Page 59 - Fundamentals of Light Microscopy and Electronic Imaging

42 ILLUMINATORS, FILTERS, AND THE ISOLATION OF SPECIFIC WAVELENGTHS

cells respond by blebbing and swelling, and particles contained in dilated vesicles begin
to exhibit vibrational movements (Brownian movements) that are not as obvious in the
living state. It is useful to observe cells without protective filters to become familiar with
these effects.
Cells may require additional chemical protection from the buildup of high concen-
trations of damaging free radicals. For well chamber slides the simplest measures are to
increase the volume of medium in the chamber to dilute the radicals or to use anti–free
radical reagents such as 10 mM ascorbate or succinate in the culture medium to neu-
tralize free radicals as they form. Alternatively, the rate of free radical formation can
be slowed by reducing the concentration of dissolved oxygen in the culture medium
using a mixture of oxygen-scavenging enzymes, such as catalase, glucose oxidase, and
D-glucose, or supplementing the medium with Oxyrase (Oxyrase, Inc., Mansfield,
Ohio), which is a commercial preparation of respiratory particles of bacterial cell mem-
branes that contains oxygen-removing enzymes. To maintain low oxygen concentra-
tions, cells must be enclosed in specially designed flow cells. If well chambers are used,
the medium should be covered with a layer of lightweight nontoxic mineral oil to pre-
vent recharging of the medium with atmospheric oxygen. The presence of these agents
is usually harmless to vertebrate somatic cells, since cells in most tissues exist in a low-
oxygen environment.