 Microscopy
is the technical field using
microscopes to view samples and objects that can
not be seen with unaided eye (objects that are not
within the resolution range of the normal eye).
There are three well-known types of microscopy:
Optical, Transmission Electron and Scanning
Electron microscopy.
 Resolution
can be defined as the least
distance between two closely placed objects,
at which they may be recognized as two
separate entities. The best resolution possible
in a LM is about 200 nm whereas a typical
SEM has 10 nm and TEM has 0.2 nm.
Resolution power





Long before , in the hazy unrecorded past, a piece of
transparent crystal thicker in the middle than at the
edges, looked through it, and discovered that it made
things look larger.
Someone also found that such a crystal would focus the
sun's rays and set fire to a piece of parchment or cloth.
Magnifiers and "burning glasses" or "magnifying glasses" are
mentioned in the writings of Seneca and Pliny the Elder,
Roman philosophers during the first century A. D.
Spectacles were invented at the end of the 13th century.
They were named lenses because they are shaped like the
seeds of a lentil.
The earliest simple microscope was merely a tube with a
plate for the object at one end and a lens which gave a
magnification ten times the actual size. These were used
to view fleas or tiny creeping things and so were dubbed
"flea glasses.
Timeline of the Microscope






4000 years ago: Use of glass lenses and water in a tube in
China
3500 years ago: Ancient Egyptians and Romans used glass to
magnify objects
14th century: spectacles were first made in Italy
1656 "an instrument for viewing what is small," from Gk.
micro- small skopion "means of viewing," from skopein
"look at.“ The Greeks gave us the
word
"microscope
1590:
Two
Dutch
spectacle-makers
father-and-son
team, Hans and Zacharias Janssen, invented first
microscope.
1665:
Robert
Hooke's
famous
"Micrographia"
was
published using the microscope.

1675: Anton van Leeuwenhoek, who used a microscope
observe insects, bacteria and other objects.
to

1830: Joseph Jackson Lister, used weak lenses together at
various distances provided clear magnification.

1878: Ernst Abbe : mathematical theory linking resolution to light
wavelength

1903: Richard Zsigmondy: invents the ultra microscope, allows for
observation below the wavelength of light.

1932: Frits Xernike’s: Transparent biological material was studied
by phase-contrast microscope.

1938: Ernst Ruska: developed the electron microscope, which
enhanced resolution.

1981: Gerd Binnig and Heinrich Rohrer: 3-D specimen images
possible with the invention of the scanning tunneling
microscope
Robert Hooks
Microscope
Made of gold
and leather and
candle as light
source

Optical or light microscopy involves passing
visible light transmitted through or reflected
from the sample through a single or multiple
lenses to allow a magnified view of the sample.
The resulting image can be detected directly by
the eye or a photographic plate or captured
digitally. The single lens with its attachments, or
the system of lenses and imaging equipment,
along with the appropriate lighting equipment,
sample stage and support, makes up the basic
light microscope. The most recent development
is the digital microscope, which uses a CCD
camera to focus on the exhibit of interest
 Optical
microscopy is used extensively in
microelectronics, nanophysics, biotechnology,
pharmaceutical research, mineralogy and
microbiology.
 Optical microscopy is used for medical
diagnosis, the field of histopathology when
dealing with tissues, or in smear tests on free
cells or tissue fragments.
 In industrial use, binocular microscopes are
common. The use of dual eyepieces
reduces eye strain associated with long
workdays at a microscopy station.
 Simple
microscope
 Compound microscope







Bright field microscope
Dark field microscope
Fluorescence microscope
Phase contrast microscope
Polarized microscope
Confocal microscope
Digital microscope
 Compound
microscope magnified an image
by a single lens can be further magnified by a
second or more lenses.
 First
microscope
of
Antonie
van
Leeuwenhoek Father of microscope): the
specimen was mounted on the top of the
pointer, above which lay a convex lens
attached to a metal holder. The specimen
was then viewed through a hole on the other
side of the microscope and was focused using
a screw (500 lenses were prepared by
grinding gave variable magnifications)
 Charles
Hall,1730s: Achromatic lens and
second lens of different shape and refracting
properties realign colors with minimal impact
on the magnification of the first lens.
 Joseph Lister 1830: solved the problem of
spherical aberration (light bends at different
angles depending on where it hits the lens)
by placing lenses at precise distances from
each other.
 Ernst Leitz 1863: Introduction of the first
revolving turret.
 Abbe
Condenser: Abbe's work on a wave
theory of microscopic imaging developed
seventeen objectives lens-three of these
were first immersion oil objectives
 First microtome was used to enabled thinner
samples.
 August Kohler 1893: Zeiss employee figured
out an unparalleled illumination system
known as Kohler illumination corrected by
using double diaphragms
 Walter Flemming 1879: cell mitosis and
chromosomes
 19th/20th
centuries Louis Pasteur invented
pasteurization while Robert Koch discovered
his famous or infamous postulates: the
anthrax bacillus, the Tuberculosis bacillus
and the Cholera vibrio
 Unstained specimen has little contrast while
stained specimen with dyes have high
contrast.
The optical microscope, often referred to
as light microscope. They are of two types:
 Simple microscope: A simple microscope is a
microscope that uses a lens or set of lenses to
enlarge an object through angular magnification
alone,
giving
the
viewer
an
erect
enlarged virtual image. Simple microscopes are
not capable of high magnification. The use of a
single convex lens or groups of lenses are still
found in simple magnification devices such as
the magnifying glass, loupes, and eyepieces for
telescopes and microscopes.




Compound microscope: In this microscope objective
lens are used close to the object being viewed to
collect light which focuses a real image of the object
inside the microscope (image 1). That image is then
magnified by a second lens or group of lenses (called
the eyepiece) that gives the viewer an enlarged
inverted virtual image of the object. The use of a
compound microscope allows higher magnification,
reduced chromatic aberration and exchangeable
objective lenses to adjust the magnification.
Now a days the compound optical microscope has
digital charge-coupled device (CCD) cameras
attached which allow to capture the digital images
showing directly on a computer screen without the
need for eyepieces.
On 8th
October 2014, the Nobel Prize in
Chemistry was awarded to Eric Betzig, William
Moerner and Stefan Hell for "the development of
super-resolved fluorescence microscopy," which
brings "optical microscopy into the nanodimension".
 Advantages



Direct imaging with no need of sample pretreatment, the only microscopy for real color
imaging.
Fast, and adaptable to all kinds of sample
systems, from gas, to liquid, and to solid sample
systems, in any shapes or geometries.
Easy to be integrated with digital camera
systems for data storage and analysis.
 Disadvantages

Low resolution, usually down to only sub-micron
or a few hundreds of nanometers, mainly due to
the light diffraction limit.

In a
conventional bright field microscope, light
from a source is aimed toward a lens
beneath the stage called the condenser,
through the specimen, through an objective
lens, and to the eye through a second
magnifying lens, the ocular or eyepiece. We
see objects in the light path because natural
pigmentation
or
stains
absorb
light
differentially, or because they are thick
enough to absorb a significant amount of
light
despite
being
colorless.
A Paramecium should show up fairly well in a
bright field.
Bright
Field
Compound
Microscope:
To view stained or naturally pigmented
specimens such as stained prepared slides of
tissue
sections
or
living
photosynthetic
organisms.
 It is useless to view live bacteria, and nonphotosynthetic protists or metazoans, or
unstained cell suspensions or tissue sections.
 Can be used to view stained bacteria, thick
tissue sections, thin sections with condensed
chromosome, stained organelles, large protists
or metazoans.
 Smears, stained blood, negative stained bacteria
 Living preparations, wet mounts, unstained pond water, algae and other microscopic plant
material.

 Darkfield
Microscope
Darkfield microscopy describes an illumination
technique used to enhance the contrast in
unstained samples. It works by illuminating the
sample with light that will not be collected by
the objective lens, and thus will not form part of
the image. This produces the classic appearance
of a dark, almost black, background with bright
objects on it.
 The light enters the sample. Most is directly
transmitted, while some is scattered from the
sample. The scattered light enters the objective
lens, while the directly transmitted light simply
misses the lens. The scattered light produces the
image, while the directly transmitted light is
omitted.

 Dark
field microscopy produces an image
with a dark background.
 It is used for live and unstained biological
samples, such as a smear from a tissue
culture or individual, water-borne, singlecelled organisms
 The
main limitation of dark field
microscopy is the low light levels seen in
the final image. This means the sample
must be very strongly illuminated, which
can cause damage to the sample.




Fluorescence microscope
Modern biological microscopy depends on the
fluorescent probes for specific structures within a
cell. In fluorescence microscopy the sample is
illuminated through the objective lens with a narrow
set of wavelengths of light. This light interacts with
fluorophores in the sample which then emit light of a
longer wavelength. This emitted light which makes
up the image.
Chemical fluorescent stains, such as 4',6-diamidino-2phenylindole (DAPI) binds to DNA, Rhodamine binds
to mitochondria, Fluorescein isothiocyanate (FITC)
and Cyanines (Cy2, Cy3, Cy5 and Cy7)
More recent develoment include immunofluorescence
which uses fluorescently labeled antibodies to
recognize specific proteins within a sample, and
fluorescent proteins like GFP which a live cell can
express making it fluorescent.





Polarized Microscope
This type microscopy uses plane-polarized light to
analyse structures that are birefringent; structures
that have two different refractive indices at right
angles to one another (e.g. cellulose microfibrils).
Plane-polarized light, produced by a polar, only
oscillates in one plane because the polar only
transmits light in that plane.
The polarized light microscope has both lenses a
polarizer, positioned in the light path somewhere
before the specimen, and an analyzer placed in the
optical pathway after the objective rear aperture.
Image contrast arises from the interaction of planepolarized light with a birefringent (double-refracting)
specimen to produce two individual wave
components that are each polarized in mutually
perpendicular planes.
 Polarized
light microscopy can be used to
measure the amount of retardation that
occurs in each direction and so give
information about the molecular structure of
the birefringent object e.g. cell wall.
Bright
Dark
Polarized




Phase Contrast Microscope
Zernicka (1941) an employ of Ziess company devised
this microscope and win a Nobel Prize in 1953.
The light slows slightly when passing through
biological specimens. Differences in the phase of
light transmitted and reflected by a specimen to
form distinct, contrasting images of different parts of
specimens.
The specimen is illuminated by a hollow cone of light
coming through a phase annulus in the
condenser. Phase contrast objectives must be used,
which have a corresponding phase plate. Light rays
passing through the specimen are slightly retarded,
and further retardation takes place in the phase
plate. When these rays combine with rays which
have not taken this path, degrees of constructive and
destructive interference occur which produce the
characteristic light and dark features in the image

Confocal microscope

In it epifluorescent illumination is used as a scanning
laser to illuminate a sample for fluorescence.
Problem of conventional light and fluorescence microscopy
not only is the plane of focus illuminated, but much of the
specimen above and below this point is also illuminated
resulting in out-of-focus blur images from these areas. This
out-of-focus light leads to a reduction in image contrast
and a decrease in resolution.
In the confocal microscope, all out-of-focus structures are
suppressed at image formation. This is obtained by an
arrangement of diaphragms, which, at optically
conjugated points of the path of rays, act as a point source
and as a point detector respectively. The detection pinhole
does not permit rays of light from out-of-focus points to
pass through it. The wavelength of light, the numerical
aperture of the objective and the diameter of the
diaphragm affect the depth of the focal plane. To obtain a
full image, the point of light is moved across the specimen
by scanning mirrors. The emitted/reflected light passing
through the detector pinhole is transformed into electrical
signals by a photomultiplier and displayed on a computer
monitor


An image of a cell stained with fluorescent dyes during metaphase.
The mitotic spindle (green) attached to the two sets of chromosomes
(blue). All chromosomes but one are already at the metaphase plate
A mouse fibroblast nucleus in which DNA is stained blue. The
distinct chromosome territories of chromosome 2 (red) and
chromosome 9 (green) are stained with fluorescent in situ
hybridization
Digital microscope
 A digital microscope is a microscope equipped
with a digital camera allowing observation of a
sample via a computer. Low-powered digital USB
microscopes are also commercially available.
These are essentially webcams with a highpowered macro lens and generally do not
use transillumination. The camera attached
directly to the USB port of a computer, so that
the images are shown directly on the monitor.
 Digital microscopy allows measurements of
distances and areas and quantitaton of a
fluorescent or histological stain.
 Dino Lite: In the 21st century Dino-Lite Digital
microscopes handheld. They offer low power
zoom capability with magnification up to 500x.
They have had a marked impact on industrial
inspection application.









Everything on a good quality microscope is unbelievably
expensive, so be careful.
Hold a microscope firmly by the stand, only. Never grab it
by the eyepiece holder, for example.
Hold the plug (not the cable) when unplugging the
illuminator.
Since bulbs are expensive, and have a limited life, turn the
illuminator off when you are done.
Always make sure the stage and lenses are clean before
putting away the microscope.
NEVER use a paper towel, a kimwipe, your shirt, or any
material other than good quality lens tissue or a cotton
swab to clean an optical surface. Be gentle! You may use
an appropriate lens cleaner or distilled water to help
remove dried material. Organic solvents may separate or
damage the lens elements or coatings.
Cover the instrument with a dust jacket when not in use.
Focus smoothly; don't try to speed through the focusing
process or force anything.
 Max
Knoll and Ernst Ruska 1931: invented
the first electron microscope
 EM transmits a beam of electrons instead of
light through the specimen. The subsequent
interaction of the beam of electrons with the
specimen is recorded and transformed into
an image
An EM uses a beam of accelerated electrons
whose wavelength can be up to 100,000 times
shorter than the photons of visible light.
 A high powered EM can achieve 100 pm
resolution and magnification of 10,000,000x
whereas a light microscopes has 200 nm
resolution and magnification below 2000x.
 EM has electrostatic and electromagnetic lenses
instead of glass lens to control the electron
beam and focus it to form an image.
 Two types of electron microscopes:

Transmission Electron Microscope (TEM)
 Scanning Electron Microscope (SEM)

 EMs
are used to investigate the ultrastructure of a wide range of biological and
inorganic
specimens
including
microorganisms, cells, large molecules,
biopsy samples, metals and crystals.
 Industrially, the EM is used for quality control
and failure analysis





In the TEM high voltage electron beam is produced by
an electron gun, commonly fitted with a tungsten
filament cathode as the electron source.
The electron beam is accelerated by an anode and
focused by electrostatic and electromagnetic lenses,
and transmitted through the specimen.
When it emerges from the specimen, the electron
beam carries information about the structure of the
specimen that is magnified by the objectives
lens system. The magnified image is projecting onto a
fluorescent
viewing
screen
coated
with
a phosphor or scintillator material such as zinc
sulfide.
Alternatively, the image can be recorded by exposing
a photographic film or a fiber optic light-guide to the
CCD (charge-coupled device) camera.
In modern TEMs the image is detected by the digital
camera and displayed on a monitor or computer.
High resolution TEM has resolution of 0.5
angstrom (50pm) and magnifications above 50
million times. The ability to determine the
positions of atoms within materials has made the
HRTEM an important tool for nano-technologies
research and development.
 The major disadvantage of the TEM is the need
for extremely thin sections of the specimens,
(100 nanometers). Biological specimens are
typically required to be chemically fixed,
dehydrated and embedded in a polymer resin to
stabilize them sufficiently to allow ultrathin
sectioning. Sections of biological specimens,
organic polymers and similar materials may
require special treatment with heavy atom like
lead in order to achieve the required image
contrast.

Electron path of TEM
Ongoing gene transcription of ribosomal RNA illustrating the
growing primary transcripts. "Begin" indicates the 5’ end of
the DNA, where new RNA synthesis begins; "end" indicates
the 3’ end, where the primary transcripts are almost
complete.



Ruska (1942) built the first scanning electron
microscope (SEM) that transmits a beam of electrons
across the specimen.
SEM can achieve magnification levels of up to 2
million times
The SEM produces images by probing the specimen
with a focused electron beam that is scanned across
a rectangular area of the specimen. When the
electron beam interacts with the specimen, it loses
energy. The lost energy is converted into alternative
forms such as heat, emission of low-energy secondary
electrons and high-energy backscattered electrons,
light emission, all of which provide signals carrying
information about the properties of the specimen
surface, such as its topography and composition.
 The
image was constructed from signals
produced by a secondary electron detector.
Generally, the image resolution of an SEM is
poorer than that of a TEM.
 SEM produces surface image of samples that
can be up to many centimeters in size and
has a great depth of field, and produce
images of three-dimensional shape of the
sample.