Exercise 1: The Compound Microscope
• Microscope
✓ an optical instrument used to view very small objects which are invisible to the naked eye
• Types
✓ Based on the illumination source
1. Light microscopes
2. Electron microscopes
✓ Other types
1. Bright field microscopes
2. Dark field microscopes
3. Ultraviolet microscopes
4. Fluorescent microscopes
5. Phase contrast microscopes
• Parts of the microscope (refer to lab manual)
• Principles of Microscopy and important features of the objectives
1. Focal length (f)
o refers to the ____________________________ to the ____________
o amount of __________ required between the objective lens and the top of your object, in order to be able
to view an image through the microscope that is in-focus.
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Relationship to magnification of the specimen image: ______________________________
Objective Focal length (f)
LPO
HPO
OIO
2. Limit of Resolution (r)
o __________________ between two closely spaced points in an object that can be seen as distinct points
of an image
o __________________ between 2 adjacent points that a microscope can distinguish
Relationship to magnification of the specimen image: ______________________________
3. Resolving Power (R)
o ability of the microscope to distinguish two points as ______________
Relationship to the magnification of the image: ___________________________________
4. Numerical Aperture (N.A.)
o an index of the _________________ of the objective
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o amount of light gathered from the specimen
o inherent property of the lens
o dependent on the index of refraction (n) of the medium between the specimen and lens as well as the
radius (r) and working distance (w) of the lens. (r) and (w) are related to the angle theta
Relationship to the magnification of the image: ___________________________________
Objective Numerical Aperture (N.A.)
LPO
HPO
OIO
5. Refractive index/ index of refraction (n)
o Ability of the medium to ____________
o Higher (n) means less deflection hence more light enters the objectives
o (n) of air= 1.0 (n) of oil= 1.5 (n) of glass= 1.5
6. Working distance (w)
o Distance from the _____ to the _________ usually expressed in millimeters
Objective Working distance (w)
LPO
HPO
OIO
7. Depth of focus
o Property of the microscope that indicates __________ the space of the microscope can bring to a clear
view at any one time
8. Field of Vision (FOV)
o ______________ under the microscope
o LPO- 1.5mm2 HPO- 0.35mm2
Laboratory work (DP Ong 2019)
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A. Operation of the Microscope
Focus the specimen on the following objectives observing proper technique:
LPO -> HPO -> OIO
1. Draw specimen that appears without oil .
2. Draw specimen that appears with oil.
3. Compare the image produced under the OIO with oil and without oil.
B. Calibration of the microscope
o Calibration of the microscope is usually done in order to be able to precisely measure the size of a specimen
being examined
o Calibration refers to the act of checking or adjusting (by comparison with a standard) the accuracy of a
measuring instrument
o Unit of measurement in microscopy is usually expressed in micrometers or microns (µm)
Ocular micrometer- mounted scale of uniform distance but must be calibrated for a particular objective eyepiece and tube
length before measurements are made.
Stage micrometer- glass slide of known intervals
1 small division = 0.01mm or 10 µm
1 big division = 0.1mm or 100µm
a. Calibration of ocular micrometer involves calculating the calibration factor (CF) afterwhich, measurement of the
specimen can be done.
Formula for computing the Calibration factor:
b. Measurement of the Specimen
Specimen size = number of spaces or divisions subtended by the specimen x CF
c. Determination of the area of the FOV
Measure the diameter of the FOV using the stage micrometer and compute for the area using the formula for
the computation of the area of a circle.
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