Download Laboratory Exercise 3: Microscopy The microscope is a tool for the

Laboratory Exercise 3: Microscopy
The microscope is a tool for the study of cells and tissues. It permits visualization of structures too small to be
seen by the unaided eye. For a microscope to reveal structural details of a specimen depends on two factors:
magnification and resolution.
Magnification – Definition: To increase the size of an object by the use of lenses. Magnification is the increase
of the ratio of image size to actual size. In a compound microscope, magnifying power is achieved by interaction
of two lenses: the ocular (eyepiece) lens and the objective lens. The total magnification is the product of the
ocular and objective magnifications.
Ocular Lens
10X
10X
10X
10X
X
Objective Lens
=
4X scanning lens
10X low power lens
40X high dry power lens
100X oil immersion 1ens
Total Magnification
40X
100X
400X
1000X
Resolution (Resolving power) – Definition: The ability to distinguish two adjacent objects as separate individual
parts in a specimen. Resolution is the capacity of the microscope to distinguish two adjacent objects set a small
distance apart. The resolving power of the compound light microscope is 0.2 micrometer (m). Two objects will
appear as distinct and separate structures if they are 0.2 m or more apart. The human eye's resolving power is
100 m.
Resolution is determined by 2 factors: 1.The physical properties of light and 2.The numerical aperture (N.A.) of
the lens. The N.A. reflects the amount of light that enters the lens. The greater the resolving power, the higher
the N.A. The N.A. value for each objective lens is printed on the lens.
Using immersion oil and the sub-stage condenser light rays are bent toward the oil immersion lens to increase
N.A. and maximize resolution.
Microscopy Terms
Field of View - circle of light seen in the microscope.
Working Distance - the amount of space between the slide and objective when specimen on the slide is in focus.
The working distance is greater under the lower powers of the microscope than under the higher powers.
Orientation of image compared to the orientation of the specimen on the slide is inverted and reversed.
Parfocal - ability to switch from one objective lens to another and the image stays focus. Focus under high
power using the fine adjustment only, since the working distance is small. Never under high power use the
coarse adjustment. Use coarse adjustment for low power.
Before switching from low power to high power center the specimen in the field of view. The high power lens
has 4 times the magnification of the low power lens; its field of view will be one-fourth the area of the low
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power field of view. An image on the edge of the low power field of view will be lost under high power unless
it is first centered.
Oil Immersion Lens - in light microscopy, optimal magnification and resolving power is achieved with the oil
immersion objective. This lens has high resolution and high N.A., since the lens gathers in a great amount of
light.
High resolving power is enhanced by clear mineral oil between the objective and the slide. The oil has a
refractive (light-bending) index similar to glass; it reduces the amount of light scattering that occurs with air
between slide and objective. As the light rays are concentrated, the resolving power is increased.
Depth of Focus
The depth of focus (depth of field) determines how much foreground and background area is simultaneously in
focus at one time. The low power lens has great depth of focus; everything in the foreground and background is
in sharp focus. High power lens has limited depth of focus; little in front of or behind the plane of focus is
sharply seen.
Binocular Dissecting Microscope
The binocular dissecting microscope has great depth of focus. It allows specimens to be viewed in 3D.
The dissecting microscope has a zoom objective lens. The zoom feature permits continuous varying of
magnification throughout the range of the lens without loss of focus. No refocusing is necessary and no image
blackout when shifting from one magnification to another.
Total magnification is equal to ocular lens power (10X or 15X) multiplied by objective lens power (3X or 7X)
for a maximum magnification of 45X or 70X.
This microscope can use either transmitted light or reflected light. There is no need for a thin specimen through
which light must pass. Large specimens are illuminated with reflected light, producing 3D images.
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