Download Microscopy

Microscope
Microscopy
compound microscopes
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Microscopes are basically classified by the type of light
source used:
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Basic Components:
1. Light source (tungsten lamp)
2. Eyepiece lens: monocular or biocular which has a
magnification of 10 X.
3. Objective lenses: may be four, five or more, 4X, 10X,
40X, and 100X.
4. The optical tube, which occupies the distance
between the eyepiece lens, and the objectives, which
is usually equal to 160 mm.
Basic Components
5. Stage
6. Sub-stage condenser, which directs the beam
of light from the source onto the specimen, it
consists of two lenses.
7. Iris diaphragm, number of leaves can be open
or closed to control the amount of light passed.
8. Microscope contains a built in light source at its
base, which usually contains a transformer for
adjusting the light intensity.
Important points around the light microscope:
1. Total magnification =
• Eyepiece magnification X Objectives magnification.
• The image seen by the eye through a compound microscope is
termed the virtual image and is upside and reversed.
2. The numerical aperture (NA):
• Is a designation of the amount of light entering the objective
from?
Lens
A
c
B
• The microscopic field
• NA = R sin 

Where:
Object
• R is the refractive index of glass
•  is the angle made by one ray passing through
the edge of the lens with the other ray passing the center of
the lens.
• Since sin  = AC/AP
• Then NA depends on the radius of the lens.
3. Resolving power:
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–
–
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Is the useful limit of magnification, it is the ability of
microscope, at specific magnification to distinguish two
separate objects situated close to one another and the
ability of the lens to reveal fine details.
The smaller the distance between the two specific
objects that can be distinguished apart, the greater the
resolution power of the microscope.
Minimal distance between two objects =
(0.612 X ) / NA
The larger NA, the smaller the resolvable distance and
hence, the more efficient the resolution power.
4. Depth of field:
• Is the capacity of the objective lens to focus in
•
•
different planes at the same time.
This is largely dependent on the NA.
Where the greater the NA, the smaller the depth of the
field, it is possible to increase the depth of the field
slightly by closing the iris diaphragm. Thus decrease
the NA.
5. Chromatic Aberration:
•
since light is formed of several wavelength then light
component are not bent in the same way as they pass
through the lens and therefore are not brought to the
same focus.
6. Spherical aberrations:
•
•
the light wave, as they travel through the lens, are bent
differently, depending in which part of the lens they
pass through, rays passing through the peripheral
portions of the lens are brought to a shorter focal point
than those rays passing through the thicker part of the
lens.
To correct chromatic and spherical aberration achromatic
(brings 2 colors) and apochromatic (brings 3 colors blue,
yellow and red) lenses may be used which are fine lenses
produced to bring rays of several colors to a common
focus
• The medium between the objective and the object
is a factor that must be taken into consideration for
the most effective use of the microscope lens.
• The low power objective 4x, 10x, and high dry
objective 40x use air.
• When oil immersion lenses are employed, a drop
of oil should be used, otherwise bending of the light
waves occurs since oil has the same RI of glass,
while air increase diffraction.
Types of compound Microscopes
1. Bright Field Microscopy:
•
2.
The light source lies in the visible region.
Ultraviolet Microscopy:
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3.
The shorter wavelength of UV can extend the limit of
microscope resolution to about 0.1  m. However, UV
light is invisible to the human eye, so the image must
be recorded on a photographic plate or fluorescent
screen. Because this light is absorbed by glass, all
lenses must be made of quartz, such microscopes
are two expensive for routine use.
Fluorescence microscopy:
–
A sample labeled with a fluorescent dye is illuminated
with UV light, the location of the dye in the specimen
is revealed by its fluorescence or emission of visible
light
Types of Microscopes
4.
Dark field Microscopy:
–
One sees a black background; against which
suspended bacteria or element appear bright. The
dark field microscope uses a special condenser that
illuminate the sample with a hallow cone of light in
such a manner the light is not directed into the
objective lens, revealing the shape of that object.
5.
Phase contrast Microscopy:
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Bacterial or animal cells are difficult to be seen using
the light microscope unless the sample is dried and
stained. This microscope enhances the slight
difference in refractive index between the cells and
the medium and thus can be used to visualize the
living bacteria and platelets, in which the slight
differences in RI are converted to differences in light
intensity.
Types of Microscopes
6.
Electron Microscopy:
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Since magnification greater than 1500X to 2000X are
not practical with the light microscope due to
decreased efficiency in resolving power. The electron
microscope has come into use, where magnification
of 50,000X may be obtained, with a high degree of
resolving power.
There are two types of electron microscope:
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1.
2.
Transmission Electron Microscope (TEM) {2 dimensional}
Scanning Electron Microscope
(SEM) {3 dimensional}