Lab 1.

Cell Diversity

Eukaryotic Cell Diversity

Eukaryotic cells from different organisms, or from different tissues of the same organism,
exhibit large differences in size, morphological complexity and biochemical
specializations. The purpose of this laboratory exercise is to demonstrate the range of
diversity among eukaryotic cells, to give you a sense of the features that these cells have
in common, and to acquaint you with several techniques in microscopy with which you
can make these observations and comparisons.

Once you have obtained a microscope from the microscope cabinet, your TA will show
you the standard operating protocols that may already be familiar, but are, nevertheless,
very important for the well-being of these precision optical instruments. As a starting
point, there is a collection of permanent slides that have been purchased from commercial
sources. On these slides, you will find examples of protozoans, algae, animal cells, plant
cells, and fungal cells. Begin by looking at the slides with the microscope in the
brightfield mode. All of these slides have cells that were rapidly killed in a way that
structure was minimally altered (these cells have been 'fixed') and then stained with dyes
that make the various cellular constituents absorb light differentially. Differential
absorption of light is visible to you as differences in intensity (amplitude), and sometimes
as differences in color. The reason that the cells are stained in these preparations is
because in general, cells do not alter amplitude of transmitted light sufficiently for our
eyes to detect structure. The point of looking at these slides is to observe the variation of
structural organization in cells from evolutionarily-diverse groups, and simultaneously, to
look for similarities in structure that cross group boundaries. Among differences, look for
the presence or absence of cell walls, the presence or absence of cilia, of microvilli, of
large ordered cytoskeletal arrays, etc. Among common features, look in particular for
nuclei, nucleoli, at the organization of chromosomes in mitotic cells if any are visible,
etc.

For all of the following observations, you must create sketches and take notes in
your lab notebook.

- Phase Contrast microscopy and the observation of living, unstained cells -

Light beams that enter unstained cells emerge with altered phase relative to those that did
not enter the biological specimen, and because of this fact, Zernicke was able to perform
a simple optical trick that transforms this phase difference into an amplitude difference
that we can perceive. The use of Zernicke's phase contrast microscope enables us to view
unstained cells and see structures that are not visible with brightfield illumination through
the same lenses. The principles underlying Zernicke's Nobel Prize-winning invention
follow in Appendix I. The TA will go through the operating principles of phase contrast
microscopy with you.

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Using phase contrast microscopy, it is rather easy to observe subtle aspects of structural
organization in unstained cells. There are several sources of living cells available for
observation.

a) With a pipette, obtain several drops of water from the aquarium in the front of
the room and place a drop of this water on a clean microscope slide. Gently place a
coverglass on top of the drop and place the slide onto the microscope stage. Using the
10X objective (phase annulus 1), observe the droplet. When the objective lens is correctly
focused, you may see a number of small organisms swimming in the field of view.
Switch to the 40 X objective (phase annulus 2). Detail in these organisms that was not
visible at the lower magnification should be apparent. Based on your observations of
prepared slides, what groups of organisms are present in the aquarium?

b) Do the same with the various cultures of protozoans provided.

c) Cut a young leaf from the stem tip of an Elodea plant growing in the aquarium.
Place the leaf in a drop of aquarium water on a glass microscope slide. Cover the leaf
with a coverglass trying to avoid the formation of air bubbles. Place the slide on the
microscope stage and observe first with brightfield microscopy and then switch to phase
contrast. What do you see in the leaf with brightfield? How does that compare with what
you see with phase contrast?

d) Using a pipette, pull a few drops of liquid from the culture of Euglena gracilis.
Place a drop of this liquid onto a glass slide and gently cover the drop with a coverglass.
Try to avoid bubbles when making this preparation. Observe the cells with the 40 X
objective lens with brightfield illumination (you should see swimming cells). How do the
cells swim? Place the phase 2 annulus into the optical path. Now, looking carefully at the
cells with phase contrast microscopy, how do you think that these cells swim? Next,
place a small piece of filter paper against the edge of the coverglass to remove excess
liquid. Drain the liquid away until it is clear that you have left only a thin film of water
beneath the coverglass. Now, look closely at the cells as they undergo oscillatory shape
changes in a kind of movement known as metaboly. If you look carefully at the cell
surface as the cell changes from spherical to elongate and back to spherical, you may be
able to resolve ridges in the plates of its pellicle, located just below the plasma
membrane. How does the cell change its shape? You can test whether metaboly requires
energy by treating cells with the respiratory inhibitor 2, 4-dinitrophenol (DNP). This
agent acts as an uncoupler of oxidative phosphorylation by causing the leakage of a
proton gradient across the inner mitochondrial membrane. (CAUTION, 2, 4-DNP is very
toxic!!!)

e) Obtain prepared slides of 10T1/2 cells from your TA. These cells have been
stained with acetocarmine. The TA mounted the cover glass on the microscope slide and
then perfused several drops of acetocarmine solution beneath the cover glass. The
staining solution is highly acidic, and it simultaneously fixed and stained the cells. To
reduce the background, the TA then perfused an excess of 70% ethanol beneath the cover
glass and then sealed the preparation with nail polish. (You will learn how to make these

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semi-permanent preparations later.) These cells grew on the coverglass as a monolayer of
a cell line that was originally cultured from fibroblasts isolated from a mouse (see
below). Look at the cells with brightfield illumination using the 40X objective lens. What
do you see? Switch the condenser to the appropriate phase contrast annulus and observe
the cells. How does this image compare with that obtained with brightfield microscopy?
What do you see?

- Differential interference contrast (DIC) microscopy -

DIC is sometime referred to Nomarski interference after its developer. Like phase-
contrast, it is a good method to give contrast to live unstained cells. DIC provides the
user with an image that has a large amount of contrast, giving a three-dimensional quality
to the image that is not seen with other types of microscopy. DIC uses polarized light
and prisms that separate the light. Light that passes through the specimen is out of phase
with the light that does not pass through the specimen due to the difference in the index
of refraction. When the light passes through the prism, this difference in phase is
increased. This appears as contrast to the human eye, and is most significant at the edges
of a cell or structures as this is where the greatest change in refractive index occurs in a
small distance. This method also leads to the “shadow” appearing on one side of the cell
or object. Observe some of the organisms or prepared slides using the DIC microscope.

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Appendix I. Principles of Microscopy
Brightfield microscopy

The microscope that is available to you for general use in this laboratory is a
sophisticated optical instrument that can provide you with high-resolution images of a
variety of specimens. Image quality is based largely on your ability to use the microscope
properly. Below you will find some basic information that you have probably heard
before, but information that is rarely presented in a thorough way. In the figure below, we
present the Olympus BX-40 microscope, as configured for brightfield microscopy (image
originally prepared by Olympus Corp.).

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- Resolution -

The magnification of small things is a necessary facet of biological research, but

the fine detail in cells and in subcellular components requires that any imaging system be
capable of providing spatial information across small distances. Resolution is defined as
the ability to distinguish two very small and closely-spaced objects as separate entities.
Resolution is best when the distance separating the two tiny objects is small. Resolution
is determined by certain physical parameters that include the wavelength of light, and the
light-gathering power of the objective and condenser lenses. A simple mathematical
equation defines the smallest distance (dmin) separating the two very small objects:

dmin = 1.22 x wavelength

N.A. objective + N.A. condenser

This is the theoretical resolving power of a light microscope. In practice, specimen

quality usually limits dmin to something greater than its theoretical lower limit.

N.A. (Numerical Aperture) is a mathematical calculation of the light-gathering

capabilities of a lens. The N.A. of each objective lens is inscribed in the metal tube, and
ranges from 0.25-1.4. The higher the N.A., the better the light-gathering properties of the
lens, and the better the resolution. Higher N.A. values also mean shorter working
distances (you have to get the lens closer to the object). N.A. values above 1.0 also
indicate that the lens is used with some immersion fluid, such as immersion oil. Your
T.A. will show you how to use immersion objectives properly, as needed, in this course.

From the equation above, you should be aware that the N.A. of the condenser is as
important as the N.A. of the objective lens in determining resolution. It is for this reason
that closure of the condenser diaphragm results in a loss of resolution. In practice, at full
aperture and with good oil immersion lenses (N.A. 1.4 for both the condenser and the
objective) it is possible to be able to resolve slightly better than 0.2 µm. From the
equation above, it should also be clear that shorter wavelength light (bluer light) will
provide you with better resolution (smaller dmin values). However, there are practical
considerations in how short the wavelength can be. In the early 1950's, a UV microscope
was designed, but required quartz objectives and a specialized imaging device. The
quartz lenses provided slightly better resolution (dmin = 0.1 µm), but image quality
suffered from an inability on the part of the manufacturers to correct for aberrations
caused by the quartz. The human eye is best adapted for green light and our ability to see
detail may be compromised somewhat with the use of blue or violet. Most manufacturers
of microscopes correct their simplest lenses (achromats) for green light.

- Magnification and Imaging -

Like most microscopes in current use, your Olympus BX-40 or BH-2 is a

compound microscope, where a magnified image of an object is produced by the
objective lens, and this image is magnified by a second lens system (the ocular or

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eyepiece) for viewing. Thus, final magnification of the microscope is dependent on the
magnifying power of the objective multiplied by the magnifying power of the ocular.
Objective magnification powers range from 4X to 100X. Lower magnification is
impractical on a compound microscope stand because of spatial constraints with image
correction and illumination. Higher magnification is impractical because of limitations in
light gathering ability and shortness of working distances required for very strong lenses.
Ocular magnification ranges are typically 8X-12X though 10X oculars are most common.
As a result, a standard microscope will provide you with a final magnification range of
~40X up to ~1000X.

Each objective lens consists of six or more pieces of glass that combine to
produce a clear image of an object. The six or more lenses in the objective lens are
needed to provide corrections that produce image clarity. The interaction of light with
the glass in a lens produce aberrations that result in a loss in image quality because light
waves will be bent, or refracted, differently in different portions of a lens, and different
colors of light will be refracted to different extents by the glass. Spatial aberrations (e.g.,
spherical aberration) can be corrected by using lenses with different curvature on their
surfaces and chromatic (i.e., color) aberrations can be minimized by using multiple kinds
of glass in combination. These corrections increase the cost of the lens to the extent that
an apochromatic objective lens exhibiting full color correction and extremely high N.A.
can cost several thousand dollars. This objective lens is about the size of your thumb.

The objective lenses in the BX-40 are achromats, and best suited for imaging with
green light. Green filters for the BX-40 microscopes are available in the lab. The
achromat lenses are not suitable for critical high-resolution imaging with white light,
because red and blue light do not focus in the same plane as green light. Color
photomicrography should be performed with totally corrected apochromatic objective
lenses. The objectives for the Zeiss microscope in the lab are fluorite lenses, best suited
for fluorescence microscopy because of their high transmittance of shorter wavelength
light. Fluorite lenses are intermediate in their level of correction, between achromats and
apochromats.

The oculars in your Olympus microscopes and the oculars in the Zeiss microscope
are designed to work specifically with the objective lenses from the same manufacturer.
Each manufacturer makes some of the color and spatial corrections in the objective and
the remainder of the corrections in the ocular. Mixing brands will usually result in a
degraded image. In addition, when you look into a microscope, the magnified and
corrected image you see through the oculars is actually a virtual image (as opposed to a
real image). The ocular, designed to provide a corrected virtual image when viewed by
eye, is not suitable for the generation of photographic or video images through the
microscope. For photography or video microscopy it is necessary to use a projection lens
that generates a corrected real image. Projection lenses for the light microscope typically
are engraved with a 'P' on the metal tube. Projection lenses are used in the Zeiss
microscope to provide an image to the video camera and in the older Olympus BH-2
microscopes outfitted with trinocular heads and the video or photographic camera. Of

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course, standard oculars are present in the viewing tubes of these trinocular microscope
heads for normal viewing of specimens.

- Illumination -

An essential factor in producing a good image with the light microscope is

obtaining adequate levels of light in the specimen, or object plane. It is not only
necessary to obtain bright light around the object, but for optimal imaging, the light
should be uniform across the field of view. The best way to illuminate the specimen
involves the use of yet another lens system, known as a condenser. The front element of
the condenser is usually a large, flattened lens that sits directly beneath the specimen. Its
placement on a movable rack provides you with the means to focus the light beam
coming past the object and maximize the intensity and control the uniformity of
illumination. Two apertures in the illumination system allow you to regulate the diameter
of the illumination beam by closing or opening iris diaphragms. One of these diaphragms,
housed within the brightfield condenser and known as the condenser diaphragm, allows
you to increase contrast, but at the cost of worsening resolution. The second of these
diaphragms, known as the field aperture diaphragm, does not affect resolution as
dramatically and is regularly adjusted for optimal illumination.

Optimal illumination of a specimen with the BX-40, BH-2 (and with most other
microscopes currently manufactured) is achieved by using a variation of Kohler
Illumination, where (for those of you are technophiles) the filament of the light source is
in focus at the rear focal plane of the objective lens. Operationally, it is easy to obtain
optimal illumination for brightfield (or phase contrast) by first placing any specimen on
the stage and focusing on the object. Next, turn the ring for the field aperture diaphragm
(the lowest aperture on the microscope) so that its edges obscure the periphery of the
field of view. Next, raise or lower the condenser until the edges of the field aperture
diaphragm are clearly focused. Do not refocus the objective on the specimen while you
are adjusting the condenser. It may be necessary to center the field aperture diaphragm,
using the condenser centering screws. When the microscope is properly illuminated, both
the object and the edges of the field aperture diaphragm should be in the same plane of
focus and the field iris diaphragm should be centered in the field of view.

Phase Contrast Microscopy

The human eye can perceive changes in light amplitude (intensity). Unstained
biological specimens, such as living cells, are essentially transparent to our eyes, but they
interact with light in a fairly uniform way, by retarding (slowing) the passage of a light
beam by approximately 1/4 of a wavelength (λ). By slowing a light beam this much
relative to another light beam that had passed though the surrounding medium, the
biological specimen alters the phase of the beams. Intensity (amplitude) is additive and
light rays that are 1/2 λ out of phase are perceived as darkness. Zernicke realized that if
he could retard the light passing through biological specimens without affecting the light
passing through the surrounding medium, he could generate changes in amplitude within
living cells. The phase contrast microscope was invented by Zernicke in the 1930's as a

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means to generate contrast in biological specimens, changing these invisible phase
differences into visible amplitude differences.

Zernicke employed an optical trick to separate the light beams interacting with the
specimen from those that do not encounter the specimen. To separate the beams of light
from each other, he placed a transparent ring (known as an annulus) in an opaque disk
and inserted this disk into the optical path of the microscope, within the condenser. He
placed a complementary ring inside the objective lens. Nearly all of the light that passes
through the sample but misses the specimen then passes through the objective lens
through this ring. Most of the light that passes through the specimen is scattered and
some of it enters the objective lens in such a way that it will not pass through the
objective lens ring, but will pass this plane in some other location. He designed the glass
plate holding the ring so that all light missing the ring would encounter an additional 1/4
λ of retardation relative to the beams of light that had not interacted with the specimen,
placing the light rays that had interacted with the specimen out of phase with rays that
had not interacted with the specimen by 1/2 λ. He found that a reduction in intensity of
the light that had not passed through the specimen would create a grey background and
increase contrast even more, with some parts of the specimen darker and other parts of
the specimen brighter than the background.

The operation of the BX-40 microscope in the phase contrast mode requires that
you first set up proper brightfield illumination, with a centered field iris diaphragm whose
edges are in focus in the specimen plane. Next, rotate the condenser turret cylinder until
the number on the condenser turret matches the number engraved on the objective lens.
Under this condition, the condenser annulus is matched to the phase ring present in the
objective. Next, remove one of the oculars and insert the Bertrand focusing telescope into
the ocular hole. This lens enables you to see the rear focal plane of the objective lens, the
plane where the ring resides. You will see a bright circle of light (the condenser annulus)
and a dark ring (present within the objective). The dark ring is stationary, but the bright
annulus is not. You may need to align the annulus with the ring so that the two are
superimposed. On the back side of your condenser, you will find two adjustment screws
that permit this alignment to be performed. When the ring and the annulus are aligned,
place the ocular back into the microscope. The difference between phase contrast and
brightfield for the observation of living cells is significant.

Fluorescence Microscopy

In certain classes of atoms and molecules, electrons absorb light, become

energized, and then rapidly lose this energy in the form of heat and light emission. If the
electron keeps its spin, the electron is said to enter a singlet state, and the kind of light
that is emitted as the electron returns to ground state is called fluorescence. If the electron
changes its spin when excited, it enters the triplet state, and the kind of light that is
emitted as the electron returns to ground state is known as phosphorescence.
Phosphorescence is much longer-lived than fluorescence. Both fluorescence and
phosphorescence emissions are of particular wavelengths for specific excited electrons.
Both types of emission are dependent on specific wavelengths of excitation light, and for

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both types of emission, the energy of excitation is greater than the energy of emission.
Described another way, λ of excitation light is shorter than λ of emission light. In
biology, we can utilize fluorescence in localization reactions, to identify particular
molecules in complex mixtures or in cells. Fluorescence has the advantage of providing a
very high signal-to-noise ratio, which enables us to distinguish spatial distributions of
rare molecules. To utilize fluorescence, we need to label the specimen (a cell, a tissue, or
a gel) with a suitable molecule (a fluorochrome) whose distribution will become evident
after illumination. The fluorescence microscope is ideally suited for the detection of
particular fluorochromes in cells and tissues.

The fluorescence microscope that is in wide use today follows the basic "incident-
light" design of Ploem, who employed a novel arrangement of filters with a chromatic
beam splitter (often wrongly called a dichroic filter both by biologists and microscope
sales people). With the incident light fluorescence microscope, the object is illuminated
with fluorescence excitation light through the objective lens. The object emits longer-λ
fluorescence in response to the shorter-λ excitation light. The objective lens then serves
both for illumination and imaging. The chromatic beam splitter transmits or reflects light,
depending on its color. For this application, shorter λ light is reflected and longer λ light
is transmitted by the splitter. Ploem placed the chromatic splitter in the optical path
between the objective lens and the ocular, at a 45o angle, so that it would reflect shorter λ
light downward toward the objective. The longer-λ fluorescence emission light would be
transmitted through the chromatic beam splitter toward the ocular.

The microscopes that you have utilized in this and other courses all operate in the
same general fashion, using transmitted illumination. Light beams pass through a
condenser lens system and provide illumination of an object at many points
simultaneously. For incident light fluorescence microscopy, the objective lens also acts as
a condenser for the excitation light beam. In its interaction with the object, some of this
light is absorbed, some of this light is scattered, some of this light is reflected, and some
of this light is slowed or retarded (relative to a beam of light that does not pass through
the object). A portion of the light that has interacted with the object then passes through
the imaging lens system of the microscope where it provides us with visual or pictorial
image information about the object. Like the process of illumination, the process of
image generation operates in a parallel fashion, where large numbers of light beams
contribute to the image simultaneously. Resolution is limited by the closeness of
overlapping points of brightness or darkness. In a practical sense, the limit of resolution is
0.18-0.2 µm with the best available objective lenses and a good specimen.

To observe cells with the fluorescence microscope, it is important to know the

spectral characteristics of the fluorochrome that has been employed. In order to excite the
fluorochrome properly and then observe its fluorescence emission, the appropriate filter
packages must be present in the microscope. The fluorochrome may not fluoresce at all if
the cells are illuminated with the inappropriate filter pack present in the optical path.
Finally, for any kind of fluorescence localizations to be performed, it is essential to have
the appropriate controls, to be sure that the cells do not exhibit excessive
autofluorescence (that is, they do not glow in the absence of the fluorochrome), and that

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the fluorochrome is responsible for the localization pattern observed. In the laboratory,
we have several microscopes equipped for incident light fluorescence microscopy. The
T.A. will guide you through the steps in visualizing fluorescence through the microscope.

Confocal Scanning Optical Microscopy

In the incident light fluorescence microscope, a light beam passes through a

chromatic beam splitter and then the objective lens to illuminate a specimen. This light
beam is used to excite electrons in fluorochrome molecules present in the object. As
some of those excited electrons return to their ground state, the emission of light is
detectable through the oculars of the microscope, or with a camera or video printer. The
image is generated continuously, across the entire field of view. A primary problem with
the fluorescence images generated in this way is that out-of-focus fluorescence appears as
'flare' in the object, and reduces the signal substantially. In addition, human eyes are not
sufficiently sensitive photodetectors for the lowest levels of fluorescence, and most
video-based imaging systems are only slightly better than your eyes. Under conditions
where there is sufficient signal for you to easily observe fluorochrome distribution
patterns, the excitation light can be of sufficient intensity to photooxidize (i.e., burn) your
specimen. Much information can be lost with just a few seconds of exposure to the
excitation lamp. The Confocal Scanning Optical Microscope, an expensive piece of
instrumentation that illuminates the object with a small beam of light in a point-by-point
(i.e., serial) fashion, eliminates most of the photoxidation problems, permitting the
observation of objects for extended periods at very high resolution with little loss of
signal. The placement of a small aperture in a special place in the beam path generates a
small depth of field, and effectively eliminates out of focus information in image
formation. In effect, this aperture reduces the depth of focus in the microscope to a
minimum, so the detector produces an image that is closely related to the thickness of the
depth of field produced by the objective lens. Light from planes above and below the
focus of the objective lens is essentially lost at this aperture.

The confocal scanning optical microscope is designed to illuminate an object in a

serial fashion, point by point, where a small beam of light (from a LASER) is scanned
across the object rapidly in an X-Y raster pattern. The raster pattern can be created in
several ways, but in one of the more popular instruments, it occurs as a consequence of
the simultaneous rotation and vibration of a polygonal mirror. The vibration is caused by
the activity of a servogalvanometer, while the rotation is caused by the activity of a small
electric motor. Thus, a bright spot of light scans across an object from top to bottom, line
by line. The image is also generated point-by-point. Image formation is translated into
intensities at each spot in the X-Y raster by a photomultiplier tube. The intensity
information is digitized and stored in a computer. A complex image processing software
package permits visualization and manipulation of the images. Resolution is limited by
spot size for the LASER and approaches 0.12-0.15 µm for an ideal specimen and with the
best available objective lenses.

The manufacturers of confocal scanning optical microscopes include a pinhole

diaphragm at a very special place in the optical path, near to the site of the

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photomultiplier tube. This pinhole is situated in a plane where the light from the in-focus
part of the image converges to a point. Light from object planes above or below that of
the focused image do not converge at the spot in the optical path occupied by the pinhole.
Because of this design, out of focus image information is darkened to the extent that it is
not detectable. The consequence is that all out of focus information is removed from the
image and the confocal image is basically an 'optical section' of what could be a relatively
thick object. The 'thickness' of the optical section may approach the limit of resolution,
but in practice, the resolution in the Z-direction is somewhat greater, approximately 0.4-
0.8 µm. The value of optical sectioning is best realized with fluorescence microscopy,
where out-of-focus information alters, distorts, or even degrades the image. Because the
confocal images are stored in a computer, it is possible to stack them up and generate
three dimensional reconstructions. The image processing programs also enable us to
rotate these images and observe three-dimensional aspects of cellular structure. It may be
clear to you that the computer responsible for these image manipulations must be fast and
powerful. The biggest problem is one of image storage, where single images can
routinely occupy >1,000,000 bytes of space. In rather short periods of use, it is easy to
accumulate sufficient numbers of images to fill the largest of hard disks.

Two of the three confocal scanning optical microscopes located on campus used
in the College of Life Sciences or the College of Agriculture were manufactured by Carl
Zeiss, located in Germany. The newest instrument has three lasers and four
photomultipliers and is designed so that we could illuminate with two or three colors of
light in rapid succession and detect as many as three superimposed signals (essentially)
simultaneously. The signals are separated from each other on the basis of color, using an
acoustical optical tunable filter (AOTF). The optical microscope is an inverted stand that
quite similar to the upright Zeiss microscope that is available to you in the Cell Biology
laboratory. The most important operational difference between this microscope and the
upright microscope in our teaching laboratory is that with this instrument, the slide is
placed in the stage holder upside-down. Like the microscopes in our lab, this microscope
is equipped for phase contrast, differential interference contrast and fluorescence
microscopy and can be used with these imaging techniques for conventional imaging.
However, unlike most of the microscopes we have in our teaching lab, this microscope is
equipped with a number of very highly corrected (read expensive) objective lenses
attached to the turret, just below the stage. These lenses are necessary for high-resolution
confocal microscopy. The confocal part of this microscope is contained in a box that is
attached to the inverted stand through an access port. As is the case with incident light
fluorescence, the laser light passes through the objective lens to illuminate the specimen.
The air suspension table is designed to eliminate vibrations present in the building.

Deconvolution Microscopy and Image Reconstruction

An alternative approach for eliminating flare from fluorescent image stacks is to

perform intensive, iterative image analysis and processing, from objects that have been
illuminated and photographed at multiple, adjacent focal planes. The images are obtained
with a high-performance CCD camera, operating at very high magnification, using
standard incident light fluorescence microscopy. The excitation source is a mercury arc

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lamp, and bandwidth for excitation and emission are controlled by filters placed in
rotating filter wheels. The lamp is stabilized and the beam is randomized for uniform
illumination of the specimen. Unlike confocal scanning instruments, the whole field of
view is illuminated simultaneously with this microscope. It is possible to perform rapid
sequential imaging (4 colors) from multiple fluorochromes with this microscope. At very
high magnification, fluorescence from any spot in a cell acts as a point source. By
knowing the image spread functions above and below the plane of focus, it is possible to
determine points of origin for fluorescence, and spreading beams of light from that point
source, above and below the plane of focus. An iterative algorithm, which is essentially a
linear combination, is performed by a computer on the adjacent pixels within a single
image plane, and in successive image planes through the thickness of the object.
Spreading light beams are subtracted from reconstructed image stack, and that light is
added back to the source, thereby reducing noise and increasing signal, respectively. We
have recently acquired a sophisticated DeltaVision microscope from Applied Precision,
Inc., which is designed to acquire these images and then perform the computer-intensive
operations. This kind of microscope is particularly well suited for generating three-
dimensional fluorescence images from small, living cells.

Polarization Light Microscopy

- Birefringence -

When light passes through an object, it interacts with some or all of the atoms and
molecules present in that object. In these interactions, sometimes light of a particular λ
(i.e., color) is absorbed by the atoms or molecules, while sometimes light is scattered.
The interaction of light with a translucent object often results in a slight reduction in the
velocity of the light beam. The extent of this reduction in velocity can be measured as the
refractive index of the object. For certain kinds of objects, especially those with high
order in particular axes of the object, such a crystalline or paracrystalline arrays, the
interaction with light beams is vastly different, depending on the orientation of the object
relative to the impinging light beam. As a result, the refractive indices are measurably
different in different axes of the object. Such an object with multiple refractive indices is
termed birefringent. Birefringence (multiple refractive indices) results from the
alignment of atoms or molecules in particular planes of an object; these atoms or
molecules interact strongly with light beams impinging on them from a particular
direction, and to a far lesser extent with light beams impinging on them from a different
direction. There are two kinds of birefringence, intrinsic birefringence, which results
from atomic or molecular order in a crystalline or paracrystalline array (i.e., calcite
crystals, membranes) and form birefringence, which results from supramolecular
associations in paracrystalline arrays (i.e., microtubules in a spindle).

- Polarized Light and Birefringent Retardation -

Any light beam shining in a particular direction vibrates in all directions around
the axis of travel. Light beams whose vibration has been restricted to a single plane, or to
a few planes are known as polarized light. Birefringence is directly observable as

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differences in intensity in different axes of crystalline or paracrystalline objects when
they are viewed with polarized light. Since birefringence results from differences in the
number of interactions between the light beam and atoms or molecules in the object in
different directions; in practice, the object is rotated around the plane of vibration for the
polarized light beam to maximize the intensity differences in the object (usually, the
dominant object axis is at a 45o angle relative to the plane of polarization). The extent of
the difference in refractive indices in different axes of the object is a measurable quantity
known as birefringent retardation (BR). BR is measured (as a distance) by placing an
object with known birefringent retardation into the light beam, and, by rotating the
calibrated object around the optic axis, extinguishing the brightness in the sample. Using
this compensation technique, BR is directly related to the number of aligned microtubules
in mitotic spindles in living cells. This principle and procedure can be of importance in
studying microtubule dynamics, where mitotic spindles of developing sea urchins can be
visualized in a totally noninvasive way.

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