Microscopy

Sub: BP3005 Pharmaceutical Microbiology

Unit I

Department of Pharmaceutical Sciences & Technology

 Microscope
▪ Microscope is an inseparable tool in a microbiology lab
▪ It’s an important instrument in identification of microorganisms
❑ Classification:
1. Light Microscope: use glass lenses to bend and focus light rays and produce
enlarged images
a) Bright-field
b) Dark-field
c) Phase-contrast
d) Fluorescence
2. Electron Microscope: using electron beams rather than visible light
a) Scanning Electron Microscope (SEM)
b) Transmission Electron Microscope (TEM)
 Lenses and the Bending of Light
❑ Refraction: bent of light at the interface of two medium

❑ Refractive index: measure of how greatly a substance slows the

velocity of light
– the direction and magnitude of bending is determined by the refractive
indexes of the two media forming the interface The Bending of Light by a Prism
– When light passes from air into glass, it is slowed and bent toward the
normal (a line perpendicular to the surface)
– As light leaves glass and returns to air, it accelerates and is bent away from
the normal.
– Thus a prism bends light because glass has a different refractive index
from air, and the light strikes its surface at an angle.

❑ Lenses act like a collection of prisms

❑ When parallel rays of light strike the lens, a convex lens will focus
these rays at a specific point, the focal point (F)
❑ The distance between the center of the lens and the focal point is
called the focal length (f)
Function of a lens
 How magnifying glasses work?
• Our eyes cannot focus on objects nearer than
about 25 cm or 10 inches
• This limitation may be overcome by using a
convex lens as a simple magnifier and holding it
close to an object.
• A magnifying glass provides a clear image at
much closer range, and the object appears larger.
• Lens strength is related to focal length; a lens
with a short focal length will magnify an object
more than a weaker lens having a longer focal
length
Light Microscope: The Bright-Field Microscope
 Microscope Resolution
• The most important part of the microscope is the objective, which must produce a clear
image, not just a magnified one
• Resolution is the ability of a lens to separate or distinguish between small objects that
are close together
• Optical theory developed by the German physicist Ernst Abbé in the 1870s

• Abbé equation: Specifies the minimum distance (d) between two objects that reveals
them as separate entities

Where λ = wavelength of light used to illuminate the specimen

n sin θ = is the numerical aperture (NA)

• As d becomes smaller, the resolution increases and finer details can be distinguished
• Abbé equation indicates the wavelength of light used is one of the major factor in
resolution
• The wavelength must be shorter than the distance between two objects or they will
not be seen clearly
• Thus the greatest resolution is obtained with light of the shortest wavelength (in the
range of 450 to 500 nm)
 Numerical Aperture & Resolution
• θ is defined as 1/2 the angle of the cone of light entering an objective
• Light that strikes the microorganism after passing through a condenser is cone-shaped
• When this cone has a narrow angle and tapers to a sharp point, it does not spread out much after leaving
the slide and therefore does not adequately separate images of closely packed objects: The resolution is
low
• If the cone of light has a very wide angle and spreads out rapidly after passing through a specimen,
closely packed objects appear widely separated and are resolved.
• The angle of the cone of light that can enter a lens depends on the refractive index (n) of the
medium in which the lens works, as well as upon the objective itself.

• The angle of the cone of light that can enter a lens depends on the refractive index (n) of the
medium in which the lens works, as well as upon the objective itself.

• The refractive index for air is 1.00. Since sin θ cannot be greater than 1 (the maximum θ is 90°
and sin 90° is 1.00), no lens working in air can have a numerical aperture greater than 1.00.

• The only practical way to raise the numerical aperture above 1.00, is to increase the refractive
index with immersion oil, a colorless liquid with the same refractive index as glass
 An increase in numerical aperture and resolution results due to immersion oil

• If air is replaced with immersion oil, many light rays that did not enter the objective due
to reflection and refraction at the surfaces of the objective lens and slide will now do so
• The resolution of a microscope depends upon the numerical aperture of its condenser as well as that of
the objective.

• The limits set on the resolution of a light microscope can be calculated using the Abbé equation.
• The maximum theoretical resolving power of a microscope with an oil immersion objective (numerical
aperture of 1.25) and blue-green light is approximately 0.2 µm.
• a bright-field microscope can distinguish between two dots around 0.2 µm apart (the same size as a very
small bacterium)
• Normally a microscope is equipped with three or four objectives ranging in magnifying power from 4× to
100 ×
 The Dark-Field Microscope
❑ A hollow cone of light is focused on the specimen in such a way that
un-reflected and un-refracted rays do not enter the objective

❑ Only light that has been reflected or refracted by the specimen

forms an image
❑ The field surrounding a specimen appears black, while the object
itself is brightly illuminated

(a)

(b)
Fig. Dark-Field Microscopy. The simplest way to convert a microscope to dark-field microscopy is to place (a) a dark-
field stop underneath (b) the condenser lens system. The condenser then produces a hollow cone of light so that the
only light entering the objective comes from the specimen
Examples of Dark Field Microscopic Image
 The Phase-Contrast Microscope
▪ Due to difference in contrast in cells and water: unpigmented living cells
are not clearly visible in the bright-field microscope
▪ Fixation and staining is required for this purpose
▪ A phase-contrast microscope converts slight differences in refractive
index and cell density into easily detected variations in light intensity
▪ The condenser of these microscopes have an annular stop: which
produces a hollow cone of light
▪ Direct light rays travel a different path than light rays reflected or
diffracted by specimen
▪ These two sets of light rays combine via ocular lens at eye
▪ The background, formed by undeviated light, is bright, while the
unstained object appears dark and well-defined.
▪ Colour Filters may be used to provide contrast:
✓ Reflected/ diffracted rays: Blue colour
✓ Direct rays: Red colour
The Phase-Contrast Microscope
• Uses:
1. Microbial motility
2. Determining shape of living cells
3. Detecting endospores and inculsion bodies (poly-β-hydroxybutyrate, poly-
metaphosphate, sulfur, or other substances)
4. Widely used in studying different eukaryotic cells
 Differential Interference Contrast (DIC) Microscope
▪ Uses differences in refractive indices and thickness
▪ Uses 2 beams of light (plane polarized light)
▪ One through the prism
▪ One through the specimen

▪ Prism splits light beams and add contrasting colour to specimen

▪ Images formed are brightly coloured and appear 3D
▪ Resolution is higher in DIC
▪ Structures visible: cell walls, endospores, granules, vacuoles, and eucaryotic nuclei
 DIC image of Amoeba proteus
Difference in image for DIC, Phase Contrast and
Bright Filed Microscopy
 The Fluorescence Microscope
▪ Fluorescence is the ability of substances to absorb short wavelength of light (UV)
and emit out longer wavelength of light (visible)
▪ Fluorescent light is emitted very quickly by the excited molecule as it gives up its
trapped energy and returns to a more stable state
▪ Light sources: UV, violet, or blue light
▪ Specimen appear luminescent, bright objects against dark background

▪ Fluorochromes
▪ Auramin O- Glows yellow under UV, strongly absorbed by
Mycobacterium tuberculosis
▪ Fluorescein isothiocyanate: Apple Green, stains Bacillus anthracis
▪ Acridine orange and DAPI (diamidino-2-phenylindole)
- used in ecological studies
 The Fluorescence Microscopy

Escherichia coli stained with Paramecium tetraurelia Crithidia luciliae stained with
fluorescent antibodies conjugating; acridine-orange fluorescent antibodies to show
fluorescence the kinetoplast
 Electron Microscopy
▪ Beam of electron of instead of light
▪ Resolving power is greater
▪ Wavelengths of electrons are 100,000 times smaller than visible light
▪ Images produced are always black and white
▪ Electromagnetic lenses are used instead of glass lenses, to form a beam of electrons
on specimen
▪ Types:
1. Transmission Electron Microscope (TEM)
2. Scanning Electron Microscope (SEM)
 The Transmission Electron Microscope
▪ Resolution 1000 times better than light microscope
▪ Magnification 100,000 and over
▪ Beam of electrons focused on small area of specimen by
eletromagnetic condenser lens
▪ Beam of electron passes through the specimen and then through
electromagnetic objective lens, which magnifies the image
▪ Electrons are then focused by electromagnetic projector lens onto a
fluorescent screen or photographic plate
▪ Final image (Transmission Electron Micrograph): Appears as many
light and dark areas depending on number of electrons absorbed by
different areas of specimen
▪ Resolve object close by 2.5 nm
▪ Stains used to absorb electron and produce
darker image:
▪ Salts of Pb, Os, W, U
▪ Fixation on specimen: Positive staining
▪ Used to increase opacity of surrounding filed:
Negative Staining
▪ Shadow casting: heavy metals like Pt or Au
sprayed at an angle of 45° so that it strikes the
microbes from only one side
▪ Metals piles up on one side of the specimen
and uncoated area on the opposite side of
specimen leaves a clear area behind it as a
shadow
▪ It gives a 3D effect to the specimen and general
idea of size and shape
 Specimen Shadowing for the TEM

Proteus mirabilis(X 42,750) T4 coliphage (X 72,000)

 Scanning Electron Microscope
• Electron gun finely focused beam of electron- Primary
Electron Beam
• Pass through electromagnetic lenses and directed on
specimen
• Knocks electron out of specimen surface and produce
secondary electron
• Secondary electrons are transmitted on electorn collector,
amplified and produces image on viewing screen on
photographic plate
• Magnifies 1000 to 10000X
• Used for surface structure of intact microscope