BIOLOGICAL

MICROSCOPY
 MICROSCOPY
 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
 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
 HISTORY OF MICROSCOPY
 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.
 TIMELINE OF THE MICROSCOPE CONT’D
 1675: Anton van Leeuwenhoek, who used a microscope to
observe insects, bacteria and other objects.
 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 HOOKE ANTHONY VAN
LEEUWENHOEK
ANCIENT SPECTACLES
ANCIENT MICROSCOPES

Robert Hooks
Microscope
Made of gold
and leather and
candle as light
source
FIRST DRAWING OF FLEA AND TS
OF CORK BY ROBERT HOOKE
OPTICAL COMPOUND MICROSCOPE
CCD CAMERA ATTACHED MICROSCOPE
CCD CAMERA ATTACHED MICROSCOPE
WITH COMPUTER
CCD CAMERA ATTACHED MICROSCOPE
WITH COMPUTER
 OPTICAL MICROSCOPY
 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
 USES OF OPTICAL MICROSCOPE
 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.
 TYPES OF OPTICAL MICROSCOPES

 Simple microscope
 Compound microscope
 Bright field microscope
 Dark field microscope
 Fluorescence microscope
 Phase contrast microscope
 Polarized microscope
 Confocal microscope
 Digital microscope
 HISTORY OPTICAL 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)
 COMPOUND MICROSCOPE (CONT’D)

 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.
REVOLVING TURRET
COMPOUND MICROSCOPE (CONT’D)
 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
 COMPOUND MICROSCOPE (CON’T)

 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.
OPTICAL MICROSCOPIC IMAGES
 TYPES OF OPTICAL MICROSCOPE
 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.
PRINCIPLE OF SIMPLE MICROSCOPE
SIMPLE LIGHT MICROSCOPE
 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".
PRINCIPLE OF COMPOUND MICROSCOPE
COMPOUND OPTICAL MICROSCOPE
 OPTICAL MICROSCOPY
 Advantages
 Direct imaging with no need of sample pre-
treatment, 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.
 TYPES OF COMPOUND MICROSCOPES
 Bright Field Compound Microscope: 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 DISSECTING/ STEREO
MICROSCOPE MICROSCOPE
OPTICAL MICROSCOPIC IMAGES
USE OF BRIGHT FIELD 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 non-
photosynthetic 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.
 IMAGE OF LIGHT MICROSCOPE

ANIMAL TISSUE ROOT TS

CELL DIVISION IN ONION ROOT TIP
CELLS
HUMAN RED BLOOD CELLS, LIKE THOSE OF OTHER
MAMMALS, LACK NUCLEI.
 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.
PRINCIPLE OF DARK FIELD LM
ADVANTAGES AND DISADVANTAGES OF DARK
FIELD LM
 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, single-
celled 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.
DARK FIELD IMAGE OF TISSUE PAPER
 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-2-
phenylindole (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.
FLUORESCENCE MICROSCOPE
IMAGES OF FLUORESCENCE
MICROSCOPE
 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 plane-
polarized light with a birefringent (double-refracting)
specimen to produce two individual wave
components that are each polarized in mutually
perpendicular planes.
PRINCIPLE OF POLARIZED LIGHT MICROSCOPE
 USE OF POLARIZED MICROSCOPE

 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.
POLARIZED LIGHT PATHWAY
 BRIGHT, DARK AND POLARIZED LM
IMAGES OF TISSUE PAPER

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
PHASE CONTRAST LIGHT
MICROSCOPE
PHASE CONTRAST IMAGE
PHASE CONTRAST IMAGE OF CELL
SHOWING NUCLEOLUS, NUCLEUS,
CYTOPLASM
 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
PRINCIPLE OF CONFOCAL MICROSCOPE
CONFOCAL MICROSCOPE
 CONFOCAL EPIFLUORESCENCE MICROSCOPIC IMAGE .

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
IMAGE OF CONFOCAL MICROSCOPE
 IN-SITU HYBRIDIZATION IMAGE OF CONFOCAL MICROSCOPE

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 high-powered 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.
DIGITAL MICROSCOPE
 CARE OF THE MICROSCOPE
 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.
 ELECTRON MICROSCOPE (EM)
 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
RESOLUTION LIMIT OF ELECTRON
MICROSCOPE
 ELECTRON MICROSCOPE
 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)
 USE OF EM
 EMs are used to investigate the ultra-
structure 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
 TRANSMISSION ELECTRON MICROSCOPE (TEM)
 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.
TRANSMISSION ELECTRON MICROSCOPE
Electron path of TEM
TRANSMISSION ELECTRON MICROSCOPE
 TEM MICROGRAPHS

CYTOPLASM NUCLEUS
TEM MICROGRAPH
 A

ELECTRON MICROGRAPH

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.
 SCANNING ELECTRON MICROSCOPE (SEM)
 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.
SCANNING ELECTRON MICROSCOPE
SCANNING ELECTRON MICROSCOPE
 SEM MICROGRAPHS

EYE OF FLY ROD SHAPED BACTERIA

 SEM MICROGRAPHS

ANT POLLEN GRAINS

SEM IMAGE OF TS OF LEAF
COMPARISON OF LIGHT, ELECTRON AND SCANNING
ELECTRON MICROSCOPES