1 Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405; and 2 Department of Kinesiology and Applied Physiology, University of Colorado at Boulder, Boulder, Colorado 80309
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We proposed and
tested the use of nontraditional excitation wavelengths
(1 and
2) and an emission wavelength
(
em) to define conditions under which free calcium
concentration and a fluorescence ratio are linearly related.
Fluorescence spectra were determined for aqueous solutions that
contained 25 µM fura 2, 125 mM K+, and either 0 mM or 0.1 mM Ca2+. Effectively linear relationships between
[Ca2+] and a fluorescence ratio, i.e., <5% bias when
[Ca2+]
5 × dissociation constant, were apparent
when
1
400 nm,
2
370 nm, and
em
510 nm. Combinations with longer
1
and
em and/or with shorter
2 reduced this
bias further. Although the method described does not obviate the
complications that surround the correction for fluorescence background,
choosing a nontraditional combination of excitation and emission
wavelengths offers several practical advantages over more traditional
fura 2 fluorescence methodologies in a variety of experimental settings.
intracellular calcium; cardiac myocyte
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MEASUREMENTS
OF FREE CALCIUM CONCENTRATION ([Ca2+]) through the
use of fluorescence dyes have grounded much of the current
understanding of intracellular Ca2+ handling and its
importance to cell function (5, 6). Of the many
intracellular calcium concentration ([Ca2+]i)
indicators that are excited by two wavelengths (1 and
2) and monitored at a single emission wavelength
(
em), fura 2 has become popular due to its favorable ion
selectivity, fluorescence yield, and bleaching characteristics
(2, 7).
The traditional fluorescence ratiometric method used to
estimate [Ca2+] with fura 2 (when changes in
[Ca2+] are slow) was presented by Grynkiewicz et al.
(2) as
![]() |
(1) |
The use of the fluorescence ratiometric method is straightforward in theory. However, determining background and the calibration constants is often difficult in practice. Background emissions due to cell autofluorescence, compartmentalization of fura 2 inside intracellular organelles, partial deestrification of fura 2-AM, and the fluorescence of fura 2-AM bound to the cell membrane can all be mistaken for the fluorescence signal of cytosolic fura 2 (7). In addition, determining values for Rmin and Rmax in an intact cell requires experimental conditions that are not easily prepared or maintained, and the chemical agents used to induce these conditions can unpredictably complicate the distinction between fluorescence and background intensities.
To avoid the complicated nature and intrinsic inaccuracies of a full
calibration procedure, it has become an increasingly common practice to
use R alone to characterize [Ca2+]. This compromise is
appropriate to the degree to which properties of the recorded R reflect
those of the actual [Ca2+]. When the traditional
excitation wavelength pair of 340 nm and 380 nm is used, there exists a
nonlinear relationship between [Ca2+] and R. Therefore,
the use of a recorded R transient to characterize [Ca2+]
is dubious. Specific temporal characteristics of
[Ca2+], such as exponential rate constants or time
derivatives, and comparisons between values of [Ca2+],
such as minimum and maximum values, are complicated by the nonlinear
relationship between [Ca2+] and R. A more valid
characterization of [Ca2+] by a recorded fluorescence
ratio would be possible if a linear relationship between
[Ca2+] and R or R1 existed.
On examination of Eq. 1, the relationship between
[Ca2+] and a fluorescence ratio would be linear if the
value for Rmax was forced to be zero. If Rmax
were zero, the calibration equation for fura 2 would simplify to the
following
![]() |
(2) |
This paper demonstrates that many combinations of 1,
2, and
em can be chosen such that an
effectively linear relationship between [Ca2+] and a
fluorescence ratio, specifically R
1, will arise
over the range [Ca2+]
5 × Kd. First, we present fluorescence emission
spectra of fura 2 in vitro over a wide range of excitation wavelengths.
We then define "an effectively linear relationship" to exist when combinations of
1,
2, and
em allow estimates of [Ca2+] using the
simplified Eq. 2 to be within 5% of the nonbiased estimate
of [Ca2+] using Eq. 1. In reference to
the recorded fura 2 fluorescence spectra, it is demonstrated that
wavelength combinations, when
1
400 nm,
2
370 nm, and
em
510 nm, satisfy
the effectively linear relationship criterion.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Spectrophotometric characterization of fura 2 fluorescence in vitro. Fura 2 pentopotassium salt (Molecular Probes, Eugene, OR) was prepared to a concentration of 25 µM in two aqueous solutions, both of which contained concentrations of (in mM) 125 KCl, 5 NaCl, 20 HEPES, 2 MgCl2 · 6H2O, and 2 ATP, pH 7.2. All chemicals were acquired from Sigma Chemical, St. Louis, MO, except where noted. One solution was termed the "zero [Ca2+] solution" after the addition of 0.1 mM EGTA, which buffered excess Ca2+, and the other was termed the "high [Ca2+] solution" after the addition of 0.1 mM CaCl2. Because Kd in this medium has been estimated between 135 and 224 nM (2), the concentration of 0.1 mM [Ca2+] in the high [Ca2+] solution was considered to satisfy the condition [Ca2+] >> Kd. Respective solutions were also prepared without fura 2 for purposes of determining fluorescence background.
Fluorescence intensities were recorded at 25°C for the zero [Ca2+] solution, the high [Ca2+] solution, and for the respective background solutions with the use of a spectrophotometer (model LS-5; Perkin-Elmer, Norwalk, CT) with an excitation bandwidth of 3 nm and an emission bandwidth of 5 nm. Fluorescence intensity was recorded at intervals of 3 nm over the range of excitation wavelengths of 330-441 nm and at intervals of 5 nm over the range of emission wavelengths of 480-560 nm. Fluorescence intensities of the zero [Ca2+] solution and high [Ca2+] solution were corrected for background with the use of the respective solutions without fura 2. The resulting fluorescence spectrum for the zero [Ca2+] solution provided a map of the fluorescence intensity emitted at a specific wavelength that was due to excitation at a specific wavelength when fura 2 was totally free of Ca2+. Similarly, the fluorescence spectrum for the high [Ca2+] solution provided a map of the fluorescence when fura 2 was fully bound with Ca2+. Figure 1 demonstrates these two fluorescence spectra as contour plots. Each contour represents a level of relative fluorescence intensity that would be recorded at any specified emission and excitation wavelength that we examined.
|
![]() |
(3) |
Criterion for an effectively linear relationship.
An effectively linear relationship between [Ca2+] and
R1 was defined to occur when estimates of
[Ca2+] using Eq. 2 were not biased by >5%
compared with the actual [Ca2+] over the range
[Ca2+]
5 × Kd. The bias
was therefore quantified as the difference between estimates using
Eq. 1 ([Ca2+]1) and those made
using Eq. 2 ([Ca2+]2) normalized
to [Ca2+]1
![]() |
(4) |
![]() |
(5) |
Demonstration of an effectively linear relationship in vivo. Cardiac myocytes were taken from a 14-mo-old male Fisher 344 rat. Myocytes were isolated from the left ventricle and septal portions of the heart as described before (4). After dispersion, the myocytes were immediately plated onto coverslips that had been lightly laminated the day before at a density of 10 mg laminin/ml of medium 199. Myocytes were then incubated in 2 ml of medium 199 at 37°C and 5% CO2-balance room air.
After 4 h of incubation, 1 µl of 1 mM fura 2-AM (Molecular Probes) was added to the media of one coverslip, thereby exposing the cells to 0.5 µM of fura 2-AM. After an additional 5 min of incubation, the coverslip served as the bottom of a custom-made flow through chamber, superfused at room temperature with normal Tyrode, that consisted of (in mM) 140 NaCl, 6 KCl, 2 MgCl2, 2 CaCl2, 10 glucose, 5 HEPES, and 2 pyruvate, pH 7.4. One myocyte of the coverslip was positioned in the field of view of an inverted microscope (Nikon, Melville, NY) with a ×40 epifluorescence lens with 1.3 numerical aperture. Optical filters were chosen with wavelengths centered at 400 nm with 20-nm bandwidth and 360 nm with 10-nm bandwidth (Omega Optical, Brattleboro, VT). A spinning wheel, which held the filters, was computer controlled such that fluorescence produced by the two excitation wavelengths was interlaced and sampled at 176 Hz (C & L Instruments, Elizabethtown, PA). Ultraviolet light was supplied by a 75 W xenon arc lamp (Ushio, Los Angeles, CA). Emitted fluorescence was filtered by an optical filter centered at 510 nm with 35-nm bandwidth and detected by a photomultiplier tube (R647-04; Hamamatzu, Bridgewater, NJ), and counts were divided by 10 such that one emission count was recorded for every 10 detected (C3866; Hamamatzu). The coverslip was field stimulated with 1.5 × threshold voltage, 0.5-ms duration pulses, using platinum electrodes at a rate of 1 Hz. The myocyte was continuously exposed to the array of excitation wavelengths for 5 min, after which fura 2 bleaching no longer occurred. Fluorescence was then recorded. The superfusate was then changed to normal Tyrode with 100 µM ouabain for 10 min. Ouabain was used to suppress Na+-K+-ATPase activity, which in turn would induce an accumulation of Ca2+ in the cell (1). Fluorescence was continuously monitored until fluorescence at peak [Ca2+] indicated saturation, i.e., [Ca2+] > Kd, and by definition, a high [Ca2+] condition. The superfusate was then changed back to normal Tyrode, and a fluorescence recording was taken. To rid the myocyte of cytosolic fura 2, but not fura 2 compartmentalized in organelles or bound to the membrane, the coverslip was exposed for 4 min to 2 µM digitonin dissolved in Ca2+-free Tyrode, which consisted of (in mM) 140 NaCl, 6 KCl, 2 MgCl2, 1 EGTA, 10 glucose, 5 HEPES, and 2 pyruvate, pH 7.4. The superfusate was then switched to Ca2+-free Tryode for 1 min, and a fluorescence recording was taken as the background emission. Fluorescence intensities were taken to be the recorded emissions minus the respective wavelength background emissions. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Spectrophotometric characterization of fura 2 salt fluorescence in
vitro.
The relative fluorescence intensities recorded along the 510-nm
emission wavelength were plotted as excitation spectra in Fig.
2. These excitation spectra demonstrated
the familiar fura 2 excitation spectra with a peak fluorescence value
at 376 nm for the zero [Ca2+] solution and at 339 nm for
the high [Ca2+] solution. The isobestic wavelength was
found at 359 nm. These values were comparable to those reported in Fig.
3 of Grynkiewicz et al. (2).
|
|
Combinations of 1,
2, and
em that satisfy linear criterion.
The calculated values for Rmin and Rmax when
em = 510 nm for all possible combinations of
excited wavelength pairs
1 and
2 are
plotted as contours in Fig.
3A. Both Rmin and
Rmax had values of unity along the diagonal that would be
expected when
1 =
2. This diagonal
was crossed at 376 nm by another diagonal of unity for values of
Rmin and also at 339 nm for values of Rmax. These crossings depicted the respective fluorescence intensity peaks
recorded for the zero [Ca2+] solution and the high
[Ca2+] solution. The contours shown in Fig. 3A
demonstrate that values for Rmin and Rmax were
found to lie along hyperbolas bounded asymptotically by the respective
diagonals of unity.
Relationships between [Ca2+] and
R1 using nontraditional wavelengths.
The actual relationships between [Ca2+] and
R
1, as defined by Eq. 1, for traditional and
nontraditional combinations of excitation and emission wavelengths,
were compared with the strictly linear relationship as defined by
Eq. 2. For the traditional combination,
1 = 380 nm,
2 = 340 nm, and
em = 510 nm were selected. It should be noted that,
although Grynkiewicz et al. (2) defined
1 = 340 nm and
2 = 380 nm, the
consequences of defining
1 = 340 nm and
2 = 380 nm on R are algebraically equivalent to
those of defining
1 = 380 nm and
2 = 340 nm on R
1, which we
demonstrate here. With the traditional combination, the linear
approximation given by Eq. 2 was found to have a 30% bias
at 5 × Kd (Fig.
4A). The effectively linear
criterion, i.e., <5% biased, was met only over the range
[Ca2+]
0.84 × Kd.
Therefore, attempts to characterize [Ca2+] using
R
1, or R with
1 and
2
defined by Grynkiewicz et al. (2), would be appropriate up
to ~0.84 × Kd.
|
Demonstration of an effectively linear relationship in vivo.
With the use of optical filters 1 = 400 nm
with bandwidth of 20 nm,
2 = 360 nm with bandwidth
of 10 nm, and
em = 510 nm with bandwidth of 35 nm,
fura 2 fluorescence was recorded from a cardiac myocyte under three
conditions illustrated in Fig. 5. First,
fura 2 fluorescence was recorded during electrical stimulation of the
cardiac myocyte. As the intracellular [Ca2+] rose
immediately after stimulation, fluorescence due to 400-nm excitation
was diminished, and fluorescence due to 360-nm excitation remained
unchanged.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The fluorescence characteristics of fura 2 are such that a
carefully chosen combination of excitation and emission wavelengths can
be used to produce an effectively linear relationship between [Ca2+] and a fluorescence ratio. We found that a
combination of 1
400 nm,
2
370 nm,
and
em
510 nm satisfied the criterion to elicit an
effectively linear relationship over the range [Ca2+]
5 × Kd. In addition, as demonstrated by a
comparison of Fig. 3, B and D, combinations that
include longer
1 and
em and/or shorter
2 better satisfy the linear relationship criterion.
There are at least three advantages that would arise from the use of
such a combination of 1,
2, and
em. 1) Time characteristics of the
fluorescence ratio reliably reflect time characteristics of the
[Ca2+] transient. Specifically, time to peak, relative
rates of [Ca2+] rise and decay, and exponential time
constants used to characterize a [Ca2+] transient rise
and decay could all be determined from the fluorescence ratio.
2) Relative changes in the fluorescence ratio would
correspond directly to those in [Ca2+]. The limit of
accuracy, with which temporal characteristics and relative changes in
[Ca2+] could be made with the use of the fluorescence
ratio, was comparable to 5%. And because full calibration was not
necessary to attain these characteristics, the constants
Kd, (Sf2/Sb2),
Rmin, and Rmax must not be determined.
3) In cases when a full calibration is warranted, only the
constants (Sf2/Sb2) and Rmin would
be necessary, because the value of Rmax could be estimated
to be zero. The only constant that must be assumed or arduously
determined is Kd, whose value does not affect
the shape or temporal characteristics of the estimated
[Ca2+] transient.
There is at least one practical disadvantage associated with the use of
a combination 1
400 nm,
2
370 nm,
and
em
510 nm proposed here. As illustrated in Figs.
1 and 2, an excitation wavelength
1 > 400 nm
produces less free fura 2 fluorescence intensity than many other
wavelengths. Therefore, fluorescence signals elicited by wavelengths
>380 nm would possess a lower signal-to-noise ratio. However, a broad
bandwidth emission filter may be used to recover some of these losses
in signal quality. In addition, some nontraditional excitation
wavelength pairs may result in a lower ratiometric dynamic range, as
illustrated in Fig. 4B. Nevertheless, if the application
permits, dynamic range may be recovered by a careful selection of
wavelength pairs, particularly a shorter
2 as presented
in Fig. 4C.
One limitation of the mathematical development presented here lies in the assumption that the rate of change in [Ca2+] is relatively low. This assumption is not unique to this study and applies to all conditions under which fura 2 is used. The relationships between [Ca2+] and R of both Eqs. 1 and 2 are accurate only when the temporal changes in [Ca2+] are relatively slow, specifically when d[fura-Ca2+]/dt << koff[fura-Ca2+] (3). Under other circumstances, the fluorescence ratio would no longer be effectively linearly related to [Ca2+].
Another limitation not unique to this study lies in the problem of accurately accounting for background emissions. When cells of any type are loaded with fura 2-AM, there is, unfortunately, a significant percentage that is trapped in noncytosolic compartments, such as in intracellular organelles (7). In the present study, we demonstrated that digitonin could be used to lyse a cardiac myocyte membrane and therefore provide a route for cytosolic fura 2 to exit. There is, however, no guarantee that this method was not complicated by some fraction of cytosolic fura 2 that did not exit before background emission was recorded. Other methods, such as recording background before dialysis of fura 2 via a patch pipette, may be more accurate in determining background emissions.
In summary, this report described conditions under which combinations of excitation and emission wavelengths can be chosen to yield a linear relationship between [Ca2+] and a fura 2 fluorescence ratio. Under these conditions, the fluorescence ratio can be used to provide reliable estimates of the temporal characteristics of intracellular [Ca2+], as well as relative changes in [Ca2+]. The reliability of these estimates, particularly of temporal characteristics, would be uninfluenced by errors in the assignment of fura 2 Kd values. In a variety of experimental settings, these represent advantages over the use of traditional excitation and emission wavelength combinations.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported in part by National Institutes of Health grants HL-40306 and AG-13981.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: R. L. Moore, Dept. of Kinesiology and Applied Physiology, Univ. of Colorado, Boulder, CO 80309 (E-mail: rmoore{at}spot.colorado.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 November 1999; accepted in final form 26 April 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Akera, T.
Pharmacological agents and myocardial calcium.
In: Calcium and the Heart, edited by Langer GA.. New York: Raven, 1990, p. 299-331.
2.
Grynkiewicz, G,
Poenie M,
and
Tsien RY.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem
260:
3440-3450,
1985[Abstract].
3.
Klein, MG,
Simon BJ,
Szucs G,
and
Schneider MF.
Simultaneous recording of calcium transients in skeletal muscle using high- and low-affinity calcium indicators.
Biophys J
53:
971-988,
1988[Abstract].
4.
Moore, RL,
Yelamarty RV,
Misawa H,
Scaduto JRC,
Pawlush DG,
Elensky M,
and
Cheung JY.
Altered Ca2+ dynamics in single cardiac myocytes from renovascular hypertensive rats.
Am J Physiol Heart Circ Physiol
260:
H349-H356,
1991.
5.
Morgan, KG.
Ca2+i versus [Ca2+]i.
Biophys J
65:
561-562,
1993[ISI][Medline].
6.
Pozzan, T,
Rizzuto R,
Volpe P,
and
Meldolesi J.
Molecular and cellular physiology of intracellular calcium stores.
Physiol Rev
74:
595-636,
1994
7.
Tsien, RY.
Fluorescence indicators of ion concentrations.
Methods Cell Biol
30:
127-156,
1989[ISI][Medline].