.  Microscope magnifies the image of the object so that we can visualize the
smallest particles.
 Wavelength in light microscopy is 300-700 nm
 During the first part of the twentieth century, the wave like property of the
electron was demonstrated, and subsequently this has been utilized in
electron microscope (EM).
 Max Knoll & Ernest Rusker- DISCOVERED EM
INTRODUCTION

4. v According to De Brogle's formula, λ=h/mv
v λ is wavelength, h = 6.626 × 10−34 (Planck’s constant), m = mass
and v = speed of the electron.
v Increase in speed of electron can decrease the wavelength-
better resolution.
v Wavelength in EM- 0.2 nm.
v EM images provide key information on the structural basis of
cell function and of cell disease

5. PROPERTIES OF ELECTRON
Electrons have shorter wavelength and
provide very high-resolution capacity.
It is easy to manipulate
Electron gives high brightness
Electron beam interacts strongly with
matter

6. ELECTRON
MICROSCOPY
TEM
BFTEM DFTEM EFTEM ED
SEM AEM STEM

7. COMPARISON BETWEEN LIGHT AND
ELECTRON MICROSCOPES

9. COMPONENTS OF ELECTRON MICROSCOPE
1. Electron source
2. Sample
illumination
3. Objective lens
4. Intermediate and
projector lenses
5. Detectors
Electron Source
Electron gun
generates the beam
of electrons.
It consists of a: (a)
Tungsten filament
(b) Wehnelt cylinder
(cathode shield) (c)
Anode plate

11. TRANSMISSION ELECTRON MICROSCOPY&
PRINCIPLES

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1 3 5
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4
2
LIGHT FROM LIGHT
SOURCE
MAGNIFIED IMAGE
BY OBJECTIVE
LENS
BRIGHTELY
ILLUMINATED
FIELD OF VISION
PASSES THROUGH
TRANSPARENT OBJECT
VIRTUAL IMAGE BY
OCCULAR LENS
LIGHT MICROSCOPY

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1 3 5
6
4
2
COUNTERPART OF
LIGHT
MICROSCOPE
50% INCIDENT
ELECTRONS
THROUGH ULTRA
THIN SECTIONS
2D IMAGES ARE
SEEN
PASSAGE OF HIGH
VELOCITY HOMOGENOUS
ELECTRONS
EMERGENT BEAM
FOCUSSED TO EML
TRANSMISSION ELECTRON MICROSCOPY

15. ELECTRON INTERACTION IN TEM

16. INSTRUMENTATION OF TEM
THE VACCUM
SYSTEM
THE
ELECTRICAL
SYSTEM
MICROSCOPE
COLUMN &
ELECTRON
OPTICS

17. THE VACCUM SYSTEM
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 Electron beam should be inside a vacum chamber for 2 reasons:
1. To prevent gas molecules colliding with electrons
2. Prevents oxidation of tungsten molecules thus, increasing
longevity
 A series of pumps are employed it includes,
MECHANICAL
ROTARY
PUMP 1x10-
2Pa
DIFFUSION
PUMP 1X10 -
5Pa
TURBO
MOLECULAR
PUMP
ION
PUMP 1x10-
7Pa

18. THE ELECTRICAL SYSTEM
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HIGH TENSION
UNIT
LENS CURRENT
SUPPLY UNIT
VOLTAGE & LENS
CURRENT
STABILISER UNIT

19. MICROSCOPE COLUMN &ELECTRON OPTICS
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ELECTRON
SOURCE
CONDENSOR
LENSES
SAMPLE
OBJECTIVE
LENS
PROJECTOR
LENSES
DETECTORS

20. SPECIMEN PREPARATION FOR TEM
FIXATION DEHYDRATION PROCESSING SECTIONING STAINING

21. FIXATION
⇨To prevent any change in the tissue and preserve the
tissue as much as possible to its living condition
⇨To prepare the tissue for the further processing so
that the tissue does not disintegrate or tear
⇨No ideal fixative- depends on the tissue type
⇨Glutaraldehyde + osmium tetroxide.
⇨Average time of fixation is 9 h by 4%
⇨ Glutaraldehyde at room temperature and 1 h for
osmium tetroxide
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22. DEHYDRATION
⇨ The sample is treated with the series of graded
alcohol:
⇨ 30% ethyl alcohol-10 min
⇨ 50% ethyl alcohol-10 min
⇨ 70% ethyl alcohol- 10 min
⇨ 90% ethyl alcohol-10 min
⇨ 100% ethyl alcohol-10 min
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23. PROCESSING
⇨ The ideal embedding medium should have the following
desirable criteria:
⇨ Easy to cut the section
⇨ Stable in electron beam and withstand higher
temperature (200 s °C) at the time of microscopy
⇨ Easy to procure the medium
⇨ Evenly polymerized
⇨ Presently the following media are used for EM: 1. Epoxy
resin 2. Acrylic media 3. Polyester resin.
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24. SECTIONING
⇨Semi thin sections and ultrathin sections are made
using ultrathin microtomy.
⇨Sections should be less than 100 nm thick section
and the optimum thickness is 80 nm.
⇨Glass knives
⇨Diamond knives are used
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25. STAINING
⇨ Reynold’s lead citrate solution is used for the
staining.
⇨ Lead rapidly reacts with the atmospheric carbon
dioxide and may form lead carbonate as
precipitate.
⇨ Therefore adequate care should be taken to prevent
such precipitation.
⇨ Alternatively uranyl acetate can be used.
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26. ADVANTAGES
⇨ Powerful magnification
⇨ Examine structure, composition, and properties of
specimens in submicron levels.
⇨ Image morphology samples
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27. DISADVANTAGES
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⇨ Size, cost, maintenance, researcher training, artefacts.
⇨ Cannot take color pictures.
⇨ Cannot image through thick samples.
⇨ Cannot image surface information.

28. “
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Diagnosis TEM+LIGHT
MICROSCOPE
Essential research tool-
life sciences& medicine
SEM are limited in
diagnostic wide spread
distribution unlike TEM.
Real space imaging of
nanoparticles
chemical, electronic and
structure of individual
nanocrystals by electron
probe.
Ultra structures of virus,
function of various viral
components.
Association of
genetically altered
proteins - vaccine
production.
Identification of unknown
virus
Bioterrorism and
emerging diseases
surveillance
APPLICATIONS OF TEM

29. “
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30. Thank you
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