Biology Reference
In-Depth Information
VIII. Concluding Remarks
References
Abstract
The use of fluorescent indicators for monitoring calcium (Ca
2
þ
)signalsandfor
measuring Ca
2
þ
concentration ([Ca
2
þ
]) in living cells is described. The following
topics are covered in detail: (1) ratiometric and nonratiometric fluorescent indicators
and the principles underlying their use, (2) techniques for loadingCa
2
þ
indicators and
Ca
2
þ
buffers into living cells, (3) calibration of indicator fluorescence intensity
measurements to yield values of intracellular [Ca
2
þ
], (4) analysis of nonratiometric
fluorescence intensity data and caveats relating to their interpretation, (5) techniques
for manipulating intracellular and extracellular [Ca
2
þ
], and (6) the use of fluorescent
indicators tomonitor Ca
2
þ
signals inmitochondria. The chapter aims to present these
fundamental topics in a manner that is practically useful and intuitively accessible.
The origins of key mathematical equations used in the article are outlined in two
appendices.
I. Introduction
In the application of any measurement technique, a body of practical knowledge is
shared by experienced practitioners. Although important for making successful
measurements, such lore, which sometimes seems arcane, often is not described
explicitly or explained in journal publications. In this respect, measuring [Ca
2
þ
]
1
with fluorescent indicators is no exception. The purpose of this chapter is to gather in
one place some of the most common and useful practical information relevant to the
useoffluorescentCa
2
þ
indicators. Such a collection of information is hoped to
alleviate the frustration of those who are novices at using fluorescent indicators.
II. Fluorescent Ca
2
þ
Indicators
The commonly available fluorescent indicators for Ca
2
þ
fall into two operation-
al classes: dual-wavelength ratiometric dyes and single-wavelength nonratiometric
dyes (
Table I
). Chemical structures of some of the indicators listed in
Table I
are
shown in
Fig. 1
. For nonratiometric indicators, a change in [Ca
2
þ
] brings about a
corresponding change in the intensity of the indicator's fluorescence excitation and
emission spectra,
2
whereas the wavelengths of the excitation and emission spectral
1
Symbols used: Ca
2
þ
, free calcium ion; [Ca
2
þ
], concentration of free calcium ions; [Ca
2
þ
]
i
, cytosolic
concentration of free calcium ions.
2
An excitation spectrum is taken by monitoring fluorescence emission intensity at a fixed wavelength
while excitation light is scanned through a wavelength range over which the sample can absorb light. The
emission intensity is plotted as a function of the excitation wavelength. To collect an emission spectrum,
excitation light at a fixed wavelength is delivered to the sample while the emission intensity is monitored
over a wavelength range. Here, the emission intensity is plotted as a function of emission wavelength.
