MCB 730: Microscopy Lab
Class: Weds 4/21 1:10 – 5 PM in Porter 305
Instructor: Janet Duerr, duerr@ohio.edu, 597-1921
YOU ARE REQUIRED TO BRING THE COMPLETED PRE-QUIZ TO CLASS
Read the included material on microscopy and C. elegans BEFORE class so
that are prepared to observe and photograph GFP-expressing C. elegans.
Learning Objectives:


Gain an understanding of the features of transmitted light microscopes.
o
Review and remember enough optics to understand how light is
altered as it interacts with lens, diaphragms, and the sample.
o
Use this understanding to learn how to adjust the features in any
standard light microscope to enhance visualization of specific samples.
Gain an understanding of how a camera and computer capture and save
images.
o
Determine the effects of changing brightness, contrast, gamma, offset,
exposure time, aperture, etc.
o
Learn how to manipulate the settings to enhance visualization of a
specific type of sample.

Review and remember enough optics to understand how fluorescence works.

Gain an understanding of the features of fluorescent light microscopes.
o
Review and remember enough optics to understand how light is
altered as it interacts with lens, FILTERS, diaphragms and the sample.
o
Use this understanding to learn how to adjust the features in a
fluorescence microscope to enhance visualization of specific samples.
o
Use this understanding to learn how to adjust the camera, computer,
and microscope to record data without damage to the samples.
o
Learn enough about fluorescent light microscopes to improve your
ability to design experiments using fluorescent tags.
The Microscope – Introduction
Adapted from: Heidcamp < http://homepages.gac.edu/~cellab>
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. 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. 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. The figure 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.
The reason for a dichotomy between magnification and resolution is the ability of the human eye to see
two objects. It is necessary that two objects be about 0.1 mm apart when held 10" from the face in order
for us to detect them as two objects. If they are closer than 0.1 mm, we will perceive them as a single
object. If two objects are 0.01 mm apart, we can not detect them unless we magnify an image of them by
10X. What has happened is that we have effectively altered our resolution ability from 0.1 mm to 0.01
mm through the use of a magnifying lens. We would say that our limit of resolution has changed from 0.1
mm to 0.01 mm, or inversely, our resolving power (resolution) has increased by a factor of 10.
Unfortunately, a lens can magnify an image without increasing the resolution. Several artifacts can be
inherent in the lens design which causes the objects to become blurry at the edges. Thus, even though they
can be made to appear 0.1 mm apart, the edges are so blurry that we lose the ability to see them as two
objects. Think of a standard eye chart: you can see the increased size of a letter, but may be unable to tell
what letter is projected.
The figure illustrates what can be seen with increased magnification and resolution. If we were to look
only at the left side of the figure, we could get the impression that the cell is filled with a homogeneous
fluid (cytoplasm). If, however, we look at the right side of the figure, it becomes apparent that the
cytoplasm is actually composed of smaller particulate components (chloroplasts, ribosomes, membranes).
As we increased the resolution of our microscopes we changed our concepts from protoplasm (the fluid of
life) to cytoplasm (the fluid of the cell outside of the nucleus) to a highly ordered machine full of
individual organelles.
It is readily apparent that while microscope lenses are usually discussed in terms of their magnification,
the most important value is their resolution. All microscopes will come with a lens that can magnify 40
times the normal size, but only a quality lens will allow you to see more than you would with a good
hand-held magnifying lens.
As mentioned, the value for resolution may be determined in one of two ways. It can be measured as the
smallest distance between two points, which allows us to see the points as distinct. With this
measurement, resolution increases as the distance decreases--that is, there is an inverse correlation
between the limit of resolution and what you actually resolve.
Limit of Resolution = (0.61 x 
Resolution = N.A./(0.61 x 
The resolution of a lens is a property of its physical properties and of the wavelength of light that is
passed through the lens. The physical properties are summed up in a value known as the numerical
aperture while the wavelength is determined by the color of light. The numerical aperture of a lens is
dependent upon two parameters, the angle of the incidence of light onto the lens (changed by the substage condenser), and the refractive index of the glass of which the lens is composed. The refractive
index is a function of the bending of light from air through glass and back again. In a microscope, the
glass of the lens is specially formulated to increase its refractive index. Once manufactured, however, this
property can not be changed. The media around the lens can be altered, however, by removing air from
between the objective and the slide, and replacing it with immersion oil.
Putting all of this to practical use, it is apparent that resolution can be increased in three ways. The easiest
method is to increase the angle of light incidence, by altering the position and/or design of the sub-stage
condenser. Second, the refractive index can be maximized by using specially manufactured lenses, and by
controlling the medium through which the light travels, i.e. using immersion oil with lenses designed for
this purpose. The third method is to decrease the wavelength of light used. For practical purposes, the
wavelength has a larger effect on resolution than either changes in the angle of incidence or the refractive
index. For maximum resolution, all three properties must be optimized.
For routine bright field microscopy, it is more convenient to work in the visible light range, and the
shortest wavelength of visible light is blue. More expensive and higher quality lenses manipulate the light
source to enhance the quality of the light and to correct for lens aberrations inherent in their design.
Resolution can be enhanced by reducing the wavelength to the ultraviolet range and yet again by levels of
magnitude to the wavelengths electrons have in motion. The use of electrons as the light source gives rise
to the electron microscope. UV light can not be seen directly by the human eye (it will injure the retina of
the eye) nor can we see electron beams. Thus, these forms of microscopy rely on photography, or upon
fluorescent screens. Visible light ranges in wavelength from the long red waves ( = 760 nm) to the short
blue/violet waves ( = 400 nm). Ultraviolet waves can be as short as 230 nm.
Alignment
Proper use of a microscope demands that the optics and light source be aligned on the optical axis. All of
the corrections for aberrations depend on proper alignment of the microscope components. There are two
general techniques used for proper alignment of the microscope. The first, and perhaps best, is known as
critical illumination. In this process an image of the light source (bulb filament) is projected into the plane
of the object, thus superimposing the light source onto the object. It has a distinct disadvantage, however,
in that it calls for a flat even light source, not really possible with a tungsten filament bulb.
The second alignment procedure is known as Koehler illumination; after a pioneer in light optics, August
Koehler. In this procedure, an image of the field diaphragm is projected onto the object plane. This
procedure requires a field condenser lens equipped with a moveable (centerable) iris or diaphragm.
Koehler illumination is the most commonly used alignment procedure.
Bright Field, Dark Field, Phase Contrast
All microscopes actually allow visualization of objects through minute shifts in the wavelength phase as
the light passes through the object. Further image forming can be had through the use of color, or through
a complete negative image of the object. If the normal phase shift is increased (usually by 1/4
wavelength), then the microscope becomes a phase contrast microscope. Phase contrast microscopes can
be designed to have medium phase or dark phase renditions, by altering the degree of additional shift to
the wavelength from 1/4 to 1/2 wavelengths, respectively.
If the beam of light is shifted in phase by a variable amount, the system becomes a differential
interference contrast microscope. The most commonly used system of interference microscopy is known
as a Normarski Interference Microscope, named for the optical theoretician, George Nomarski. Once used
nearly exclusively by parasitologists, this type of microscopy has increased in use because of the work
currently done on the nematode C. elegans; interference microscopes are superb for both observation and
measuring thickness of embryos within specimens with little or no contrast.
Web Links on Fluorescence Microscopy:
http://www.invitrogen.com/site/us/en/home/support/Tutorials.html
http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-TheHandbook/Introduction-to-Fluorescence-Techniques.html
http://swehsc.pharmacy.arizona.edu/exppath/micro/fluortech.php
MCB 730: Microscopy Lab PRE-Quiz
Use the information from the website resources provided above or by Dr. Berryman, Wikipedia, or other
sources to answer the following questions. Your completed PRE-quiz is due at the START of class on
Wednesday, May 11.
Fluorescence microscopy:
1. Excitation light has (higher/lower) energy and (longer/shorter) wavelength than emission light.
2. To see green fluorescent protein, we illuminate it with a ____________ (what color) light. This
excitation is optimum at a wavelength of __________ nm. Green fluorescent protein looks
______________ (what color); its maximum emission is at a wavelength of ________ nm.
3. UV light has (higher/lower) energy and a (longer/shorter) wavelength than infrared light. Which
wavelength of dye tends to be more damaging to unstained tissue?
4. What is a dichroic mirror?
5. Why do we use epi-fluorescence rather than transmitted light fluorescence?
6. What is the difference between fluorescence and bioluminescence?
7. What is autofluorescence? What is one common sources of autofluorescence in biological tissues?
8. Luciferase and green fluorescent protein are both used experimentally. What aspects of their chemistry
suit them for examining different processes?
9. You can use laser excitation of GFP containing cells to selectively kill those cells. Why?
10. Why do we need to use ‘anti-fade’ agents? What do these agents do?
What is C. elegans and why study it?
Adapted from <http://www.biotech.missouri.edu/Dauer-World/Wormintro.html>
Caenorhabditis elegans is a nematode - a member of the phylum Nematoda, the roundworms and
threadworms. These simple animals are smooth-skinned, unsegmented worms with a long cylindrical
body shape tapered at the ends; they may be free-living or parasitic. C. elegans is small (~ 1 mm) and
lives in the soil - especially rotting vegetation - where it survives by feeding on microbes such as bacteria.
C. elegans is about as primitive an animal that exists which nonetheless shares many of the essential
biological characteristics of humans. The worm has a nervous system with a ‘brain,’ capable of
rudimentary learning. It produces sperm and eggs, mates and reproduces. After reproduction, it gradually
ages, loses vigor and finally dies. In C. elegans, we can examine genetic regulation of embryogenesis,
morphogenesis, development, nerve function, behavior and aging. All 959 somatic cells of its transparent
body are identical from animal to animal and are visible with a microscope.
C. elegans has two sexes: a self-fertilizing hermaphrodite and a male. Like other animals, the adult
essentially comprises a tube. The epidermis is surrounded by a tough cuticle (consisting of collagen and
other proteins) and is lined with muscle. The interior body contains a pharynx (eating apparatus)
connected to the gut, and the reproductive system. The 302 neurons include sense organs in the head that
mediate responses to taste, smell, temperature and touch. Neurons in the head and ventral and dorsal
nerve cords running back down the body control the contraction of the 81 muscle cells. C. elegans moves
by means of waves of contraction of four longitudinal bands of muscle. The 22,000 genes have been
extensively studied and thousands of mutants are available. Furthermore, the consequences of disruption
of the expression of most genes by RNAi have been examined.
Figure 1: Life cycle of C. elegans (from www.wormatlas.org/handbook/anatomyintro/anatomyintro.htm)
Figure 2: An adult hermaphrodite seen with a light microscope. The pharynx and developing embryos are
visible (compare with diagram below).
Figure 3: Diagrams of the major tissues in C. elegans: Anterior (head) to the left (from WormBase.org)
Top (left to right): The yellow pharynx pumps rhythmically to bring bacteria into the gut (orange), which
runs the length of the animal to the anus in the tail. The central region of the body also contains the
reproductive system (cream), developing oocytes (pink) and young embryos (black).
Middle: The body is lined with bands of muscles (pink) which contract in waves to cause movement.
Bottom: The nervous system (blue) is concentrated in the head, with dorsal and ventral nerve cords
running the length of the body to control muscle contraction and body movement.
------------------------------------------------------------------------------------------------------------------------------For more information on C. elegans, check the following websites:
WormAtlas: Introduction to C. elegans anatomy
<www.wormatlas.org/handbook/anatomyintro/anatomyintro.htm>
WormBase: C. elegans genes, proteins, strains, reagents, etc
<www.wormbase.org>
MCB 730: Microscopy Lab IN-CLASS Quiz
Use the information from the web resources to begin to answer the following questions. We will discuss
these aspects of microscopy during class – you can continue to work on this quiz during class. Your
completed quiz is due at the END of class on Wednesday, May 11.
White light microscopy: Starting from the white light source, we can follow the light path and adjust
features in the light path:
A) To limit the area of our sample that is illuminated by the white light, we can adjust the
_______________. This feature is located (after/before) the condenser.
B) The microscope condenser is located in the light path just (after/ before) the sample. What does it do?
EXPLAIN.
C) If you are going to use phase or Nomarski optics, you need to have additional lens (before/after/before
and after) the sample.
D) What is the difference between regular transmitted white light microscopy and Nomarski? Which
would be better for viewing low contrast clear samples and why? EXPLAIN.
E) For optimal viewing, we usually mount our specimen on a glass slide, place it in a special solution
(mounting media), and then cover it with a glass cover slip. Why do we use a glass slide and cover
glass (rather than plastic)?
Why does mounting media often contain glycerol instead of just saline?
Camera: Adjusting brightness/contrast/gamma/offset:
a) Your sample is very dim – you should try changing _______________
b) Your unstained sample is bright, but hard to see – you should try changing_______________
c) Your sample is too bright – you should try changing _______________
d) Your sample is has a very bright background, making it hard to see your sample – you should try
changing _______________
e) Your sample is very bright, but the image looks ‘noisy’ - you should try changing _______________
f) Your sample starts to fade - you should try changing _______________
g) When you change the objective to a different magnification, the intensity of the white light goes
______ with higher magnification and the intensity of fluorescence goes ____________ .