Chapter 2
Microscopy
Dr Sonal
Saxena
Dr Arpita
MICROSCOPY
The size of microorganisms is expressed in
• Microns (micrometer or µm) for bacteria, parasite and fungi
• Nanometers for viruses
1 micron (µm) = one-thousandth of a millimetre
1 nanometre (nm) = one-thousandth of a micron
• The limit of resolution of the unaided eye is 200 µm
• Bacteria of medical importance measure 0.2–1.5 µm in
diameter and 3–5 µm in length
• Antony van Leeuwenhoek first visualized
bacteria through hand-ground lens
MICROSC • In 1590, Hans and Zacharias Janssen developed
the first compound microscope
OPY • Over years, different types of microscopes were
developed
Terms
Used in
Microscopy
Resolution power: Minimum
distance between two
objects placed close to each
other that can be
distinguished as separate
images
That of the unaided eye is
about 1200 microns
The resolution power of a
light microscope is 1200 nm
Inversely proportional to the
wavelength of the light used
Magnification: Degree of
visual enlargement of an
observed object
Fig. 2.4 Resolution power of light and electron microscopes and the
microorganisms visualized through them
Total magnification = Magnification by the objective lens ×
magnification by the eyepiece (in an oil-immersion lens,
magnification is 100 × 10 = 1,000 times)
MAGNIFICA Empty magnification refers to the degree of magnification of
TION an object that can be achieved without any additional details
being visible.
Contrast is the difference between the light intensities of the
image and the adjacent background.
It is the most commonly used method
1) Principle
Two series of lenses, objective and ocular
OPTICAL Light rays passing through the condenser illuminates the object
on the stage.
OR LIGHT These rays then pass through the lens of the objective to the
eyepiece, and then to the observer.
MICROSC Magnification: 1000 times
Resolving power: 300 nm
OPY
LIGHT
MICROSCO
PE
FIG. 2.2 LIGHT MICROSCOPY
LIGHT
MICROSCOPE
• Bacteria are examined under light
microscope
⁻ In living state (hanging drop for
motility)
⁻ In fixed stained smears (to examine
shape, size and arrangement)
USES OF 1) Gram staining of clinical specimen
• To identify bacteria in smears, based on Gram stain
LIGHT • Helps in rapid presumptive identification of the organism for
starting empiric antimicrobial treatment
MICROSC • Aids in detecting presence of inflammatory cells
OPE 2) Stool microscopy : to detect ova, cysts, RBCs, pus cells, etc.
3) To visualize fungal elements in clinical material
USES OF 4) Peripheral blood smears to detect parasites like
LIGHT Plasmodium, Leishmania, and microfilariae
MICROSC 5) Identification of an organism from a culture medium
OPE It is a rapid and presumptive diagnostic method
PHASE CONTRAST
MICROSCOPY
Improves contrast in the field
Structures within the cell are clearly seen
Visualised as high-contrast images
Phase difference viewed as contrast
Principle
Small variations in phases of light waves are
converted into corresponding changes in amplitude
This is translated into differences in image contrast
PHASE CONTRAST
MICROSCOPE
• Refractive index (RI) different for bacterial cells and the surrounding medium
• Annular ring: Produce phase difference between the two types of rays by retarding the rays
passing through the object
• The phase plate (diffraction plate) between the objective lens and the eyepiece enhances
the difference in the diffracted rays which produce three- dimensional effect
• Depth and thickness of structures of bacteria are highlightened
Fig. 2.3 (a) Pathway of light
waves in a phase-contrast
microscope and (b) image of
Saccharomyces cerevisiae with
a 3D effect (Source: Masur,
Public domain, via Wikimedia
Commons)
Living microbes, cells and
tissues – visualized as high- Uses of
contrast images phase
contrast
microscop
For research purposes e
DARK- Uses reflected light instead of transmitted light
Principle
FIELD/ Dark-field condenser with central circular stop directs a
DARK- cone of light to the object
GROUND Light falling on the object is scattered and only these rays
reaches objective lens
MICROSCO Object appears bright against a dark background
PY Slender organisms like spirochetes are seen
USES OF DARK-
FIELD MICROSCOPE
• To detect treponemes from syphilitic ulcer
discharge
• To detect Leptospira from urine of suspected
patients
Fig. 2.4 (a)Pathway of light rays passing through a dark-field microscope and (b)
Treponema pallidum visualised by dark-field microscopy (source: (b) CDC, PHIL, Image
ID:21116)
FLUORESCEN
CE
MICROSCOPY
Principle
High intensity light excites a fluorescent agent
Agent emits a longer wavelength light, thereby
producing the image
Filters: for blocking surrounding radiation
Fluorescent dyes (fluorochromes) – auramine O,
rhodamine
Fig. 2.5 (a) Pathway of light in a fluorescence microscope and (b) fluorochrome- stained
Mycobacterium tuberculosis (Source: CDC, PHIL, Image ID22206
FLUORESCEN
CE
MICROSCOPE
• Immunofluorescence:
Uses antibody labelled
with fluorescent dye
• FITC (fluorescein
isothiocyanate):
Conjugate to specifically
stain a particular bacterial
species or infected cells
Fig. 2.6 Fluorochrome staining and immunofluorescence
Uses of fluorescence microscope
• Direct fluorescence (LED microscope):
- Sputum smears for Mycobacterium tuberculosis
- Fungal filaments
• Rapid diagnosis of infections like
- Diphtheria
FLUORESC - Viral respiratory infections
ENCE •
- M. tuberculosis in sputum or CSF smear
Peripheral blood smear: Malaria parasite
MICROSCO • Early detection of bacteria in blood cultures
PE •
•
Cell organelles can be visualised
Quantitative measurement of lipids, proteins and nucleic acids is possible
• Used in specialised areas of cell biology
Principle
CONFOCA Two lenses focus on the same point
Confocal pinholes are present: Allows only light from plane of
L focus to reach detector
Thick specimen — various layers are seen by adjusting the
SCANNIN plane of focus of the laser beam
Fluorescent stained — more visible
G LASER Uses
MICROSC Examine tissue sections
Stem cell research
OPE Fluorescence in-situ hybridisation (FISH)
ELECTRON MICROSCOPY
Discovered by Max Knoll and Ernst Ruska in 1931
Uses beam of electrons instead of light rays
Electron beam is focused by electromagnets
Object scatters electrons
Viewed on fluorescent screen
Resolving power is about
0.1 nm
Fig. 2.7 Electron microscope (Source: Miloš Jurišić, CC BY- SA
3.0 <[Link] via
Wikimedia Commons)
ELECTRON
MICROSCO
PE
Fig. 2.8 A line diagram showing the
path of electron beams and the
electromagnets, which function as a
series of condenser lenses in an
electron microscope
MODIFICATION OF
ELECTRON
MICROSCOPE
Shadow-casting: using vapourised heavy metals; gives
three dimension with contrast
Negative staining: phosphotungstic acid staining; used in
study of fine structures
Freeze-etching: Deep-freezing and forming carbon-
platinum replicas of the material; to study cellular
ultrastructure
Scanning electron microscopy: cell surfaces seen with
greater contrast and higher resolution
ELECTRON MICROSCOPE
Uses of electron microscope
Mainly for research purposes to study
- Intracellular structures
- Pathogen-host structural
interactions
- Virus structures
Fig. 2.9 Scanning electron micrograph of the Covid- 19
virus in the cytoplasm of an infected cell (Source
[Link]
• AFM or scanning force microscopy (SFM)
• Very- high- resolution type of scanning probe microscopy (SPM)
• Resolution power: Fractions of a nanometer
• Principle: Topographic differences in sample translated to different amount of reflected light
• Uses:
Molecular biology
Cell biology
Medicine
ATOMIC FORCE MICROSCOPY (AFM)
