Dear Calcium Imagers,

To say that ratiometric measurements are
quantitative and single-wavelength measurements
are not is, at best, an oversimplification.

Certainly, single wavelength indicators provide
numbers, hence quantitation, but the issue is,
what is being quantitated?

            Ratio-dye users might claim that
ratio indicators provide absolute levels of
free calcium, but they simply do not.  The
uncertainty in the intracellular Kd and dynamic
range are too great for them to provide precise
measurements of resting calcium, and
consequently, any changes in calcium from
rest are also uncertain.  Given this uncertainty,
is there any advantage over single wavelength
indicators?

Single wavelength dyes cannot easily detect
CHANGES  in resting calcium levels because they
are not ratiometric.  But can ratio indicators do
this?  There are many reasons why ratio
measurements can change in time and in space.
There are alterations of the dye itself, compartmentalization
[where you now have a mix of two ratios: the cytosolic
ratio and, e.g., the ER ratio: how are you going to
resolve that?], spectral shifts, alterations in Kd,
and other problems that have all been addressed
in the literature (Thomas et al. article; earlier articles
by myself, Baylor and others).  In addition, if one
mixes two dyes together and they bleach at different
rates (which is quite common) the resulting ratio
will of course also change.  It is important to note,
that if one is using AM loading, there will always
be some degree of compartmentalization, and this
may severely distort ratio measurements.

If one assumes that resting calcium is relatively
constant inside a cell (i.e. pre-stimulation), and
this usually is not a bad assumption, and one
estimates resting calcium, then one can get
numbers that are just as quantitative with a
single wavelength dye as with a ratio dye.
Indeed, one "ratio's" the calcium increase against
the resting fluorescence in that volume, so
non-uniformity in volumes ARE taken into account.
[Also see article by Maravall et al., 2000,
Biophys. J, 78:2655.  Estimating intracellular
calcium concentrations and buffering
without wavelength ratioing.  This article
provides additional strategies for quantitating
single wavelength calcium measurements,
and does not require any assumptions about
resting calcium.]

In fact, the single wavelength dyes are, I think, better:
if one looks at many of the advances made over
the past 10 years with calcium imaging, most
were made with either single wavelength dyes
or with ratio dyes used in single wavelength
mode.

Because single wavelength dyes provide greater
dynamic range,  are easier to use, and don't require
a UV laser, I think that anyone planning to do
calcium imaging experiments should give them
full consideration. This said, I am not "anti-ratio"--
if we ever find an instance in our experiments where
ratios provide reliable additional information, we shall
certainly make use of this approach!

A book chapter of ours from 2000 reviews
much of this earlier literature:
"Fluorescent calcium indicators: subcellular
behavior and use in confocal imaging"
p. 261-303, O'Malley, Burbach and Adams
in Confocal Microscopy: Methods and
Protocols, 2000, Humana Press.
I still have some reprints available.

I think our review nicely complements the
Thomas et al. article.  But the Thomas
article provides more up to date information
on the newer calcium indicators.

My two cents,

Donald M. O'Malley, Ph.D.
Assistant Professor
Department of Biology
414 Mugar Hall
Northeastern University
Boston, MA 02115
[log in to unmask]
www.omalleylab.neu.edu