© 1999 by CRC Press LLC
Optical microscopy provides one of the most powerful techniques for studying
intracellular second messengers in living cells. In contrast to flow cytometry and
spectrofluorometry, optical microscopy can be used to make measurements on
individual cells, providing subcellular spatial information in addition to temporal
and quantitative information. Unfortunately, there are currently only a modest num-
ber of fluorescent indicators of physiologically important intracellular regulators
, and cAMP) that can be used for microscopy studies.
less, the advent of even this small group of indicators has been instrumental in
broadening our understanding of intracellular signaling.
Studies in our laboratory have utilized various fluorescent indicators of
) to investigate the role of this ubiquitous second messenger
in cell migration. Specifically, we have used optical microscopy to determine the
spatial and temporal characteristics of changes in [Ca
in motile polymorphonu-
clear leukocytes (neutrophils),
to correlate these characteristics with motility
parameters such as speed and persistence,
and to illustrate a possible mechanism
for the regulated adhesion necessary to support motility.
In this chapter, we will
present a practical guide to designing and setting up optical microscopy experiments
for studying [Ca
signaling during cell migration. We will discuss available [Ca
indicators, methodological approaches to single-cell measurements, imaging instru-
mentation, and quantification using digital image analysis.
II. Is Optical Microscopy the Right Choice
for Your Studies?
Optical microscopy experiments can be expensive and time consuming, so it is
worthwhile to consider carefully whether this is the best method to use. Methods
such as flow cytometry and spectrofluorometry are relatively easy to use to monitor
changes in [Ca
. For certain experiments, flow cytometry has several advantages
over fluorometry. First, in flow cytometry, measurements are made on suspended
cells, and only cell-associated fluorescence and the fluorescence of a very small
volume of buffer contribute to the measurement. For this reason, the need for
extensive, time-consuming washing of the sample is eliminated. This feature of the
flow cytometer is important for experiments in which cells are loaded with an