Light Microscope Principle & Uses
(By: Dr. Akalesh K Verma, Asst. Prof., Cotton University)
Fig 1: Components of Light Microscope.
Parts of Light Microscope
a. Illuminator: the light
source in the base of the microscope;
b. Abbe Condensor: a two lens
system that collects and concentrates light from the illuminator and directs it
to the iris diaphragm;
c. Iris diaphragm: regulates
the amount of light entering the lens system;
d. Mechanical stage: a
platform used to place the slide on which has a hole in the center to let light
from the illuminator pass through. Often contains stage clips to hold the slide
in place;
e. Body tube: houses the lens
system that magnifies the specimens;
f. Upper end of body
tube—oculars/eye pieces: what you view through;
g. Lower end of body
tube—nose-piece: revolves and contains the objectives.
h. Essentially, a light microscope
magnifies small objects and makes them visible. The science of microscopy is
based on the following concepts and principles:
i.
Magnification
is simply the enlargement of the specimen. In a compound lens system, each lens
sequentially enlarges or magnifies the specimen;
j.
The
objective lens magnifies the specimen, producing a real image that is then
magnified by the ocular lens resulting in the final image;
k. The total magnification can
be calculated by multiplying the objective lens value by the ocular lens value.
KEY TERMS IN MICROSCOPY
Magnification
In most light microscopes,
the objective lens, which is closest to the specimen, magnifies objects 100×
(times), and the ocular lens, which is nearest the eye, magnifies 10×. Using
these two lenses in combination, organisms in the specimen are magnified 1000×
their actual size when viewed through the ocular lens. Objective lenses of
lower magnification are available so that those of 10×, 20×, and 40×
magnification power can provide total magnifications of 100×, 200×, and 400×,
respectively. Magnification of 1000× allows for the visualization of fungi,
most parasites, and most bacteria, but it is not sufficient for observing
viruses, which require magnification of 100,000× or more (Electron Microscopy needed).
Total
magnification:
The total magnification obtained in a compound microscope is the product
of objective magnification and ocular magnification.
Mt = Mob X Moc
Where,
Mt = Total magnification,
Mob = Objective magnification and
Moc = Ocular magnification
Resolution
In microscopy,
the term 'resolution' is used to describe the ability
of a microscope to distinguish in details. In other words,
this is the minimum distance at which two distinct points of a specimen can
still be seen - either by the observer or the microscope camera
- as separate entities.
Fig 2: Difference between high and low resolution image of an object.
To optimize visualization,
other factors besides magnification must be considered. Resolution, defined as
the extent to which detail in the magnified object is maintained, is also
essential. Without it everything would be magnified as an indistinguishable
blur. Therefore, resolving power, which is the closest distance between two
objects that when magnified still allows the two objects to be distinguished
from each other, is extremely important. The resolving power of most light
microscopes allows bacterial cells to be distinguished from one another but
usually does not allow bacterial structures, internal or external, to be
detected.
To
achieve the level of resolution desired with 1000× magnification, oil immersion
must be used in conjunction with light microscopy. Immersion oil has specific
optical and viscosity characteristics designed for use in microscopy. Immersion
oil is used to fill the space between the objective lens and the glass slide
onto which the specimen has been affixed. When light passes from a material of
one refractive index to a material with a different refractive index, as from
glass to air, the light bends. Light of different wavelengths bend at different
angles creating a less distinct distorted image. Placing immersion oil with the
same refractive index as glass between the objective lens and the coverslip or
slide decreases the number of refractive surfaces the light must pass through
during microscopy. The oil enhances resolution by preventing light rays from
dispersing and changing wavelength after passing through the specimen. A
specific objective lens, the oil immersion lens, is designed for
use with oil; this lens provides 100× magnification on most light microscopes.
Lower
magnifications (i.e., 100× or 400×) may be used to locate specimen samples in
certain areas on a microscope slide or to observe microorganisms such as some
fungi and parasites. The 1000× magnification provided by the combination of
ocular and oil immersion lenses usually is required for optimal detection and
characterization of bacteria.
Contrast
The another key component
to light microscopy is contrast, which is needed to make objects stand out from
the background. Because microorganisms are essentially transparent, owing to
their microscopic dimensions and high water content, they cannot be easily
detected among the background materials and debris in patient specimens. Lack
of contrast is also a problem for the microscopic examination of microorganisms
grown in culture. Contrast is most commonly achieved by staining techniques
that highlight organisms and allow them to be differentiated from one another
and from background material and debris. In the absence of staining, the
simplest way to improve contrast is to reduce the diameter of the microscope
aperture diaphragm increasing contrast at the expense of the resolution.
Setting the controls for bright field microscopy requires a procedure referred
to as setting the Kohler illumination.
Fig 3: High and low contrast images.
The resolving power
The resolving
power is the inverse of the distance between two objects that can be just
resolved. On the other hand it is the ability of an optical instrument or type of film to
separate or distinguish small or closely adjacent images.
It
is given by Abbe's criterion
Resolving power = 1/delta 'd' = 2a / 'λ'
where a is the numerical aperture
d
is distance
λ is the wavelength of the objective lens used
(a)
When wavelength of incident light is decreased, the resolving power
increases.
(b)
When aperture of the objective lens is decreased, the resolving power
decreases.
Numerical Aperture
The numerical aperture (NA) of an optical system
(e.g. an imaging system) is a measure for its
angular acceptance for incoming light. It is defined based on geometrical considerations and is thus a
theoretical parameter which is calculated from the optical design. It cannot be
directly measured, except in limiting cases with rather large apertures and
negligible diffraction effects.
Numerical Aperture of an Optical System
Numerical aperture is
a numerical value concerned with the diameter of the objective lens in relation
to its focal length. Thus, it is related to the size of the lower aperture of
the objective, through which light enters into it. In a microscope, light is
focused on the object as a narrow pencil of light, from where it enters into
the objective as a diverging pencil.
Numerical aperture (n.a.) = n sin θ
Where,
n = Refractive index of the medium between the object and the objective
and
θ = Half aperture angle
NA of a Lens
A simple case is that of a collimating lens:
Fig 4: A collimating lens
can theoretically accept light from a cone, the opening angle of which is
limited by its size.
PRINCIPLES
The light microscope is an important
tool in the study of microorganisms,
particularly for identification purposes. The light microscope is
an instrument for visualizing fine detail of an object. It does this by
creating a magnified image through the use of a series of glass lenses, which
first focus a beam of light onto or through an object, and convex objective
lenses to enlarge the image formed. In the majority of light microscopes,
the image is viewed directly through binocular eyepieces that act as a
secondary lens in the form of a magnifying glass to observe the projected
image. Such instruments are termed ‘compound microscopes,’ and the total
magnification is the sum of the objective magnification and the eyepiece
magnification. The magnification range extends from ×10 to ×1000, with a
resolving power of the order of 0.2 μm, depending on the type and
numerical aperture (area available for passage of light) of the objective
lenses.
Fig 5: Path of light in Light microscope.
Fig 6:
Principle of Light Microscope
APPLICATIONS OF LIGHT MICROSCOPE
1. For details study of cells or tissues
section under high magnification and contrast.
2. For histopathological study to
understand toxic effect of drugs/chemicals.
3. For studying anatomical structures of
bones, tissues (Liver, kidney, spleen etc).
4. For counting WBC, RBC and platelets
Counts in patients sample.
5. Rapid final identification of certain
organisms by direct visualization in patient specimens
6. Detection of different organisms or
contaminations present in the specimen.
7. Detection of organisms not easily
cultivated in the laboratory.
8. Evaluation of patient specimens for
the presence of cells indicative of inflammation (i.e., phagocytes) or
contamination (i.e., squamous epithelial cells).
9. Determination of an organism’s
clinical significance; bacterial contaminants usually are not present in
patient specimens at sufficiently high numbers (×105 cells/mL)
to be seen by light microscopy.
10. Provide preculture information about
which organisms might be expected to grow so that appropriate cultivation
techniques are used.
11. Determine which tests and methods
should be used for identification and characterization of cultivated organisms.
12. Provide a method for investigating
unusual or unexpected laboratory test results.
