27-09-2023

MICROSCOPY
The universal realm of microorganisms was
anonymous until the microscopic
instruments were invented.

Development and sophistication of

microscopes lead to the development of
microbiological sciences.

1
 27-09-2023

HISTORICAL DEVELOPMENTS:

ZACCHARIAS JANSEN:
 Dutch spectacle maker
 In 1609, he accidently discovered concept of microscopy.

ANTON VAN LEEUWENHOEK:

 1st to observe live microorganisms through magnifying glass lenses
(simple microscope).
 In 1676, he presented his observations to the Royal Society of London.

ROBERT HOOKE:
 He constructed microscopes with two lenses (compound microscope).
 1st to coin the term ‘cell’ using the microscopic observations.

HISTORICAL DEVELOPMENTS:
ATHANASIUS KIRCHER:
 Discovered the causative microorganisms of plague applying
primitive microscopes.

JOSEPH JACKSON LISTER:

 In 1830, he improved the quality of microscopes which paved
the way for development of modern-day microscopic
techniques

2
 27-09-2023

BASIC TERMS AND CONCEPTS:

MICROSCOPY:
It is a study of optical instruments (microscopes)
used in enlarging images of extremely small objects
for proper visualization by human eye.

WORKING DISTANCE: Distance between the front surface of

lens and the surface of cover slip / the specimen to be
observed.
Objective lens with larger numerical apertures (NA) and higher
resolving power have shorter working distances.

RESOLUTION/ RESOLVING POWER:

• Ability of lenses to separate small objects that are close

together for distinguishing their fine detail and structure.
• Microscope with a resolution power of a 0.2 nm indicates that it
can distinguish between two points only if they are minimum
0.2 nm distant apart.
• 3 factors affect the resolving power of a microscope:
1. Shorter the wavelength of light used in the microscope, greater
is its resolving power.
2. Larger the size of objective lens, greater is its resolving power.
3. More is the refractive index, greater is the resolving power

3
 27-09-2023

MAGNIFICATION/ MAGNIFYING POWER:

 Degree of enlargement of an image.

 Magnification can be enhanced by making the lens more convex or by
bringing the object closer to the lens.
 Total magnification of a specimen can be calculated by multiplying the
magnification of all lenses present in the microscope.
 E.g.: Maximum magnification of compound microscope (1000X) =
Magnifying Power of oil immersion objective lens (100X) x magnifying
power of oculars (10X)

NUMERICAL APERTURE (NA):

 Ability of lens to gather light.
 Defined by two components – refractive index of the
medium in which the lenses work (n) and the angle of the
cone in which light enters an objective (θ).
 NA is calculated by following formula:-

Where, λ is the wavelength of light used

4
 27-09-2023

REFRACTIVE INDEX (N):

 Measure of the light-bending ability of a medium.
 Helps to achieve contrast and better resolution.
 Staining the specimen helps to improve its refractive
index.

SIGNIFICANCE OF OIL IMMERSION LENS:

 For higher magnification and greater resolution, objective lens with higher NA is
recommended.
 The refractive index (n) of air is 1.00 and θ cannot be greater than 1 (since
maximum value for θ cannot exceed 90° and sin 90° is 1.00).
 Thus, no objective lens in air can have a NA greater than 1.00.
 So, replacing air with colorless immersion oil (cedar wood / microscopy oil)
possessing higher refractive index 1.5 (same as glass) will help to enhance NA and
thus, magnification as well as resolution of the image.
 Using oil immersion lens will prevent the detrimental reflection and refraction of
the light rays at the surfaces of slides or lenses.

5
 27-09-2023

CLASSIFICATION:

6
 27-09-2023

BRIGHT FIELD MICROSCOPY [COMPOUND

MICROSCOPE]:
 All modern laboratory microscopes are compound
microscopes.
 PRINCIPLE: Dark image is form against a bright
background.
 A compound microscope is made up of series of
lenses and utilizes light as the source of illumination.
 It contains three major systems
 Magnification System: Objective lens and eyepiece/ oculars
 Illumination System: Light source or mirror and condenser
(iris diaphragm and filter)
 Support System: Arm, base, nosepiece, body tube and
specimen stage.

7
 27-09-2023

OBJECTIVE LENS:
Forms the initial image of specimen on the slide and has various

ROLES:
1. Collect light rays coming from any point of object and unite
them at a point of the image.
2. Real image formation and magnification.

Parfocal Objective Lens:

 Significant feature of compound microscope.
 In these, when a specimen is focused with one objective lens,
it continues to be in focus after changing over to other
objective lens on the same nosepiece.
 Such parfocal lenses reduce the amount of damage to slides
and objective lenses.

TYPES OF OBJECTIVE LENS

• Achromatic objective lenses are simple, economic and can
eliminate color and spherical aberrations.
• Fluorite and apochromatic lenses are advanced objective
lenses with highest degree of perfection, magnification and
can correct all types of aberrations.
• Objective lenses with different magnifications are present on
nose piece.

8
 27-09-2023

Type of objective lenses

Properties
Low power High power Oil immersion
Numerical
0.25 0.55-0.65 1.25-1.4
aperture
Magnification 10X 40-45X 100X
Focal Length
(approximate) in 16 4 1.8-2.0
mm
Resolution with
blue light
0.9 0.35 0.18
(approximate) in
µm
Working distance
4-8 0.5-0.7 0.1
in mm

EYEPIECE/ OCULARS:
Ultimate part of a compound microscope through which the final
image of the specimen can be visualized by human eye.

ROLES
1.To remagnify the image formed by objective lens and create a
final virtual image.
2.To rectify the remaining aberrations in the image missed by
objective lens.

TYPES OF OCULARS
• Huygenian or negative oculars are the basic oculars with focus
present within the eyepiece.
• Ramsden and compensating oculars are advanced, positive
oculars wherein the diaphragm is placed externally below the
lower lens.

9
 27-09-2023

Positive oculars are more accurate than negative oculars.

Most standard compound microscopes come with 10X

eyepieces and 1000X is the highest possible magnification.

Even 15X eyepieces can be used with maximum 1500X

magnification.

Higher magnifying oculars suffer from resolution

inadequacy. They will simply magnify the blurred images.

Only electron microscopes would be useful for further

magnification and better resolution.

CONDENSERS:
• Series of lenses mounted in series under the
stage.
• It contracts light rays into a strong beam and
pass them through the opening of stage.
• It is made up of two parts: Iris diaphragm, a
shutter-like apparatus mounted on the filter.
• Iris diaphragm controls the light intensity which
is screened by the filters.

10
 27-09-2023

TYPES OF CONDENSERS:
• Abbe and variable-focus condensers have two lenses
and are used for general microscopic applications but
fail to correct spherical and chromatic aberrations.
• Achromatic condenser with more than two lenses
fixes both aberrations and thus, more degree of
perfection in the image is obtained.
• Paraboloid and cardioid condensers are used in dark
field microscopy.

APPLICATIONS of Compound Microscope:

1. Light microscopes are useful in material science,
pharmaceutical research, medicine, size analysis as
well as microbiology.
2. Staining techniques combined with light microscopy
can allow scientists to identify different kinds of
prokaryotes.
3. Bright field microscopes have applications in
diagnostics and histopathological studies.

11
 27-09-2023

DARK FIELD MICROSCOPY

• Few microorganisms are invisible in the ordinary light

microscope or are difficult to stain.
• In such cases, dark field microscopy is useful.
• It is based on the principle of scattering. A dark field
condenser containing an opaque disc is placed instead of normal
condenser. The disc blocks the light rays from entering the
objective lens directly and redirects it to pass through the
specimen. The light rays strike the specimen, scatter and reach
the objective lens. As no other light rays enter the objective lens,
the specimen image appears bright against a dark
background.
• Useful technique to observe unstained microbial suspensions.
• E.g. Identification of thin spirochetes viz. Treponema palladium.

12
 27-09-2023

PHASE CONTRAST MICROSCOPY

• Invented by Frits Zernike – awarded Nobel Prize in 1953.
• Observe unstained living cells without fixing them to slide.
• Its PRINCIPLE is based on the wave nature of light rays. Light
rays can be in phase (their peaks match) or out of phase (their
peaks and troughs match). If all the light rays coming from a
source are not interrupted, they will reinforce each other and
produce relatively higher brightness (background image).
However, if they strike the specimen, they will be diffracted or
reflected and go out of phase giving rise to interference pattern
and thus, relative darkness.

13
 27-09-2023

PHASE CONTRAST MICROSCOPY

• When the reinforcement and interference patterns are

combined together, they form an image with various black and
white shades on a bright background.
• In this, even the internal structures of a cell are visible.
• Phase contrast microscopy is useful in studying the
morphology of microbes, their motility, detecting their internal
components, endospores, cell division and viral cytopathic
effects.

DIFFERENTIAL INTERFERNCE CONTRAST (DIC)

MICROSCOPY

• DIC is similar to phase contrast microscopy except that

it is based on the differences in the refractive indices
and thickness.
• In this, minute differences in refractive index and cell
density aid in detecting variations in light intensity
and hence, the cell morphology in detail.

14
 27-09-2023

DIC MICROSCOPY
• As compared to phase contrast microscopy, DIC
microscope uses two beams of light, perpendicular to
each other and generated by prisms; one passes
through the background and other across the
specimen.
• After passing, the two beams combine and interfere
with each other to form a brightly colored 3D image.
• Resolution of DIC microscope is better than phase
contrast microscope.
• Cell wall, spores, granules, capsule, vacuoles and other
cell inclusions are easily visible by DIC.

15
 27-09-2023

FLUORESCENCE MICROSCOPY
• Principle: Fluorescence instead of absorption and
diffraction.
• Fluorescence is the ability of substances to absorb
shorter wavelength light (UV) and give off longer
wavelength light (visible).
• Few microbes possess fluorescence inherently. If the
microbial culture is devoid of fluorescence, they are
stained with fluorescent dyes known as fluorochromes.

Diamidino-2-phenyl indole
Stains DNA, fluoresces green
(DAPI) dye

Tetramethyl rhodamine Attaches to specific

isothiocyanate (TRITC) dye antibodies, fluoresces red

Attaches to specific
Fluorescein isothiocyanate
antibodies or DNA probes,
(FITC) dye
fluoresces green

16
 27-09-2023

 After staining, the microbial cultures are detected as

luminescent, bright objects against a dark background.
 Intense beam of light from a mercury arc lamp or any
other source is made to pass through the excitation
filter so as to transmit only blue light (shorter
wavelength) towards dichromatic mirror.
 This mirror reflects light of shorter wavelength but
allows longer wavelength light to pass through it.
 The reflected light passes through the objective lens
towards the fluorescent specimen.

 The specimen absorbs the light and emits bright

fluorescence, giving off longer wavelength light.
 The emitted fluorescent light passes through the
objective lens and the dichromatic mirror towards a
barrier filter.
 The barrier filter blocks any residual excited light and
allows only the fluorescent beam to reach the
observer.

17
 27-09-2023

• Fluorescent-Antibody (FA) technique/

Immunofluorescence: Specific FA is prepared by
tagging the desired antibody with specific
fluorochrome.
• Then the FA is added into the plasma sample wherein it
forms complex only with the specific corresponding
pathogenic antigen and gives off a typical
fluorescence.
• It is a useful tool to detect bacterial pathogens, study
immunological principles, identify mechanical
pathways, in cancer research studies and toxicological
profiling.

ELECTRON MICROSCOPY
 Light microscopes with a resolution limit of 0.2 µm are
useful to study microorganisms.
 However, there is use is limited when viruses or cell
inclusions are to be studied in detail.
 This restriction originates from the nature of light
waves and not from the insufficiency of light
microscopes.
 Hence, there was a need for alternative source to light
and glass lens which gave rise to electron microscopy.

18
 27-09-2023

 Electron microscopes (EM) utilize a beam of

electrons and electromagnetic lens instead of light
waves and glass lens, respectively.
 A beam of electrons used for illuminating the objects
can be focused as light waves but their wavelength is
approximately 0.005nm, around 1000 times smaller
than visible light.
 Thus, electron microscopes have 1000 times better
resolution than light microscopes and 1,00,000X
magnification. They can resolve points as closer as 0.5
nm.

There are TWO TYPES of electron microscopes:

1.Transmission Electron Microscope (TEM)

2.Scanning Electron Microscope (SEM)

19
 27-09-2023

TRANSMISSION
ELECTRON
MICROSCOPE
(TEM)

• PRINCIPLE: TEM creates an image from electromagnetic

radiations that have passed across the specimen.

• Focused beam of electrons from an electron gun passes

through the ultrathin sections of the specimen via
electromagnetic condenser lens which direct the beam
in a straight pathway to illuminate the specimen.

• Electromagnetic lenses aid in controlling magnification,

illumination and focus.

• Specimen is positioned on a copper mesh grid.

20
 27-09-2023

• After passing across the specimen, the electron beam

passes through the electromagnetic objective lens wherein
the image is magnified.
• Further, the electrons are focused by electromagnetic
projector lens on a fluorescent screen or photographic
plate to form the final image showing grey and white
areas.

1. TEM has higher limit of resolution i.e. 2.5 nm and

magnifying power is in the range of 10,000X to 1,00,000X

2. To overcome the challenge of weak contrast in ultrathin

specimen sections, salts of various metals like lead,
osmium, uranium and tungsten are commonly used as
stains.

3. TEM of specimens can be imaged by positive or

negative staining techniques. Shadow casting method
gives a 3D image of cell morphology.

21
 27-09-2023

TEM Limitations:
• As electrons have inadequate penetrating power,
ultrathin sections of specimen are required.
• Thus, 3D imaging is not feasible except for shadow
casting method.
• Dehydration, high vacuum conditions are required to
overcome electron scattering effect.
• Expensive analysis and formation of undesirable
artifacts.
• Large instrumentation with special housing and
maintenance requirements.
• Only trained personnel can operate it.
• Only black and white images obtained.

TEM Applications:
In the field of nanotechnology, life sciences, medical,
biological and material research, forensic analysis,
gemology, metallurgy as well as pharmaceutical industry
and education.
Viral particles, cancer cells and their mechanism,
molecular pathways and cell inclusions can be studied in
depth.
In detecting flaws, fractures & other damages to micro-
sized objects.
Provides topographical, morphological, compositional
and crystalline information about nano formulations.
In the study of crystals and metals.
Valuable for semiconductor analysis and production.

22
 27-09-2023

SCANNING
ELECTRON
MICROSCOPE
(SEM)

PRINCIPLE: SEM forms an image owing to electrons released

from atoms on the object’s (specimen) surface.
 Resolving power of 7 nm or less
 1000X-10,000X magnification
 Specimen preparation is comparatively easy; air dried
samples are directly observed. However, microbial
specimens need to be fixed, dehydrated and dried for
structural preservation under high vacuum conditions. There
is no need of ultrafine sections.
 Prior to imaging, specimen samples are dried, mounted and
coated with thin metallic layer (gold or platinum) to
overcome the charge build up problems and for better
imaging.

23
 27-09-2023

 In this, a narrow, tapered electron beam in scanned

over the specimen.
 Striking the beam on any specific area of specimen
activates the surface atoms to discharge secondary
electrons which are trapped and amplified by a
detector.
 Signals are sent to cathode ray tube (CRT) for imaging.
 Number of secondary electrons defines the image of
specimen. Dense areas give large number of
electrons. Thus, specimen 3D image appears light on a
dark background.

SEM Limitations:
 Applicable for solid specimens only
 Specimens must be stable in vacuum
 SEMs are expensive, large and must be housed in
an area free of any possible electric, magnetic or
vibration interference.
 Live microbial specimens are difficult to observe.

24
 27-09-2023

APPLICATIONS:

 In life sciences, pharmacy, biology, gemology,

medical and forensic science, metallurgy
 Examination of microbial and viral particle presence
in ecological niches, such as human skin and gut
lining without need for ultrathin sections.
 Unique tool for analyzing solid samples at
molecular level.
 Analyze chemical composition (EDX) and
crystallinity of microbial cells.

Parameters Light microscope Electron

microscope
Lens Glass Electromagnet
Source of radiation Visible light Electron beam
Specimen Glass slide Metal grid (usually
mounting platform copper)
Contrast Differential light Scattering and
mechanism absorption excitation of
electrons
Medium of travel Air, immersion oil High vacuum

25
 27-09-2023

Parameters Light microscope Electron

microscope
Focusing Mechanical Adjustment of
mechanism adjustment of lens current to the
position electromagnetic
lens
Method of Switch the objective Adjustment of
changing lens or eyepiece current to the
magnification magnetic lens
Best limit of 0.2µm 0.5 nm
resolution
Highest 1,000-1,500X 1,00,000X
magnification

LM SEM TEM

26