Microscopy is the technical field of using microscopes to view objects that cannot be seen with the naked eye. The diameters of most cells fall between 0.2 and 50 μm. There are two main types of microscopes used: light microscopes, which use lenses and visible light, and electron microscopes, which use electron beams. Light microscopes have two main lenses - an objective lens closest to the object and an ocular or eyepiece lens closest to the observer's eye. Increased magnification without improved resolution results only in a blurred image. Modern microscopes allow viewing of very small structures through techniques like phase contrast, fluorescence, dark field, and electron microscopy.

1. MICROSCOPY
Ref: Microbiology
5th Edition
Lansing M. Prescott

2. • Microscope invented by Francis Janssen and
Zacharias Janssen
• Microscopy is the technical field of using
microscopes to view objects and areas of objects
that cannot be seen with the naked eye.
• The diameters of the majority of cells fall within
a range of 0.2 and 50 μm.
• When a ray of light passes from one medium to
another, refraction occurs—that is, the ray is
bent at the interface.
• The refractive index is a measure of how greatly
a substance slows the velocity of light, and the

3. • a prism bends light because glass has a
different refractive index from air.
• Lenses act like a collection of prisms
operating as a unit.
• When the light source is distant so that parallel
rays of light strike the lens, a convex lens will
focus these rays at a specific point, the focal
point.
• The distance between the center of the lens and
the focal point is called the focal length

4. Bright Field
Microscope

5. • The ability of an observational instrument such
as a human eye or a microscope to reveal
details of structure is expressed in terms of
limit of resolution (l).
• Resolving Power; The smallest distance that
may separate two points on an object and
still permit their observation as distinct
separate points.
• Magnification, the increase in size of optical
image over the size of the object being viewed.
• Increased magnification without improved
resolution results only in a large blurred image.

6. • The human eye has no power of magnification.
• The limit of resolution (l) of any optical
instrument (i.e., eye or microscope) is given
approximately by the Abbe’s relationships :
• Where, λ (lambda) is the wavelength
• n is refractive index (a function of density) of the
material (i.e., mostly air or water) between the
specimen and the first lens (or objective lens), and
• The quantity “n sin α” - the numerical aperture
(NA).

7. • Abbe’s relationships make it clear that high
resolution in a microscope can only be
achieved by manipulating a small number of
variables: the wavelength of the illuminating
radiation, the refractive index and the
aperture.
• The Numerical aperture is limited to
something less than 90° since that would have
the lens and specimen in contact with one
another.
• In fact, 85° is about the limit in good optical
microscopes.

8. • Refractive index is easy to alter, but only
within narrow limits.
• It can be increased by using oils to fill the space
between the specimen and the objective lens
(oil immersion lens).
• In order to increase resolution, ultraviolet rays
can be used instead of normal light and
specialized lenses like quartz may be used for
UV source.
• Thus, a good light microscope, with a
numerical aperture of 1.4 and using light of
short wavelength (0.4 μm) will resolve two

9. • The following two types of microscopes are
most extensively used :
a. Light Microscopy
b. Electron Microscopy
• Light Microscopy; The compound light
microscope uses visible light for illuminating
the object and contains glass lenses that
magnify the image of the object and focus the
light on the retina of the observer’s eye.
• Electron Microscopy; a microscope with high
magnification and resolution, employing
electron beams in place of light and using

10. • It consists of two lenses, one at each end of a
hollow tube;
i. The lens closer to eye is called ocular lens or
eyepiece and
ii. The lens closer to the object being viewed is
called objective lens.
• Usually objective lenses of various magnifying
powers are mounted on a revolving turret at
the lower end of the tube.
• The object, supported by a glass slide under
the objective lens, is illuminated by light
beneath it.

11. • In some microscopes, a third lens, called
condenser lens, is located between the object
and the light source and serve to focus the
light on the object.
• Certain improved types of light microscopes:
i. Bright-field microscope
ii. Dark-field microscope
iii. Phase-contrast microscope, and
iv. Fluorescence microscopes
• Electron Microscopes:
i. Transmission Electron Microscope (TEM)
ii. Scanning Tunneling Microscope (SEM)

12. Dark- Field Microscopy
Treponema pallidum, the spirochete
that causes syphilis
Volvox and Spirogyra

13. Phase-contrast Microscopy
Spirillum volutans Clostridium botulinum, the bacterium
responsible for botulism, with
subterminal oval endospores;

14. Fluorescence Microscopy
Escherichia
coli
Paramecium
tetraurelia;
conjugating
The flagellate
protozoan
Crithidia
luciliae
A mixture of
Micrococcus
luteus and
Bacillus cereus
(the rods). The
live bacteria
fluoresce green;
dead bacteria are
red.

15. Bright- field Microscope
Rhodospirillum rubrum
TEM (Transmission
Electron Microscope)
STM (Scanning Tunneling
Microscope)