Terminologies in microscopy
TERMINOLOGIES IN MICROSCOPY
1. Reflection:
when
a beam of light strikes the smooth surface of a non-transparent material (e.g., the Polished surface of a mirror), it is bounced back, i.e., reflected. The return
of light or sound waves from a surface.
2. Refraction: when
a beam of light strikes a smooth interface separating two transparent media
(e.g. air and glass; air and water; glass and water), it is partly reflected
and partly transmitted, i.e., refracted, into the second medium.
Reflection
and refraction of light would help us in understanding the principles of
microscopy.
3.
Lens:
A lens is an optical system with two
refracting surfaces. There are two categories of lenses
Ø Converging - convex
Ø Diverging – concave
Convex
lenses converge parallel rays going in and convex lenses diverge rays.
4.
Magnification:
Magnification the size of the image produced by a lens
where is according to the position of the object. The ratio of the height of
the image to the height of the object is called linear magnification, m.
5.
Resolving
power
·
The
resolving power of our I is its ability to see the too closely place objects
as 2 distinct objects.
·
The
resolving power of our eye is its ability to see two closely placed objects as
2 distinct objects in other words, it is the minimum distance between 2 objects
that we can see as separate objects.
·
Our
eye can resolve 2 objects separated by a distance of at least 20 micrometers.
·
For the microscope is determined by its optical system used in it and the wavelength of
light used.
6.
Numerical
aperture
·
The
NA of a system of lenses is an index of its light-gathering capacity and is measured in terms of the angle rays entering into the objective lenses.
·
The
resolving power of the microscope depends on the numerical aperture of the
objective and the substage condenser.
7.
Illumination
·
An
important factor in determining the resolving power of a microscope is the
proper illumination of the object. Illumination involves a light source, a
mirror, a filter, and a condenser.
8.
Kohler
illumination (Optimum illumination)
·
The
Kohler illumination is the precise control of the light path before it reaches
the specimen by proper alignment of the light/lamp, mirror, and adjustment of the
condenser.
·
By
this work, has several advantages including low power input, elimination of
unnecessary irradiation, increasing contrast and depth of focus, and at last
brilliant image.
9.
Aberration-
An optical defect. The
lens does not bring all the rays of light to an exact focus. There are several
different types of aberrations each having a contributing factor to image
quality
10.
Chromatic
aberration
·
This
occurs because the lens refracts various colors present in the illuminating
white light at different angles according to the wavelength of the colors.
11.
Spherical
Aberration:
a. All spherical lenses, having spherical surfaces,
cause spherical aberration of the image of the specimen observed in a microscope.
b. It is an optical defect by which the
lens fails to form a sharp image.
c. Rays
of light that pass through a lens near its edge are converged to a point
nearer the lens than those rays passing through the center of the lens.
12.
Substage
condenser
·
The
condenser of a light microscope functions to condense the scattering light rays
from the light source into a cone of bright light, align and focus onto a tiny
spot of the specimen.
·
This
illuminated part is the area that is perceived by the objective lens.
13.
Fluorochromes
a. Fluorochromes stains are acidic and
basic dyes that are mostly yellow or orange in color. They fluoresce intensely
when exposed to lights of shorter wavelengths such as ultraviolet, violet, and
blue lights.
b. For DNA staining – auramine O, acridine
yellow, and acriflavine are used.
c. Lights used in fluorescence microscope –
mercury vapor arc lamp, quartz-halogen tungsten filament lamp; these two
produce blue light.
d.
14.
Exciter
filter:
a. Designed to transmit only wavelengths
required for the excitation of a fluorochrome, and to absorb or prevent other
wavelengths.
15.
Barrier
filter:
a. Placed between the objective and ocular
lens.
b. Designed to absorb any remaining
exciting radiation, such as UV radiation, which is harmful to the eye, or blue
and violet lights.
16.
Dichromatic
mirror:
a. The other names are – dichroic mirror, partial
mirror, dichromatic beam splitter, and chromatic beam splitter.
b. It is designed to reflect lights of
lower wavelengths and to transmit the lights of higher wavelengths.
c. Therefore, this mirror can isolate the emitted
light from the excitation wavelength.
17.
Photoluminescence
a. Photoluminescence
is a process in which a molecule absorbs a photon in the visible region,
exciting one of its electrons to a higher electronic excited state, and then
radiates a photon as the electron returns to a lower energy state.
18.
Photomultiplier
tube (PMT):
a. Extremely sensitive detectors of light
in the ultraviolet, visible, and near-infrared range. It multiplies the signal
produced from the incident light from the incident light from which single photons
are detectable.
b. It is a device for the quantification of
radiation (light) of very low intensity.
19.
Photons:
a. Discrete particles that carry the energy of
light waves, also known as quanta.
b. In other words - a minute energy packet
of electromagnetic radiation.
20. Birefringence:
a. The double refraction of light in a
transparent, molecularly ordered material.
b. Birefringence is caused by the
anisotropic forces that bind the atoms of a crystal.
c. Anisotropy refers to the non-uniform
distribution of properties in different directions.
d. Some mineral crystals have two different
refractive indices, showing birefringence.
21. Electrons:
a. Electron is one of the three elementary
particles along with proton and neutron in an atom.
b. Used in the electron microscope, cathode
ray- oscilloscope, and television picture tube.
c. The wavelength of the electrons in an Electron microscope is about 0.005nm, which is approximately 100,00 times shorter
than that of visible light.
d. Since electrons cannot pass through the glass, the electrical fields and magnetic fields (electromagnets) are used as
condensers and lenses.
22. Phosphorescent screen:
a. Used in TEM to create images of incident
electrons.
b. Since electrons are not visible to human
eyes, the image is focused onto a phosphorescent screen, which is excited by
the incident electrons to emit visible light.
23. Negative staining in TEM:
a. Negative staining involves the spreading
of a thin film of the specimen with heavy metals, which do not penetrate the specimen
but provide a dark background. The specimen appears bright in photographs
against a dark background.
b. Superior way to study the structure of
virus, bacteria and its internal organelles.
24.
Shadowing:
a. In TEM microorganisms are coated with a thin
film of platinum or other heavy metal at an angle of 45o from
horizontal, so the coating metal strikes the microorganisms only on one side.
b. The metal-coated area scatters electrons
and appears much more brighter than the uncoated area, which appears darker.
25.
Freeze-etching:
a. In TEM to get 3D images of the
intercellular structures.
b. Cells are frozen with liquid nitrogen,
then warmed to -100oc in a vacuum chamber, which becomes brittle.
c. Then using a precooled knife, the brittle
cells are fractured down the internal membranes.
d. Now the specimen Is allowed to sublimate
under a highly vacuum chamber, so the ice is removed now to uncover more internal
structures of cells.
26.
Piezoelectricity:
a. Piezoelectric Effect is the ability of
certain materials to generate an electric charge in response to applied
mechanical stress.
b. The word Piezoelectric is derived from
the Greek piezein, which means to squeeze or press, and piezo, which is Greek
for “push”.
c. When piezoelectric material is placed
under mechanical stress, a shifting of the positive and negative charge centers
in the material takes place, resulting in an external electrical
field.
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