Studying Living Cells
It's not always easy, but studying living cells
is how to find out what makes them tick
The following is a manuscript for an article published in R&D
magazine. R&D magazine holds the copyright for the finished
C.G. Masi, Contributing Editor
If you want something easy to image in a microscope, pick something
that is dead. With dead things, you have plenty of time to set up
your lighting, fiddle with positioning, add stains and so forth.
The problem is that studying dead things tells you very little.
You can observe their structure. You can sometimes learn about the
channels they used to take up stains just before they died. That,
however, is pretty much it. Mostly, you are looking at morphology.
While morphology and structure are important things to know about
cells, it doesn't tell you much about how they work. To answer many
of the important questions in microbiology, you have to see cells
at work. That means observing living cells.
In Cells: A Laboratory Manual, Spector et al list two motivations
for studying with live cells. Some studies attempt to establish
natural behavior of cells as part of a living organism. Others attempt
to determine changes brought about by adjusting a controlling factor.
Obviously, both types of study are important to advancing our understanding
of the genetic and protein mechanisms of living organisms--in both
normal and diseased states--and for developing intervention strategies.
What makes live cell microscopy difficult is that researchers have
to consider the needs of their subjects as well as the needs of
their instruments. Live cells, even differentiated cells from complex
organisms, are living organisms themselves. They need to be fed,
watered and sheltered if they are to perform as expected throughout
A live cell dropped on a slide under a microscope is equivalent
to a human staked out in the desert. It won't function normally
and it won't function long.
While providing something close to normal care and feeding for
your microscopic subjects is one aspect of the live cell problem,
being able to see the things is another. Living cells are not designed
to provide good optical contrast for microscopists, which is why
stains are such an important part of cell-morphology studies. Most
of the traditional stains, however, can prove more or less toxic
to your experimental subjects. Thus, your choices for contrast media
are much more limited when studying live cells.
Finally, live cells move. Flagella flail. Pseudopods reach out.
Organelles run chemical processes. Cytostructures bend and flex.
Whole cells scurry about. External and internal movement is the
hallmark of life, and it is these movements that we want to study.
Care and Feeding of Live Cells
Just as whole-organism researchers have developed enclosures to
maintain their experimental subjects, microscopists have developed
a wide range of microenvironmental chambers to contain and maintain
live cells. These range from simple open petri dishes to continuous-flow
perfusion chambers capable of tightly controlling the cells' physical
and chemical environment for indefinite periods.
The old drop of pond water on a glass slide maybe fine for some
studies, but the water quickly heats up under the microscope light,
and evaporates (which rapidly changes its chemistry). The limited
fluid volume means that essential dissolved gases are used rapidly,
while wastes from cellular metabolism have nowhere to go.
Putting the drop in a well slide and adding a cover slip helps
by preventing evaporation, but it also leads to faster heat build
up. It also stops the only hope cells have of respiration. It's
like putting a plastic bag over the head of the human you staked
out in the desert.
Fig. 1 shows the structure of an FCS2 closed-system cooled chamber
available from Bioptechs in Butler, Penn. Cells are plated on a
40-mm coverslip and placed into the chamber, which provides a perfuseable
laminar-flow optical chamber with user-modifiable flow characteristics.
The upper glass element (microaqueduct slide) is then used to remove
heat from the specimen cavity. The heat is absorbed in a cooled
fluid being circulated through the cavity formed by the addition
of an O-ring sealed window adapter. The cells remain safely enclosed
in the separate optical enclosure. A separate ring is used to cool
the microscope's objective lens if the study is to be done at below
|Fig. 1: Environmentally controlled specimen chambers can
keep live cells functioning normally for indefinite periods.
Courtesy Bioptechs, Butler, Penn.
Contrast in any optical system can be obtained by manipulating
the intensity, color and phase of light as it passes through or
reflects from a target. This applies to living cells as well as
anything else. Most of the contrast we see in macroscopic systems
comes from manipulating either the color or intensity (more often
both) of the light we see with. Living cells, however, generally
absorb very little light and have little or no color. That is why
stains are so important to cell-morphology studies.
The internal structures of living cells, however, affect the speed
of light as it passes through them. That leads to the possibility
of using phase contrast to image them (See Fig. 4). There are several
phase contrast techniques available, such as differential interference
contrast (DIC) and Nomarski DIC.
|Fig. 4: Phase contrast uses the optical properties of the
materials making up living cells to create image contrast. Courtesy
Carl Zeiss, Thornwood, N.Y.
Phase contrast exploits one physical phenomenon: indices of refraction
differ in the different materials making up the cell. It would be
useful to have a contrast mechanism that keys on cellular chemistry,
which is what interests researchers most. Fluorescence is such a
Many biologically important molecules fluoresce. That is, they
absorb light photons of one wavelength, hold the energy for a short
time, and then expel that excess energy by reemitting another photon
of a longer wavelength.
By illuminating cells containing fluorescent molecules with the
light at the appropriate excitation wavelength and observing them
through a filter that isolates the emission wavelength, researchers
have long exploited this phenomenon to obtain microscopic image
contrast. Examples include fluorescent phalloidin for imaging actin
filaments, fluorescent phospholipids for imaging plasma membranes,
fluorescent ceramide for imaging Golgi apparati, and many others.
This fluorescence technique has the decided advantage that it generally
provides contrast without disturbing the normal operation of the
cells' biochemical processes.
The disadvantage, of course, is that natural fluorescence is largely
serendipitous--at least for microscopy. It would be useful to have
a fluorescent molecule available that could be used to tag various
cellular chemical components more or less at will.
Aequorea victoria is a luminescent jellyfish whose bioluminescence
machinery includes a photoprotein, aequorin, which is activated
by Ca++ ions and emits blue light, along with an accessory green
fluorescent protein (commonly referred to simply as "GFP"),
which accepts energy from aequorin and sheds it by emitting green
Genetically modified GFP expressed in transgenic plants, fungi
and animals has proven to be useful as a safe and bright fluorescent
marker (see Fig. 2). Genes for producing GFP can be fused with the
entire coding region of a particular protein, making it possible
to follow functional, full-length proteins as they are used by living
cells. Since GFP-tagged proteins can be followed in living cells,
it is now possible to study dynamic systems.
|Fig. 2: Green fluorescent protein (GFP) provides a flexible
contrast medium that does not interfere with normal cellular
processes. Courtesy Applied Precision, Issaquah, Wash.
Imaging Cellular Motions
Motions of live cells present two challenges to microscopists:
obtaining clear images in the face of rapid cellular motions, and
capturing the temporal development of cellular processes. Incorporation
of digital video technology helps researchers meet both challenges.
CCD cameras are roughly two orders of magnitude more sensitive
than photographic films. This higher sensitivity allows microscopists
to capture images with simultaneously lower illumination levels
and shorter exposures. Motion-induced blur is proportional to speed
of movement across the image divided by the exposure time. Thus,
CCD cameras' increased sensitivity allows them to "freeze"
cellular motions much more successfully in still images.
To follow dynamic cellular processes, microscopists employ variable-frame-rate
video. The limiting factor in CCD technology is frame-transfer rate,
so, researchers generally have to off between speed and resolution.
High-speed systems are available that can capture up to 100 frames
per second, but their resolution is limited to roughly a quarter
of a million pixels. Megapixel-resolution cameras are, of course,
available, but they are limited to frame rates of 10 frames per
Employing digital video technology also brings with it the advantages
of image processing. Images can easily be combined (as shown in
Fig. 3), enhanced and published electronically.
|Fig. 3: Digital video technology opens up the whole realm
of image processing for live cell microscopy. Here, images using
three fluorescence wavelengths are combined. Red highlights
actin filaments, green highlights mitochondria and blue highlights
cell nuclei. Courtesy Carl Zeiss, Thornwood, NY.
These advances in specimen chambers, contrast methods and digital
imaging are making live cell microscopy an increasingly important
part of the biotechnologist's tool kit.
Spector, David L., Goldman, Robert D., and Leinwand, Leslie A.,
Cells: A Laboratory Manual,Cold Spring Harbor Laboratory Press,
Plainview, N.Y., ISBN0-87969-522-6, 1998.