Steps involved in Electrophoresis, Light Microscope, Scanning and Transmission Electron Microscope. Differences among them, Advantages and Limitations.

1. PROFESSOR JAYASHANKAR TELANGANA STATE
AGRICULTURAL UNIVERSITY
PRESENTATION ON : Electrophoresis, Use of Light Transmission and Scanning Electron
Microscopy.
COURSE TITLE : Techniques in Plant Protection (0+1).
Submitted by :
Prajwal Gowda M.A
RAM/2020-50
Msc-Ag , 2nd Year
Dept of Entomology.

2. ELECTROPHORESIS
“Electrophoresis is a common genetic lab technique used to separate charged particles
such as DNA or RNA based on their size.”
• DNA has no color or smell and so we can’t see it. If we can’t find it, how can we analyze
and process it? It’s hard, right! But what if somehow we paint our DNA or fragments of
DNA, use its negative charge to run under an electrical field?
• We can see DNA, not like the typical spiral/helical structure but we can observe it like a
sharp band. That’s what electrophoresis serves.
• It is a process to identify, separate, and characterize biological molecules like DNA,
RNA, or proteins, under the influence of the constant electrical current.
Electrophoresis is used to separate and the protein or gene of interest is excised out from
the entire sample (Purification).

3. AIMSANDOBJECTIVESOFELECTROPHORESIS
• To identify biomolecules like DNA, RNA or proteins.
• To determine the size of biomolecules.
• To run biomolecules under electrical current.
• It is used to identify microorganisms in agriculture.
Principle of Agarose gel Electrophoresis.
-If a mixture of electrically charged biomolecules is placed in an electric field, they will
freely move towards the electrode of opposite charge. However, different molecules will move
at quite different and individual rates depending on the physical characteristics of the
molecule.
Three of the important factors that affect DNA migration rate are,
• Agarose concentration
If the concentration of agarose is lower, the DNA can migrate easily and faster and vice versa.

4. • Confirmation of DNA
The supercoiled DNA migrates faster than the linear DNA and circular DNA.
The supercoiled form of DNA is more compact than other forms causing speedy migration.
• Size of DNA
Larger DNA fragments run slower while smaller DNA fragments run faster.
Commonly followed is Gel Electrophoresis.
METHODOLOGY
It’s a 3 step process.
1. Preparing a gels and other materials.
2. Running a power-on electrophoresis.
3. Placing a gel under U.V light for observation.

5. GelElectrophoresis
Gel is a cross linked polymer.
Types of Gel:
 Agarose gel
Agar is a jelly-like substance consisting of polysaccharides obtained from the cell walls of
red algae, primarily from Gracilaria and Tengusa.
The use of agar gel around its ability to form gels. The large difference between the temperature
at which gel is formed and the temperature at which it melts is unusual, and unique to agar.
 Polyacrylamide gel components:
Acrylamide monomers, Ammonium persulphate, Tetramethylenediamine (TEMED),
N,N’-methylenebisacrylamide. These free radicals activate the monomers inducing them to react
with other monomers forming long chains.
Used to separate proteins and small oligonucleotides because of the presence of small pores.

7.  Red is a positive node and black is negative node.
 The EtBr makes DNA visible.
The rate of DNA migration is inversely proportional
to the size of it & to the concentration of agarose gel.
Why do we use agarose in Gel electrophoresis?
The agarose powder used in the AGE process provides a
matrix for DNA or RNA to pass through, under the
influence of an electric current.
Pores of agarose polysaccharides allow DNA to go
through.

8. MaterialPreparation.
Gels and Buffers are the most important materials.
Agarose gel: 50mM Tris-base, 30mM sodium acetate, 3mM EDTA in deionized water, pH of nearly
7.8 with 96% acetic acid.
TAE buffer: 2M sucrose, 100mM EDTA, 0.2mM bromophenol blue.
Indicator: As gels and macromolecules are colorless and transparent, the original strips are invisible
after being exposed to U.V.
Thus, their presence is identified either by staining or by using gene probes. Thereby
revealing the presence of the target fragment or gene.
Procedure
 Place the agarose gel in the electrophoresis apparatus.
Fill the chamber with 1x TAE buffer to cover the gel.
 Use a separate nozzle for each sample, and insert the sample into the well.

9.  Place the lid and switch on 100V power supply and electrify for 30min.
 Expose the gel to U.V (254nm) and collect the images.
OUTCOME.
Different bands of gel are visible under U.V light, representing different DNA fragments. The same
position indicates the same DNA fragments as molecules of the same type move at equal speed.
APPLICATIONS OF ELECTROPHORESIS.
• gDNA isolation,
• PCR amplification,
• Mutation detection,
• Restriction digestion & Gene cloning,
• DNA sequencing, Gene amplification.

10. LIGHT MICROSCOPE
Optical microscope, also referred to as a light microscope.
A type of microscope that commonly uses visible light &
a system of lenses which first focus a beam of light onto or
through an object, and convex objective lenses to enlarge
the image formed to generate magnified pics of small objects.
These are used to study living cells and for regular use when
relatively low magnification & resolution is enough.
Advantages of LM
a. Less expensive & easy to operate.
b. Both living and dead specimens can be viewed.
c. The original colour of the specimen can be viewed.
Disadvantages of LM
can be used only in the presence of light & has lower resolution. Light Microscope

11. Parts of Light Microscope.
 Base - the flat structure that serves as its foundation
 Arm - connects the base of the microscope to the nosepiece and eyepiece; used to carry the microscope around
 Stage - the flat platform where the slide is put in place for viewing; can be adjusted via the coarse and fine
adjustment knobs
 llluminator - steady light source
 Diaphragm - an adjustable apparatus located below the stage which can be manually modified to vary the
intensity of light that reaches the specimen
 Body Tube - connects the eyepiece to the objective lenses
 Condenser Lens - collects the light from the illuminator and focuses it on the specimen
 Ocular Lens- lenses that the viewer looks through
 Eyepiece Adjustment Knobs - used to bring the specimen into focus
 Stage clips - used to keep the slide in place

13. Difference between Simple & Compound Microscope.
Simple Microscope Compound Microscope
Only single lens is present 3 to 5 objective lenses
Only one lens for magnifying objects Has two sets of lens(eyepiece and objective)
Condenser lens is absent Condenser lens is present
Light source is natural Source: llluminator
Has only one adjustment screw that is used for
focusing an object.
Has coarse adjustment screw (for rapid
focusing an object) and fine adjustment screw
(for fine and sharp focusing).
Mirror is concave-reflecting type Mirror is plane at one side and concave at other
side
Total magnification is limited Higher magnification
Usage of coarse, hooks, and knobs is not that
much.
Use of knobs is much, which help in focusing
and as a result, a clear and concise image is
seen.

14. ELECTRON MICROSCOPY (EM)
An EM uses a beam of accelerated electrons as a source of illumination. It was invented by Knoll & Ruska in the year
1931.
Principle of Electron Microscopy
o Electrons are such small particles that, they act as waves. A beam of electrons passes through the specimen, then
through a series of lenses that magnify the image.
o Since the resolving power is inversely proportional to the wavelength, so it has high resolving power because of its
shorter wavelength.
The image results from a scattering of electrons by atoms in the specimen.
o The biggest advantage is that they have a higher resolution and are therefore also able of a higher magnification (up to 2
million times).
o It’s helpful in viewing surface details of a specimen.
Parts of Electron Microscope
1. Electron gun: The electron gun is a heated tungsten filament, which generates electrons.

15. 2. Electromagnetic lenses
 Condenser lens focuses the electron beam on specimen. A second condenser lens forms the electrons
into a thin tight beam.
 Objective lens, which has high power and forms the intermediate magnified image.
 The third set of magnetic lenses called projector (ocular) lenses produce the final further magnified
image.
 Each of these lenses acts as an image magnifier all the while maintaining an incredible level of detail
and resolution.
3. Specimen Holder
 The specimen holder is an extremely thin film of carbon or collodion held by a metal grid.
There are 2 main types of electron microscope – the transmission EM (TEM) and the scanning EM
(SEM).
The transmission electron microscope is used to view thin specimens (tissue sections, molecules, etc)
through which electrons can pass generating a projection image.

16. o If you need to look at a relatively large area and only need surface details, SEM is ideal.
o If you need internal details of small samples at near-atomic resolution, TEM will be necessary.
1. SCANNING ELECTRON MICROSCOPE.
SEM uses a fine beam of focused electrons to scan a sample’s surface. This records information about
the interaction between the electrons and sample, creating a magnified image.
How does SEM work?
 To get a high-resolution, an electron source (known as an electron gun) emits a stream of high-energy
electrons towards a sample. The electron beam is focused using electromagnetic lenses.
 Once the focused stream reaches the sample, it scans its surface in a rectangular raster.
The interaction between the electron beam and sample creates secondary electrons, backscattered
electrons, and X-rays. These interactions are captured to create a magnified image.
SIMILARITIES BETWEEN SEM & TEM.
 Each has an electron gun that emits an electron stream towards a sample in a vacuum, and each
contains lenses and electron apertures to control the electron beam and capture images.

17. SCANNING ELECTRON MICROSCOPE.

18. TRANSMISSION ELECTRON MICROSCOPE.
A TEM is a type of electron microscope that uses a broad beam of
electrons to create an image of a sample’s internal structure.
It is used to identify and characterize viruses, bacteria, protozoa and
fungi and to study the morphology of cells and their organelles.
How does a TEM work?
An electron source sends a beam of electrons through an ultrathin
sample. When the electrons penetrate the sample, they pass
through lenses below.
• The specimen to be examined is prepared as an extremely thin
dry film or as an ultra-thin section on a small screen and is
introduced into the microscope at a point between the
magnetic condenser and the magnetic objective.
• Section must be less than 80 nm thick which allows at least 50%
of the electron beam to penetrate the sample.

19. DIFFERENCES BETWEEN SEM & TEM.
SEM TEM
Electron stream Fine, focused beam Broad beam
Image taken Topographical/surface Internal structure
Resolution Lesser Higher
Magnification Up to 2,000,000 times Up to 50,000,000 times
Image dimension 3-D 2-D
Sample thickness Thin and thick samples Ultrathin samples only
Penetrates sample No Yes
Sample preparation Less preparation required More
Cost Less expensive More
Speed Faster Slower
Operation Easy to use Complex, needs training.

20. Sample preparation for TEM
For sample preparation three important steps are Processing, Embedding and Polymerization.
Processing further includes fixing, rinsing, post fixation, dehydration and filtration.
1. Fixation of tissues.
Double fixation is the most popular method, as it involves primary fixation in an aldehyde followed by
post fixation in osmium tetroxide. Glutaraldehyde (dialdehyde) preserves ultrastructure well but
penetrates slowly than the monoaldehyde, paraformaldehyde. Glutaraldehyde is used alone for small
pieces of material, but a mixture of the two aldehydes may be used for fixation of larger items.
2. Rinsing of specimen TEM.
In order to remove excess glutaraldehyde from samples, the specimens are washed in 0.1 M cacodylic
acid buffer whose pH is 7.3.
3. Secondary/ post fixation of the specimen.
Secondary fixation is done by osmium tetroxide for 1.5 hrs at room temperature.

21. Dehydrating Percentage of Ethanol.
Dehydration in Time
50% ethanol 5 minutes
70% ethanol 10 minutes
80% ethanol 10 minutes
90% ethanol 15 minutes
99.9% ethanol (twice) 20 minutes
4. Dehydrating the Specimen.
Organic solvents (methanol, ethanol or
acetone) are usually used. Acetone is
preferred as there is less lipid loss than with
ethanol dehydration.
The specimen can be dehydrated in a graded
series of ethanol/acetone.

22. 5. Alcohol substitution
It involves replacement of the dehydration solution (ethanol/ acetone) by another intermediary solvent
(propylene oxide), and it involves infiltration of the specimen with a transitional solvent.
It involves overnight immersion of specimen in propylene oxide (1:1, v/v) at room temperature.
Next day the freshly prepared durcupan ACM mixture (pure) should be made and specimens should
be immersed in it and left for 2 h at room temperature.
6. Embedding media
The embedding media for EM are resins, methacrylates. For general electron microscopy epoxy resins
have most properties that are desired for electron microscopy.
Addition of curing agents is required to convert them to a tough, extremely adhesive and highly
inert solid. Labelling should be done at this point.
7. Polymerization
Polymerization of the epoxy material can be achieved by placing the specimen in a drying cabinet for
2 days (40º C).

23. 8. Sectioning
In order to cut ultra-thin sections glass knives/ diamond knives are used.
Getting an ultra-thin section is the prerequisite for TEM.
9. Staining of the specimen
Semi-thin sections (0.5 µm to 2 µm) are stained with toluidine blue on a hot plate
(70ºC to 90ºC) and is examined by LM. Ultrathin sections (70 nm to 90 nm) are
then stained with uranyl acetate followed by lead citrate.

24. Sample preparation for SEM
It involves cleaning the surface, stabilizing, rinsing, dehydrating, drying, mounting and
coating the specimen.
1.Cleaning the surface of specimen.
The proper cleaning of the surface of the specimen is important as the surface of the
specimen may contain dust, mud, soil etc. depending on its source of collection.
2.Stabilizing the specimen.
Fixation is usually performed by incubation in a solution of a buffered chemical fixative
like glutaraldehyde, sometimes in combination with formaldehyde and post fixation by
osmium tetroxide or tannic oxide.
3.Rinsing the specimen.
In order to remove excessive fixative, samples must be rinsed properly.

25. 4. Dehydrating the specimen.
This is to remove free water in the fixed tissue in a graded series of acetone or ethanol.
5. Drying the specimen.
The specimens must be dry in case of SEM, since the specimen chamber is at high vacuum
otherwise the sample will be destroyed in the electron microscope chamber.
6. Mounting the specimen.
The specimens should be mounted on a tub using an adhesive (epoxy resin) or adhesive
tape, and sputter-coated with gold or palladium before examining in the microscope.
7. Coating the specimen.
Specimens are coated with a thin layer of approximately 20 nm to 30 nm of a conductive
metal like gold, palladium or platinum. The main objective of coating the specimen is to
increase its conductivity and to provide the buildup of high voltage charges on the
specimen.

26. LIMITATIONS OF EM.
 The live specimen cannot be observed.
 As the penetration power of the electron beam is very low, the object should be ultra-thin.
 As the EM works in a vacuum, the specimen should be completely dry.
 Expensive to build and maintain.
 Requiring researcher training.
 Cumbersome extremely sensitive to vibration and external magnetic fields.

27. References
Asthagiri, A.R and Arkin, A.P. 2016. Computational methods in cell biology. Methods in Cell Biology.
110(3): 1–10.
Bisma Ayub and Mudasir Ali. 2017. Specimen preparation for electron microscopy: an overview. Journal
of Environment and Life Sciences. 2 (3): 85-88.
Chin, C.S and Beow, C.Y. 2013. Electrophoresis: What does a century old technology hold for the future of
separation science?. International Research Journal of Applied and Basic Sciences. 7 (4): 213-221.
Gel-Electrophoresis and Its Applications by P. Rabindra Reddy and N. Raju.
https://microbenotes.com/scanning -electron-microscope-sem/
Yiran cai. 2019. Analysis on Gel Electrophoresis in Biology. The Journal of Chemical Physics. 119 (1):
44-51.

28. THANK YOU