Download Chapter 1: The Microscope – Introduction

Microscope Lab
Objectives:
After doing this lab you should be able to
1. Properly use both a compound microscope, including setting critical illumination, bringing objects
into focus, and estimating sizes
2. Make a wet mount
3. Make, scale, and label scientific drawings
Since its invention, the microscope has been a valuable tool in the development of scientific theory.
Magnifying lenses have been known for as long as recorded history, but it was not until the advent of the
modern compound light microscope that the device was used in biology. A compound microscope is
composed of two elements; a primary magnifying lens and a secondary lens system, similar to a telescope.
Light is caused to pass through an object and is then focused by the primary and secondary lens. If the
beam of light is replaced by an electron beam, the microscope becomes a transmission electron
microscope. If light is bounced off of the object instead of passing through, the light microscope becomes
a dissecting scope. If electrons are bounced off of the object in a scanned pattern, the instrument becomes
a scanning electron microscope.
The function of any microscope is to enhance resolution. In particular, they increase resolving power, the
ability to distinguish 2 separate objects as 2 distinct objects (or points of light). Resolution is the distance
between these 2 points. Unaided the human eye has a resolution of about 100 μm. The microscope is
used to create an enlarged view of an object such that we can observe details not otherwise possible with
the human eye. The microscope we will be using, if used properly can resolve points separated by as little
as 0.1 μm. Because of the enlargement, resolution is often confused with magnification, which refers to
the size of an image. In general, the greater the magnification, the greater the resolution, but this is not
always true. There are several practical limitations of lens design which can result in increased
magnification without increased resolution.
Figure 1.1 illustrates this point. If an image of a cell is magnified from 10X to
45X, the image gets larger, but not necessarily any clearer. The image on the left
is magnified with no increase in resolution. The image on the right is magnified
the same, but with increasing resolution. Note that by the time the image is
magnified 10X (from 10X to 100X), the image on the left is completely
unusable. The image on the right, however, presents more detailed information.
Without resolution, no matter how much the image is magnified, the amount of
observable detail is fixed, and regardless of how much you increase the size of
the image, no more detail can be seen. At this point, you will have reached the
limit of resolution or the resolving power of the lens. This property of the lens is
fixed by the design and construction of the lens. To change the resolution, a different lens is often the only
answer.
PreLab
Read pages 95-97, and Apendix C in Campbell and visit and explore the sites, http://www.microscopyuk.org.uk/index.html?http://www.microscopy-uk.org.uk/primer/basics.htm;
http://www.unl.edu/CMRAcfem/temoptic.htm ; http://www.mos.org/sln/SEM/seminfo.html;
http://www.purdue.edu/REM/rs/sem.htm before answering the following questions in your lab book.
1. Distinguish between resolution and magnification.
2. What is the function of every microscope?
3. How do Compound light microscopes, Dissecting light microscopes, Transmission electron
microscope (TEM) and a Scanning electron microscope (SEM) differ in terms of resolving power
and magnification. Cite an example of when each may be used in science.
4. Complete the diagram of the compound light microscope and state the function of each part of the
microscope. Cut and Paste into your notebook.
Mag vs Resolution Large Pic
Complete the diagram of the compound light microscope and state the function of each part of the
microscope in the table. Cut and paste both into your lab notebook (yellow page)
Name
Ocular
Objective
Arm
Base
Illuminator
Condenser
Lever for iris
Diaphragm of
condenser
Stage
Stage clip
Coarse adjustment
knob
Fine adjustment
knob
Nosepiece
Description
Using the Microscope:
General Microscope Care:
*Before plugging in the compound microscope make sure that the lamp switch is off
* Should any reagents spill on a microscope, they should be cleaned off immediately
Part I Setting optimal illumination and optimal resolution:
Perhaps one of the most misunderstood and often neglected concepts in optical microscopy is proper
configuration of the microscope with regards to illumination, which is a critical parameter that must be fulfilled
in order to achieve optimum performance. The intensity and wavelength spectrum of light emitted by the
illumination source is of significant importance, but even more essential is that light emitted from various
locations on the lamp filament be collected and focused at the plane of the condenser aperture diaphragm.
** Go to http://www.microscopyu.com/tutorials/java/kohler/index.html and explore the tutorial to learn
how each component of the microscope can affect the image resolution
Class Microscopes
1.
2.
3.
4.
Proper illumination of the specimen is essential for effective and optimum microscope use.
Rotate the low power objective, 4X, into place
Set the iris diaphragm adjustment disc to its largest setting, 5.
Lower the stage all the way down using the coarse focus knob
Obtain a parachute cloth and make a wet mount with water and cover-slip. DO NOT USE
MORE THAN 2-3 DROPS OF WATER.
To make a wet mount, add 1-2 drop of dH2O to the center of your slide and place the wet
fabric on top. Carefully place a cover-slip over your ‘specimen’. The figure below
demonstrates how to place the cover-slip to avoid view obstructing air bubbles.
5. Place the slide so that the specimen (cloth) sits directly over the condenser. Turn on the power
6. Focus by using the coarse focus knob. Once in focus use only the fine focus knob.
7. Close the iris diaphragm, move through the settings until you see the most number of trapped air
bubbles in the cloth. This is showing you the illumination that gives the most information.
Watching the changing quality of the image you will notice that closing the diaphragm increases contrast
but may introduce “artifacts”, opening the diaphragm decreases “artifacts” but decreases contrast.
Continue to adjust the diaphragm back and forth until your image is bright, detailed and sharp.
SAVE THIS SLIDE
Estimating the size of objects in the “field of view”
Use the Campbell CD to investigate the relevant size of objects:
Chapter 6: A Tour of the Cell
Concept 6.1 To study cells, biologists use microscopes and the tools of biochemistry
Investigation: What Is the Size and Scale of Our World? [Do the investigation]
Make a Titled table in your notebook that shows your estimations for each of the 10 specimen slides in the activity
Your drawings in microscopy should be accurate and should reflect size relationships (pay close
attention to the specimen slides in the CD activity. Determining and making a scale bar will be important
and will provide your viewer with a perspective of the size of your drawn object.
1. Using your premade silk fabric slide, with the fibers focused using the 10X objective, look at the
illuminated area (field of view)
2. Move the slide so that the left edge of a fiber bundle lines up with the left edge of the field of
view. (see figure 2)
3. The distance between the leading edge of one fiber
bundle to the leading edge of the next is ~275
micrometers (microns). Use the fibers to measure the
diameter of the field of view by counting the number
of leading edges in the diameter. (see example fig. 2,
the # of leading fiber edges is 4)
4. Determine the diameter of the “field of view” with 4X
and 10X Make a Table in your lab book
Objective
Total magnification
Field Diameter (m)
4X
10X
Total Magnification = objective mag x ocular mag
5. Make a wet mount from the piece of newsprint with the letter ‘e’.
6. Draw the image using the 4X objective and make a scale bar. (Use your field diameter estimate)
….See Drawing Guidelines
Drawing Guidelines:
1. Drawings should reflect what you see!
2. Include a title
3. Clearly label all of the important details but do not include
things you cannot see
4. Include a scale bar
5. You need not and should not draw each and every cell or
organism in the field of view. Instead, draw only an overview of
the specimen and the details of only a few representative cells of
interest