Page 216 - Fundamentals of Light Microscopy and Electronic Imaging

EXAMINING FLUORESCENT MOLECULES IN LIVING CELLS 199

lipids and proteins, including ion pumps, channels, and gates, which lead to loss of ion
balance, loss of transmembrane potential, and rapid cell death. We discussed the conse-
quences of phototoxicity on cell shape and cell behavioral responses in Chapter 3.
Briefly, cells cease movement; intracellular organelle trafficking ceases; cells round up
and form vacuoles and eventually lyse. Excitable cells such as neurons and free-living
amoebae are among the most sensitive to light exposure in the microscope. Just 3 sec-
onds of continuous exposure to the blue excitation wavelengths from a mercury lamp
filtered by a standard fluorescein filter set is sufficient to cause Acanthamoeba and Dic-
tyostelium amoebae to stop moving and round up. The requirement to minimize the
exposure of living cells to light can place extreme demands on photography, because
short exposures reduce the signal-to-noise ratio and result in grainy images. Control of
the exposed dose is especially difficult during the acquisition of time-lapse sequences.
The following approaches reduce the effects of phototoxicity:

• Additional UV and IR cutoff filters should be inserted near the illuminator, since
the majority of the spectral output of mercury and xenon lamps is, respectively, in
the UV and IR regions of the spectrum, and because most fluorescence filter sets
are not completely effective in removing these unwanted wavelengths.
• Minimize exposure to light to allow time for dissipation and degradation of free radi-
cals. Phenol red-free basal salt solution is recommended to reduce photon absorption.
• Addition of millimolar concentrations of anti–free radical reagents to live cells and
in vitro preparations of purified cell organelles and filaments minimizes free radi-
cal damage, because the reagents readily react with free radicals and help spare
endogenous molecules. Sodium ascorbate, reduced glutathione, or Trolox (a water-
soluble form of vitamin E) at 10 mM concentrations are effective, particularly for
in vitro systems.
• To retard the rate of free radical formation, the concentration of dissolved oxygen
can be reduced to 4% of the saturating value for buffer in room air by adding
oxygen-depleting enzymes to the medium. Low oxygen concentration is usually
not damaging to tissue culture cells—in fact, cells exist at similarly low values of
oxygen tension in body tissues. One effective commercial product is Oxyrase
(Oxyrase, Inc., Mansfield, Ohio), a preparation of respiratory particles from
Escherichia coli membranes. Alternatively, an oxygen-scavenging system can be
constructed based on a combination of catalase, glucose oxidase, and D-glucose.
Cells in well chambers open to the air can be covered with a layer of embryo-grade
mineral oil such as that available from Sigma Chemical Company (St. Louis, Mis-
souri) to prevent resaturation of the medium with oxygen.

Exercise: Fluorescence Microscopy
of Living Tissue Culture Cells

The purpose of this exercise is to learn how to: Prepare and examine live cells
whose organelles are labeled with specific fluorescent dyes; Become familiar
with the action of narrow band-pass and long-pass filter sets that are used to
examine double-stained specimens; Evaluate and select filter sets based on their
transmission profiles; Learn how to balance the intensities of different fluo-
rochromes to minimize bleed-through in inappropriate filter sets. Note: Read the