BIO 208L Study Guide - Midterm Guide: Semipermeable Membrane, International System Of Units, Digital Image
BIO 206L: Exercise 2 Analysis
Please answer the following questions during lab. Keep in mind that you need to work independently. Write in
complete sentences, using proper grammar and spelling when an explanation is required. Be complete yet
concise in your answers. Submit your report as a pdf file on Canvas before the beginning of the next lab.
1. What is Köhler illumination and why is important?
Kohler illumination ensures that the shape of the beam of light passing through the specimen will make the best
contrast. The specimen will absorb as much light as possible. To do this the condenser must be properly
adjusted, if done correctly this only needs to be done once during use. The condenser will shape the direction of
light before it reaches the specimen.
2. How does the “field of view” change with magnification? Relate this to the rule that one should always use
the low-power scanning objective first to locate a specimen. How does the “depth of focus” change as
magnification increases? Are thick (e.g., Elodea) or thin (e.g., prepared Paramecium) samples of specimens
preferred at high magnifications?
As magnification increases, the field of view decreases. Only a portion of the specimen can be viewed at a
higher magnification. By starting with a low-power scanning objective, you are able to view the entire
specimen, focus, and then magnify the area of interest. This is why it’s necessary to begin with low power
objective. The depth of focus decreases as magnification increases, you would not be able to focus on the entire
stack of plates, becoming more isolated. Thick samples of specimen are preferred, more dense specimens can be
viewed at greater detail.
3. How does one improve resolution? What does a numerical aperture (N.A.) indicate? What is the relationship
between resolving power and numerical aperture?
To improve resolution the wavelength of light would need to be decreased. Numerical Aperture indicates the
ability of the lens to resolve an image, or its ability to gather light. The higher the Numerical Aperture, the
lower the resolution will be. The relationship between resolving power and numerical aperture is that the
resolution is dependent on wavelength of light and numerical aperture to distinguish between to objects.
4. What is a stage micrometer? How is it different from an ocular reticle? How are a stage micrometer and
ocular reticle used together?
A stage micrometer measures objects on the microscope’s stage using millimeters. Whereas the ocular reticle
uses a special measuring ruler with “reticle units”, this ruler can be seen in the field of view over the specimen.
Each reticle unit is equivalent to 25 micrometers, or 0.025 millimeters. Together they are used to calibrate the
reticle, and determine the absolute length of the specimen by comparison.
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5. Report in the table below for each of the objectives (4x 10x, 40x, & 100x), the corresponding N.A, total
magnification, and the calculated theoretical resolution using Abbe’s equation (show your work, indicate the
wavelength of light chosen).
Wavelength of light chosen is 500 nm.
Objective Magnification
Total Magnification
(10x ocular)
Numerical Aperture
(NA)
Theoretical Resolution
)
4x Objective
x
0.10 NA
3050 nm
10x Objective
x
0.25 NA
1220 nm
40x Objective
x
0.65 NA
469.23 nm
100x Objective
x
1.25 NA
244 nm
6. Based upon the steps you performed to determine the size of the Paramecium specimen using the ocular
reticle and the stage micrometer, list a set of brief instructions to determine the actual size (in SI units) of any
object. Report the calibration of reticle units you determined for each objective in the table below as well.
To determine the actual size (in SI units) of an object, you will begin by adjusting/rotating the ocular that
contains the reticle so that it aligns with the specimen. This should be done with the 10x objective. Once the
reticle ruler has been aligned with the long axis of the specimen determine the length by counting the units to
the nearest 05. units. Repeat the aforementioned steps and rotate the ocular to determine the width of the
specimen in reticle units.
Objective Magnification
mm/RU
µm/RU
4x
0.025
25
10x
0.01
10
40x
0.002
2
100x
0.001
1
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Document Summary
Keep in mind that you need to work independently. Write in complete sentences, using proper grammar and spelling when an explanation is required. Kohler illumination ensures that the shape of the beam of light passing through the specimen will make the best contrast. The specimen will absorb as much light as possible. To do this the condenser must be properly adjusted, if done correctly this only needs to be done once during use. Relate this to the rule that one should always use the low-power scanning objective first to locate a specimen. As magnification increases, the field of view decreases. Only a portion of the specimen can be viewed at a higher magnification. By starting with a low-power scanning objective, you are able to view the entire specimen, focus, and then magnify the area of interest. This is why it"s necessary to begin with low power objective.
