Magnification and

Electron Microscopy
By Ishani and Varuni
Table of contents
01 Magnification, Resolution, and Contrast

02 Electron Microscopy
 Magnificati
01 on,
Resolution,
and
contrast
Microscopic slide preparation
Dry mount:
https://youtu.be/MDTXJAmhTf0?si=SBLO0HncsSkX3QNjhttps://youtu.be/MDTXJAmhTf0?si=SBLO0H
ncsSkX3QN
Wet mount:
https://youtu.be/yxTFgDe5CEE?si=ecR7zfKJ_eM0PcWb
Squash slides:
https://youtu.be/c2h0J-0wnzE?si=hYeJdWAVyykO9h0I
Smear slides
https://youtube.com/shorts/hcoygUP58ss?si=-C9Fs3M_-kJcDzFm
● Stained → transparent(cytosol) / cell structures that are difficult
to distinguish
● To stain a slide the sample needs to be first air-dried and then
heated by passing it through a Bunsen burner flame – this will
allow the sample to be fixed to the slide and to take up the stain
● Type of stain depends on the type of specimen used
● Common types:
Eyepiece graticules and stage
micrometers
● Eyepiece graticule:
○ A disc that fits in the eyepiece with a fine scale (100 divisions on it)
○ It has to be calibrated for every magnification and microscope
● Stage micrometer
○ Scale fitted on the cover slip (Placed on the stage of the
microscope)
○ Exactly 1mm long, divided into 100 divisions →
1 division= 0.01mm = 10µm
○ Like a small ruler on the cover slip
● 40 graticule divisions = 100 µm
1 graticule division = number of micrometres ÷ number of
graticule division
100 ÷ 40 = 2.5 µm this is the magnification factor
graticule divisions x magnification factor = measurement (µm)
● https://youtu.be/PW4s42K-zio?si=4NYR2Ecqn4LDfEBW
Magnification
● Ability of a lens
to enlarge an
object when
compared to the
real object
● Magnification is
not limited in
microscopes,
resolution is.
● A light microscope has 2 types of lens:
○ Eyepiece lens: X10
○ Series of objective lens (usually 3) each with a different
magnification
○ Total magnification = magnification of eyepiece lens X
magnification of objective lens

Contrast
● Required to distinguish between small structures +
transparent microorganisms
● Done by using…
- Features of light/electrons
- Stains
Resolution
● Ability to distinguish between 2 separate points.
● Factors affecting resolution
○ Numerical aperture: maximum angle of light coming from
the sample that the lens can collect
○ Wavelength
● Diffraction limits the ability of light microscopes to resolve small
objects. As wavelength increases, diffraction increases.
● High energy waves (UV and X rays) have a high frequency and
low wavelength → Electrons have a lower wavelength than light
so resolution is higher
● Light microscope: 200 nm; Electron microscope: 0.5 nm
● Rule: Limit of resolution (smallest distance between two points
that can still be distinguished as separate entities in an image)
is ½ the wavelength of the radiation used
How
microscopes
work
The objective lens focuses
diffracted light and forms an
image
If the angle at which light is
diffracted is too high, the lens
cannot collect all the diffracted
light so the image is blurred
 02
Electron Microscopy
Introductio
n
The maximum theoretical resolution of
images created by light microscopes is
ultimately limited by the wavelengths of
visible light, which does not begin to
approach the magnifying power of an
electron microscope (EM), which uses
short-wavelength electron beams rather
than light to increase magnification and
resolution.
Electrons, like electromagnetic radiation,
can behave as waves, but with
wavelengths of 0.005 nm, they can
produce much better resolution than visible
light. An EM can produce a sharp image
that is magnified up to 100,000⨯.
 Preparation of
Samples
Samples to be analysed using a TEM must have
extremely thin slices. However, cells are too soft
to cut thinly, even with diamond cutters. To cut
cells without causing harm, they must be
imbedded in plastic resin and subsequently
dehydrated using a succession of ethanol soaks
(50%, 60%, 70%, and so on). Ethanol replaces
water in the cells, and the resin dissolves and
enters the cell, solidifying. Then, tiny slices are
cut with a specialised apparatus known as an
ultramicrotome. Finally, samples are mounted
on tiny copper wire or carbon-fiber grids and
stained with electron-dense heavy metal atoms
such as uranyl acetate or osmium tetroxide,
rather than coloured dyes.
continued…
When samples are prepared for
viewing using an SEM, they must also
be dehydrated using an ethanol
series. However, they must be even
drier than is necessary for a TEM.
Critical point drying with inert liquid
carbon dioxide under pressure is used
to displace the water from the
specimen. After drying, the specimens
are sputter-coated with metal by
knocking atoms off of a palladium
target, with energetic particles.
Sputter-coating prevents specimens
from becoming charged by the SEM’s
electron beam.
How a TEM
works:
A TEM uses an electron beam from above
the specimen that is focused using a
magnetic lens (rather than a glass lens)
and projected through the specimen onto
a detector. Electrons pass through the
specimen, and then the detector captures
the image. For electrons to pass through
the specimen in a TEM, the specimen
must be extremely thin (20–100 nm
thick). The image is produced because of
varying opacity in various parts of the
specimen. This opacity can be enhanced
by staining the specimen with materials
such as heavy metals, which are electron
dense. Since it is not possible to see an
electron beam, the electron beam has to
be projected onto a fluorescent screen.
Notes
● Electron microscopes, both scanning and transmission, are used for specimens
above 0.5 nm
● Electron microscopes fire a beam of electrons at the specimen either a broad
static beam (transmission) or a small beam that moves across the specimen
(scanning)
● The electrons are picked up by an electromagnetic lens which then shows the
image
● Due to the higher frequency of electron waves (a much shorter wavelength)
compared to visible light, the magnification and resolution of an electron
microscope is much better than a light microscope and electrons are selected
also due to the fact that they can be easily focused using electromagnets
● Electron microscopes are useful for looking at organelles, viruses and DNA as
well as looking at whole cells in more detail
● Electron microscopy requires the specimen to be dead however this can
provide a snapshot in time of what is occurring in a cell eg. DNA can be seen
replicating and chromosome position within the stages of mitosis are visible
continued…
● Requires a vacuum (electrons cannot be focused without a vacuum as they will
collide with air molecules and scatter)
● Water boils at room temperature in a vacuum, so the sample must be dehydrated
(specimen has to be dead)
● It is not possible to see an electron beam so to make the image visible it is
projected onto a fluorescent screen
● The areas hit by electrons shine brightly, giving an overall black and white picture
● Staining the tissue improves the contrast of biological specimens as the staining
agents contain heavy metal atoms which stop the passage of electrons
● The resulting image is like an X-ray photograph where more densely stained parts
appear blacker
● ‘False-colour’ images appear by coloring the image using a computer
● The detailed structure of a cell as revealed by an electron microscope is called its
ultrastructure
Exercise

1. What are the limits of

resolution of a light and
electron microscope?
2. Why do electron microscopes
have a higher resolution?
3. Give an example of a
negative stain
4. What is 1 division in a stage
micrometer equal to
(micrometers)
5. 4 types of microscopic slide
preparation
Exercise: True or false?
Facts True or false?
A TEM uses a typical glass lens

Samples prepared for an SEM need to be more dehydrated

than ones for a TEM

Samples are stained with electron dense heavy metal ions

For electrons to pass through a sample in a TEM, the sample

must be 120-200 nm thick
 Thanks
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CREDITS: This presentation template was created by Slidesgo, and
includes icons by Flaticon, and infographics & images by Freepik