Dr M.K.Bedi, Deptt.

of Microbiology, DIBNS 1

4/26/2017
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Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

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 Microscopy: The science dealing with the betterment of

microscope for studying the microorganisms is known as
microscopy.

 A microscope may be defined as an instrument

consisting of a lens or a combination of lenses for viewing
magnified images of minute objects. It is an instrument
that magnifies and resolves the image seen through it.

 Most photographs of cells are taken using a microscope,

and these pictures can also be called micrographs

Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

 Van Leeuwenhoek
4

 Simple microscope: The simple

microscope used by van
Leeuwenhoek in the seventeenth
century had only one lens and
was similar to a magnifying glass

 Leeuwenhoek’s single lens could

magnify a microbe 300X. His
simple microscopes enabled him
to be the first person to see
bacteria

 Normally the human eye cannot

see objects less than 100μm in
diameter, thus cannot see a
bacteria (0.2 – 1.5μm diameter
and 3 – 5 μm length)
Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017
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Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

 Compound microscope
6
 ZacchariasJanssen, is credited with making the first compound microscope
around 1600.
 Robert Hooke, built compound microscopes, which had multiple lenses.
 He made an observation that if the second lens is used to magnify the image
formed by the primary lens then the image obtained subsequently reveals the
fine structures of the object
 Compound microscope has two sets of lenses, the objective and the eyepiece.
 The basic principle behind it is to visualize the enlarged image of the object by
the help of these lenses.
 When the beam of light passes through the object and then convex lense of
objective, it forms a real inverted and enlarged image of the object in the focal
plane of eyepiece (by adjustment).
 This image now acts as object for the eyepiece.
 Eyepiece lenses finally forms a further enlarged virtual image of the object.
 Thus, magnifying power of a compound microscope is the multiplication
product of magnifying powers of objective and eyepiece.
 A compound microscope differs from a simple one in having two sets of
lenses:
 Objective : near Dr
theM.K.Bedi,
object Deptt. of Microbiology, DIBNS 4/26/2017
 Eye piece / ocular : near the eye
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 In the microscope beam path (A), the

object (←) (1) is recorded by the
objective (2) and is first projected at
infinity. Therefore, the light rays
originating from one point of the object
run parallel behind the objective. The
tube lens (3) now functions in a similar
way to a camera and produces a
magnified intermediate image (4), which
is captured by the eyepiece (5) and
shown to the eye (6)

 The resulting viewing angle δ1 is much

larger than δ2 in case B, where the
object is seen directly from a distance of
approx. 25 cm

Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

 Types of microscopes
8
 On the basis of method of magnification there are two types
 Optical / light microscope : uses optical lenses and light waves to obtain
magnification. In a light microscope, visible light passes through the specimen (the
biological sample you are looking at) and is bent through the lens system, allowing
the user to see a magnified image. They include
 Bright field microscopy
 Dark field microscopy
 Phase contrast microscopy
 Interference microscopy
 Fluorescence microscopy
 Electron microscope : Electron microscopes differ from light microscopes in that
they produce an image of a specimen by using a beam of electrons rather than a
beam of light. Use electromagnetic lenses and an electron beam to obtain
magnification. They include
 TEM : Transmission electron microscopy
 SEM : Scanning electron microscope
 HVEM : High/ voltage electron microscope

Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

 Types of microscopes
9

 Most microscopes can be classified as one of three basic types: optical,

charged particle (electron and ion), or scanning probe.

 Optical microscopes are the ones most familiar to everyone from the high
school science lab or the doctor ’s office. They use visible light and
transparent lenses to see objects as small as about one micrometer (one
millionth of a meter), such as a red blood cell (7 μm) or a human hair (100
μm).

 Electron and ion microscopes, use a beam of charged particles instead of

light, and use electromagnetic or electrostatic lenses to focus the particles.
They can see features as small a tenth of a nanometer (one ten billionth of a
meter), such as individual atoms.

 Scanning probe microscopes use a physical probe (a very small, very sharp
needle) which scan over the sample in contact or near/ contact with the
surface. They map various forces and interactions that occur between the
probe and the sample to create an image.
Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017
 Focal length
10

 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.

Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

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Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

 Magnification
12

 Magnification is the ratio of the size of an object seen under microscope to

the actual size observed with unaided eye
 Magnification is a measure of how much larger a microscope (or set of
lenses within a microscope) causes an object to appear. For instance, the
light microscopes typically used in high schools and colleges magnify up
to about 400 times actual size. So, something that was 1 mm wide in real
life would be 400 mm wide in the microscope image.
 The total magnification compund microscope is calculated by multiplying
the magnification of objective by the magnification of ocular lens or eye
piece

 The following objectives are generally used;

 Scanning: 4 X
 Low power : 10 X
 High power : 40 X
 Oil immersion : 100 X
 Ocular lenses are generally of 10 X. So if 45 X objective is used with a 10
X eyepiece, theDroverall magnification
M.K.Bedi, of the DIBNS
Deptt. of Microbiology, specimen will be 450 X.
4/26/2017
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Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

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Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

 Magnifying power
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 It is the ability to enlarge the image of an

object

 It is also called angular magnification and it is

the ratio of the angle subtended by an object
and image

Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

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Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

 Resolution (resolving power)
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 The ability of the lenses to distinguish fine detail and structure.

 The resolution of a microscope or lens is the smallest distance by which two points
can be separated and still be distinguished as separate objects. The smaller this
value, the higher the resolving power of the microscope and the better the clarity and
detail of the image. If two bacterial cells were very close together on a slide, they
might look like a single, blurry dot on a microscope with low resolving power, but
could be told apart as separate on a microscope with high resolving power
 The most important part of the microscope is the objective lens which produces a
clear image, not just a magnified one.
 Resolution is extremely important.
 Specifically, it refers to the ability of the lenses to distinguish two points a specified
distance apart.
 For example, if a microscope has a resolving power of 0.4 nm, it can distinguish two
points if they are at least 0.4 nm apart.
 A general principle of microscopy is that the shorter the wavelength of light used in
the instrument, the greater the resolution.
 The white light used in a compound light microscope has a relatively long
wavelength and cannot resolve structures smaller than about 0.2 μm.

Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

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 Given sufficient light, the unaided human eye can distinguish two
points 0.2 mm apart

 A modern light microscope (often abbreviated to LM) has a

magnification of about 1000x and enables the eye to resolve
objects separated by 200 nm

 light of shorter wavelenght (blue light or UV) and Immersing the

specimen and the front of the objective lens in a medium with a
high refractive index (such as oil) gave another small improvement,
but these measures together only brought the resolving power of
the microscope to just under 100 nm

Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

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 Much of the optical theory underlying microscope design was

developed by the German physicist Ernst Abbé in the 1870s.

 The minimum distance (d) between two objects that reveals

them as separate entities is given by the Abbé equation, in
which lambda (λ) is the wavelength (the distance between
two corresponding points on a wave) of light used to
illuminate the specimen and n sin θ is the numerical aperture
(NA)

 The numerical aperture (NA): NA = (n sin θ)

 Limit of resolution (d) is indirectly proportional to resolution
Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017
 Numerical aperture
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Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

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Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

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 Theta 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 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, and therefore achieve
higher resolution, is to increase the refractive index with immersion oil, a colorless liquid
with the same refractive index as glass.
 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.
Dr M.K.Bedi,
An increase in numerical apertureDeptt.
and of Microbiology,
resolution DIBNS 4/26/2017
results
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Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

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 The resolution of a microscope depends upon the numerical aperture of

its condenser as well as that of the objective. This is evident from the
equation describing the resolution of the complete microscope

 Most microscopes have a condenser with a numerical aperture between 1.2

and 1.4. 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 (450 to 500 nm) is approximately 0.2 μm

 At best, a bright/ field microscope can distinguish between two dots around
0.2 m apart (the same size as a very small bacterium)

Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

 Refractive index
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 It is a measure of the light­bending ability of a medium or it is a measure

of how greatly a substance slows the velocity of light
 the ratio of the velocity of light in a vacuum to its velocity in a specified
medium
 Refractive index (n)= c/ν
 We change the refractive index of specimens by staining them
 Light rays move in a straight line through a single medium
 After the specimen is stained, when light rays pass through the two
materials (the specimen and its medium) with different refractive indexes,
the rays change direction (refract) from a straight path by bending or
changing angle at the boundary between the materials and increase the
image’s contrast between the specimen and the medium
 As the light rays travel away from the specimen, they spread out and enter
the objective lens, and the image is thereby magnified. To obtain a clear,
finely detailed image under a compound light microscope, specimens must
be made to contrast sharply with their medium (substance in which they
are suspended). To attain such contrast, we must change the refractive
index of specimens from that of their medium.
Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017
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Material/medium Refractive index at 25ºC

Air 1

Water 1.33

Glass 1.51

Paraffin oil 1.47

Cedarwood oil 1.51

Sandalwood oil 1.51

Crown oil 1.55

Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

 Working distance
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 Working distance of an objective is the distance between the front

surface of the lens and the surface of the cover glass (if one is used) or
the specimen when it is in sharp focus

 Objectives with large numerical apertures and great resolving power have
short working distances

 The largest useful magnification increases the size of the smallest

resolvable object enough to be visible. Our eye can just detect a speck
0.2 mm in diameter, and consequently the useful limit of magnification is
about 1,000 times the numerical aperture of the objective lens. Most
standard microscopes come with 10X eyepieces and have an upper limit
of about 1,000X with oil immersion. A 15X eyepiece may be used with
good objectives to achieve a useful magnification of 1,500X. Any further
magnification increase does not enable a person to see more detail. A
light microscope can be built to yield a final magnification of 10,000X, but
it would simply be magnifying a blur. Only the electron microscope
provides sufficient resolution to make higher magnifications useful

Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

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Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

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Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

 The Bright Field Microscope
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 Produces a dark image against a brighter

background

Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

 Preparation and Staining of Specimens
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 increases visibility of specimen

 accentuates specific morphological features

 preserves specimens

Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

 fixation
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 process by which internal and external

structures are preserved and fixed in position
 process by which organism is killed and firmly
attached to microscope slide
 heat fixing
preserves overall morphology but not
internal structures
 chemical fixing
protects fine cellular substructure and
morphology of larger, more delicate
organisms
Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017
 dyes
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 dyes
 make internal and external structures of cell
more visible by increasing contrast with
background
 have two common features
chromophore groups
chemical groups with conjugated
double bonds
give dye its color
ability to bind cells

Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017

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 The basic design of

b r i g h t f i e l d
microscopes has
been modified for
special uses

 I n v e r t e d
microscopes allow
viewing of cells in
flasks, welled/
plates, or other deep
containers that do
not fit between the
Inverted microscope
objectives and stage
of standard BF Position of light source and objectives
microscopes is “inverted”/ / light source is above
specimen and objective lenses are
located beneath the stage
Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017
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Dr M.K.Bedi, Deptt. of Microbiology, DIBNS 4/26/2017