Introduction to Microscopy

Shailesh R.Dave
UGC Emeritus Fellow, Adjunct faculty, XRF, Loyola Centre for R & D,
St. Xavier College Campus, Ahmedabad – 380009, Gujarat, India
shaileshrdave@yahoo.co.in
Why we need microscope?
• If you want to actually visualize microbes, you’ll need the ability to
magnify – you’ll need a microscope of some type. And, since “seeing
is believing,” it was the visualization of microbes that got people
interested in them in the first place.
Antonie van Leeuwenhoek
• Antonie van Leeuwenhoek, often called the “Father of
Microbiology,” wasn’t a scientist
by profession. He was a cloth merchant from Holland who was
believed to be inspired by
Mr. Hooke’s work, probably with the original intention of examining
textiles to determine quality.
• He was using what is called a simple microscope, a microscope with
just a single lens.
The first MICROBIOLOGIST
and his MICROSCOPE

Anton van Leeuwenhoek and his microscope

Leeuwenhoek’s Microscope
• Consist of only single biconvex lens
• Stage for sample
• Adjustment screws
• Magnification about 200x
Detail of Leeuwenhoek’s lens system
How microbes were first described?
• First to describe the microbes in 1676, his 8th letter sent to Royal
society of London in 1676 and published in 1677.
• He wrote 200 letters in the 50 years time.
• Drawing (sketch) of microbes he sent in 1683.
• He referred them as “animalcules”
Leeuwenhoek’s description of bacteria
Letter sent to Royal society 17/9/1683
Microscopy in the 1600s
• It is believed that Robert Hooke was one of the first scientists
to actually observe microbes, in 1665. His illustrations and
observations from a variety of objects viewed under a
microscope were published in the book Micrographia.
• Hooke used a compound microscope, meaning it contained
two sets of lenses for magnification: the ocular lens next to
the eye and the objective lens, next to the specimen or object.
• The magnification of a compound microscope is a product of
the ocular lens magnification and the objective lens
magnification.
• Thus a microscope with an ocular magnification of 10x and an
objective magnification of 50x would have a total
magnification of 500x.
Abbé equation:
Resolution power
• R.P.= /2xN.A.
• Numerical aperture of
• low power objective is 0.25
• High power objective is 0.65
• Oil immersion objective is 1.25
Resolution power
• Low power objective
• R.P. = 550nm/2x0.25 = 1100nm = 1.1 m (micro meter)

• Oil immersion objective

• R.P. = 550/2x1.25 = 220nm= 0.22 m
 Microscopy
• Optical / Light: microscopy depends upon lenses that refract light rays,
drawing them to a focus to produce a magnified image.
• A simple microscope consists of a single magnifying lens, whereas a
compound microscope relies on two lenses: the ocular lens and the
objective lens. The objective lens is responsible for the real image, and
the ocular lens forms the virtual image.
Bright-Field Microscope
Major parts of the microscope
• Base of microscope
• Light source
• Light control
• Iris diaphragm
• Diaphragm aperture knob
• Condenser
• Stage adjustment knobs
• Stage
• Course focus knob
• Fine focus knob
• Objectives
• Nosepiece
• Arm
• Body
• Ocular /eyepiece lenses
Numerical aperture
• Numerical aperture, which is a function of the objective lens and its
ability to gather light.
• The numerical aperture value is actually defined by two components:
n, which is the refractive index of the medium the lens is working in,
and
• sin θ, which is a measurement of the cone of light that enters the
objective.
• A lens can typically work in two media: air, with a refractive index of
1.00, or oil, with a refractive index of 1.25. Oil will allow more of the
light to be collected, by directing more of the light rays into the
objective lens.
• Resolution is a somewhat subjective value in optical microscopy
because at high magnification, an image may appear unsharp but still
be resolved to the maximum ability of the objective. Numerical
aperture determines the resolving power of an objective, but the
total resolution of the entire microscope optical train is also
dependent upon the numerical aperture of the substage condenser.
The higher the numerical aperture of the total system, the better the
resolution
• Shorter wavelengths are capable of resolving details to a greater
degree than are the longer wavelengths.
Dark-field microscope
• The dark-field microscope is really just a slightly modified bright-field
microscope. In fact, you could make this modification to the
microscope you have at home! It makes use of what is known as a
dark-field stop, an opaque disk that blocks light directly underneath
the specimen so that light reaches it from the sides. The result is that
only light that hit from different directions give light to objects only.
 Dark field microscopy
• Simple and popular method for rendering unstained and
transparent specimens clearly visible.
• Good candidates for dark field observation often have refractive
indices very close in value to that of their surroundings and are
difficult to image with conventional bright field techniques.
Dark-field microscope
• The dark-field microscope is really just a slightly modified bright-field
microscope. In fact, you could make this modification to the
microscope you have at home! It makes use of what is known as a
dark-field stop, an opaque disk that blocks light directly underneath
the specimen so that light reaches it from the sides. The result is that
only light that
 Dark field microscopy
• Simple and popular method for rendering unstained and
transparent specimens clearly visible.
• Good candidates for dark field observation often have refractive
indices very close in value to that of their surroundings and are
difficult to image with conventional bright field techniques.
Phase Contrast Microscope 1/2
• Fritz Zernike developed the Phase contrast Microscopy and was
awarded the Nobel prize in physics for the same in 1953.
• Here light rays are in phase or out of phase
• One come direct from light source and second one is reflected or
diffracted from a particular structure.
 2/2
• The phase-contrast microscope is also a modified bright-
field microscope, although the modifications are getting
more complex, as well as more expensive. This
microscope also uses an opaque ring or annular stop, but
this one has a transparent ring that only releases light in a
hollow cone. The principle of this microscope gets back to
the idea of refractive index and the fact that cells have a
different refractive index than their surroundings,
resulting in light that differs slightly in phase. The
difference is amplified by a phase ring found in a special
phase objective. The phase differences can be translated
into differences in brightness, resulting in a dark image
amidst a bright background. This allows for the
observation of living, unstained cells, once again useful to
observe motility or eukaryotic organelles.
Phase Contrast Microscope
 Phase contrast microscopy
• An optical microscopy technique that converts phase shifts in light
passing through a transparent specimen to brightness changes in the
image.
• Phase shifts themselves are invisible, but become visible when
shown as brightness variations.
Other type of microscopes
• Fluorescence
• Differential Interference Contrast
• Confocal
Fluorescence Microscope
Types of electron microscope
• Transmission Electron Microscope (TEM)
• Scanning Electron Microscope (SEM)
• Scanning Probe Microscope
• Scanning Tunneling Microscope
• Atomic force Microscope
TEM
• A heated tungsten filaments in the electron gun generates a beam of
electrons is used
• The beam focus on the specimen by condenser
• Electromagnetic lenses are used to focus the beam
• Lenses and specimen are under high vacuum
 Conti….
• The specimen scatters electrons passing through it.
• The beam is focused by magnetic lenses to form an enlarged, visible
image on a fluorescent screen
 Conti…
• Fixation Glutaraldehyde/ Osmium tetroxide
• Dehydration by acetone /alcohol
• Soaked in unpolymerized liquid epoxy plastic
• Allowed to hardened Section by glass or diamond knife
• Stained by soaking in the heavy metal solution like lead citrate and
uranyl acetate
• Specimen also stain by negative staining with phosphotungstic acid or
uranyl acetate
• Mounted on tiny copper grids
Light and Electron microscope
Light and TEM
Transmission Electron Microscope.
SEM and it’s working
• Examines the surface of the specimen
• Resolve 7nm or less
• Produce image by emitted electron
• Air dried sample can be examined directly
• If want the sample may be fixed, dehydrated and dried these
treatment will preserve surface structure
 Conti……..
• Emitted electron trapped by detector
• Electron enter in the detector strike the scintillator and emit light
flashes, which are converted to electric current and ampliphied by a
photomultiplier.
• The signal is sent to a cathode ray tube which produce an image,
which can be seen as television picture.
• Image produce is three dimensional
Scanning Electron Microscope