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Department of Biomedical Engineering of the School of Engineering, The University of Alabama at Birmingham, Alabama 35294-0019
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Transmembrane voltage-sensitive
fluorescence measurements are limited by baseline drift that can
obscure changes in resting membrane potential and by motion artifacts
that can obscure repolarization. Voltage-dependent shift of emission
wavelengths may allow reduction of drift and motion artifacts by
emission ratiometry. We have tested this for action potentials and
potassium-induced changes in resting membrane potential in rabbit
hearts stained with di-4-ANEPPS [Pyridinium,
4-(2-(6-(dibutylamino)-2-naphthalenyl) ethenyl)-1-(3-sulfopropyl)-, hydroxide, inner salt] using laser excitation (488 nm) and a
two-photomultiplier tube system or spectrofluorometer (resolution of
500-1,000 Hz and <1 mm). Green and red emissions produced upright
and inverted action potentials, respectively. Ratios of green emission
to red emission followed action potential contours and exhibited larger fractional changes than either emission alone (P < 0.001). The largest changes and signal-to-noise ratio (signal/noise)
were obtained with numerator wavelengths of 525-550 nm and
denominator wavelengths of 650-700 nm. Ratiometry lessened drift
56-66% (P < 0.015) and indicated decreases in
resting membrane potential. Ratiometry lessened motion
artifacts and increased magnitudes of deflections representing
phase-zero depolarizations relative to total deflections by
123-188% in intact hearts (P < 0.02). Durations
of action potentials at different pacing rates, temperatures, and
potassium concentrations were independent of whether they were measured
ratiometrically or with microelectrodes (P 
0.65). The
ratiometric calibration slope was 0.017/100 mV and decreased with time.
Thus emission ratiometry lessens the effects of motion and drift and
indicates resting membrane potential changes and repolarization.
potentiometric dye; Pyridinium, 4-(2-(6-(dibutylamino)-2-naphthalenyl) ethenyl)-1-(3-sulfopropyl)-, hydroxide, inner salt; potassium; action potential
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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TRANSMEMBRANE voltage-sensitive fluorescent dyes have been used to monitor fast events such as action potentials and membrane polarization produced by electric stimulation. However, the fluorescence emission and fractional fluorescence change depend on variables, including dye concentration, dye internalization to the inner leaflet of the membrane, dye photobleaching, and possible changes in excitation light intensity, which are effects that can occur slowly and are not related to transmembrane potential. Thus fluorescent dyes may not be suitable for measuring slow changes of the resting membrane potential such as those produced by hyperkalemia or ischemia.
However, optical mapping studies in hearts have not used all of the information contained in the emitted light of a dye. The emission spectrum of the dye di-4-ANEPPS [Pyridinium 4-(2-(6-(dibutylamino)-2-naphthalenyl) ethenyl)-1-(3-sulfopropyl)-, hydroxide, inner salt] undergoes a reversible shift toward shorter wavelengths in response to membrane depolarization, i.e., electrochromism (10). Cardiac optical mapping studies have frequently measured the decrease in emitted light for wavelengths longer than ~600-650 nm (red portion of the emission spectrum). Our recent measurements of di-4-ANEPPS emission spectra in hearts indicated that with an appropriate excitation wavelength, both red and green portions of the emission spectrum can be measured. The emitted light for shorter wavelengths (green) increases simultaneously with the decrease in red emission during depolarization (17).
The availability of both red and green emission signals may allow improvements in recording of changes in the membrane potential by using the ratio of the green and red signals in hearts, as was developed by Bullen and Saggau (5) for measurements in hippocampal neurons during voltage-clamp pulses. Changes in fluorescence that are common to both signals should theoretically cancel in a ratio. Therefore, effects of dye photobleaching and variations in dye concentration, which should affect both red and green signals, may be lessened.
Also motion artifacts may be lessened, which was described for transmembrane voltage changes in arterioles and heart cell cultures and calcium signals from hearts (2, 4, 23). Motion is a limitation of optical mapping of transmembrane potentials in the heart, requiring mechanical stabilization, pharmacological electromechanical uncoupling, or use of temporal derivatives of the optical signal to detect repolarization of the action potential (6, 8, 9, 12, 13).
This study considers ratiometry of epicardial di-4-ANEPPS emission in isolated rabbit hearts with excitation by a laser beam. Ratiometry was performed for action potentials and resting membrane potential changes produced by potassium infusion. We measured emission in two optical bands and, in separate experiments, 16 bands using a spectrophotometer to determine the impact of the selection of emission wavelengths used for the numerator and denominator.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Heart preparation. Hearts (n = 11) were removed from pentobarbital-anesthetized New Zealand White rabbits in accordance with Institutional Animal Care and Use Committee guidelines. Hearts were initially perfused with a solution containing (in mmol/l) 129 NaCl, 5.4 KCl, 1.8 CaCl2, 1.1 MgCl2, 26 NaHCO3, 1 Na2HPO4, 11 glucose, and 0.04 g/l bovine serum albumin, at an aortic pressure of 60-80 cmH2O and a temperature measured with a miniature probe (Physitemp) in a ventricular chamber of 35-36°C. Later studies used injection of a 5- to 10-ml bolus of solution with potassium concentration of 40 or 100 mM into the perfusing tubing or 1 min of perfusion with a solution in which the potassium concentration was increased to 20 mM by the addition of KCl. All solutions were equilibrated to 95% O2-5% CO2 and pH of 7.3-7.4 with gas exchangers fabricated from silicone tubing. Optical recordings were obtained from all hearts. In the first five hearts, of which simultaneous microelectrode recordings were obtained in two hearts, diacetyl monoxime (15 mM) was present in the solution during all recordings to reduce cardiac movement (3, 21, 25). Two other hearts did not receive diacetyl monoxime and were endocardially prefrozen with a 10-mm left ventricular catheter containing liquid nitrogen for 6 min. This produced an epicardial layer (~1 mm) of viable cardiac fibers that exhibited action potentials and contractility (1, 20). Four other hearts were not endocardially prefrozen and were studied both with diacetyl monoxime (20 mM) during simultaneous optical and microelectrode recordings and with no diacetyl monoxime during optical recordings to allow analysis of effects of heart motion. These four hearts had atrioventricular block produced by thermal ablation at the beginning of the experiment and were paced for at least 10 min at approximately twice the threshold strength and 3-ms pulse duration from a bipolar electrode on the ventricular epicardium at intervals from 150 to 1,250 ms. One of the hearts fibrillated during pacing at 150 ms. Orders of perfusion with and without diacetyl monoxime and of different pacing rates were varied. Three of the hearts were further studied at a temperature of 22-23°C by turning off the perfusate heater.
Potassium change. Because the resting membrane potential in cardiac tissue is depolarized by the addition of extracellular potassium, we used potassium bolus injections or steady perfusion of hyperkalemic solution to alter the resting membrane potential (18). In preliminary tests, the delay from potassium bolus injection to the solution change at the aortic cannula was determined with blue dye and with measurement of transmittance of laser light through the cannula. Also elevated potassium concentration in effluent from hearts after bolus injection was verified with an electrolyte analyzer. In some experiments, a custom-fabricated aortic cannula containing a potassium ion-selective electrode (TIPK, World Precision Instruments, Sarasota, FL), reference, and ground electrodes was used to indicate timing of the potassium change just above the left ventricle. The potassium signal was recorded simultaneously with optical signals.
Ratiometric fluorescence emission measurements.
Hearts were stained with di-4-ANEPPS dissolved in ethanol that was
gradually injected into the perfusion tubing (total 0.2-0.3 ml in
15 min). The heart gently contacted a transparent plate through which
excitation and emission light passed. In some experiments the plate
contained a 1-cm diameter hole to allow positioning of an intracellular
microelectrode near the optical recording site. Two optical recording
systems were used with different hearts (Fig.
1). In both systems, a 488-nm argon laser
excited the di-4-ANEPPS. This wavelength is near the excitation peak or
crossover wavelength (480 nm in bilayer), which minimizes transmembrane
voltage dependence of absorption (5, 10). Thus the
fractional change of red fluorescence is expected to be less than that
found when a longer excitation wavelength is used (5).
With a two-photomultiplier tube system, the laser scanned 63 spots in a
1-cm2 region of epicardium. Red fluorescence was collected
with a photomultiplier tube that had its photocathode covered with a
long-pass glass filter (>610 nm), whereas green fluorescence was
collected with another photomultiplier tube covered with an
interference filter (half-height bandwidth of 534-546 nm). The
photomultiplier tubes were positioned side by side and sufficiently far
from the heart so that the optical path from the heart was essentially
the same for both tubes (maximum angular difference <3°) and
incident fluorescence was approximately perpendicular to the filter
surface.
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Data analysis. Fluorescence signals and ratios were calculated and graphed with a computer (Sparcstation 20, Sun Microsystems, Mountain View, CA) using PV Wave or Matlab. So that differences after versus before action potential phase-zero depolarization or potassium infusion represent all available data from the two-photomultiplier tube system, measurements were performed on averaged recordings for all laser spots. Recordings were not temporally smoothed. Changes in fluorescence and ratios produced by phase-zero depolarizations or potassium-induced depolarizations were determined from the mean values in 6-ms segments before and after depolarization. The RMS noise (i.e., positive square root of the average of the squares of the deviations of the flourescence samples about their mean) was determined for 6-ms segments before depolarization. Comparisons of action potential duration measured with the microelectrode versus that measured ratiometrically were performed with a single laser spot near the microelectrode. Summaries are given as means ± SD. Statistical significance was determined with the two-tailed t-test or analysis of variance. A P value <0.05 was considered significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Dual-emission wavelength measurements.
Figure 2 shows results from the
two-photomultiplier tube system and a simultaneous signal from an
intracellular microelectrode. The green signal (wavelengths of 540 ± 6 nm), the red signal (wavelengths >610 nm), and their ratio are
shown. The red signal contains an inverted action potential,
i.e., downward deflection corresponding to the phase-zero
depolarization and upward deflection corresponding to the gradual
repolarization of the action potential. The green signal contains an
upright action potential. These recordings indicate a shift toward
shorter emission wavelengths when the membrane depolarizes.
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F/F)
during the action potential phase-zero depolarization was 2.1 ± 0.8% for green fluorescence and 
3.8 ± 0.5% for red fluorescence. The fractional change of the ratio of the green fluorescence to the red fluorescence (Ratio/Ratio) was 6.1 ± 1.1%. Magnitudes of fractional changes were significantly larger for
the ratio versus either of the individual fluorescence signals (P < 0.001, paired 2-tailed t-test).
Figure 3 shows an example of the ratio of
the green and red signals during potassium infusion and the
simultaneous recording from a potassium ion-sensitive electrode located
in the aortic cannula ~1 cm above the heart. Data were recorded
without pacing the heart. Baseline drift in the ratio, which was not
negligible, has been canceled with linear subtraction. Resting membrane
potential decreased and the heart became quiescent when potassium
concentration was raised. The action potential amplitude and duration
also decreased during the first 20 s of hyperkalemia. Recovery
occurred after returning to the initial potassium concentration. In a
different heart that was paced continuously to prevent the ventricles
from becoming quiescent, again the emission ratio after subtraction of
drift followed depolarization and recovery of the resting membrane potential.
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Spectrofluorometric measurements. To gain insight into the role of the emission wavelengths chosen for ratiometric measurements, the emitted fluorescence was measured with a spectrograph (77400, Oriel, Stratford, CT). Discrete wavelength bands having a width of 21.9 nm were measured for wavelengths just above the laser light wavelength of 488 to 817 or 838 nm (17). Of six hearts with no diacetyl monoxime that were used to test the ability to lessen motion artifacts with ratiometry, two were endocardially prefrozen and four were not prefrozen.
Figures 4 and 5 show examples of the motion artifacts and their reduction by emission ratiometry in endocardially prefrozen hearts. In Fig. 4, simultaneous recordings from two diodes that collected green and red emission wavelengths and their ratio are shown. The green signal (Fig. 4A) and red signal (Fig. 4B) exhibited a rapid increase and decrease, respectively, that corresponded to phase-zero depolarization. The rapid changes were followed by motion artifacts that obscured repolarization in signals from both diodes. The ratio of the green signal to the red signal (Fig. 4C) canceled the artifact and revealed the action potential repolarization. Figure 5 shows an example in which the numerator and denominator signals represented wider bands of the green and red emission spectrum, such as those that may be sampled with bandpass or longpass filters. The bands were produced by summing signals from the spectrophotometer's diodes. The green signal (Fig. 5A) represents emission wavelengths in the band of 510-576 nm. The red signal (Fig. 5B) represents emission wavelengths of 598-838 nm. Both green and red signals contain motion artifacts. The ratio of the green signal to the red signal (Fig. 5C) eliminated motion artifacts and revealed action potentials.
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p). This analysis
was performed on the first recording obtained in each heart without
diacetyl monoxime. The Ph0/p p will have a maximum
possible range of 0-1, will have a small value when motion causes
the signal to exceed the upper or lower values that occur at the
beginning and end of the phase-zero depolarization, and will have a
larger value when the effects of motion are reduced. Wavelength bands
were 510-576 nm and 598-751 nm for numerator and denominator.
In the two hearts that were endocardially prefrozen and received no
diacetyl monoxime, the Ph0/pp was 0.386 ± 0.104 for
the numerator, 0.700 ± 0.311 for the denominator, and 0.941 ± 0.083 for the ratio (P < 0.02 for ratio vs.
numerator, paired 2-tailed t-test). In the four hearts that
were not endocardially prefrozen and received no diacetyl monoxime, the
Ph0/pp was 0.273 ± 0.099 for the numerator,
0.351 ± 0.164 for the denominator, and 0.785 ± 0.144 for
the ratio (P < 0.02 for ratio vs. denominator or
numerator, paired 2-tailed t-test).
Effects of photobleaching, i.e., a slow decrease in emission, were
evident in long-duration recordings obtained with the
two-photomultiplier tube system and with the spectrofluorometer system
as seen in Fig. 5. Results from both systems indicated that the
ratiometry decreased drift but did not fully eliminate it. This may
occur if the fractional decrease in emission due to drift is different for different wavelengths. In all seven trials with the
spectrofluorometer in two endocardially prefrozen hearts that did not
receive diacetyl monoxime, the emission at wavelengths of 532-554
nm (green) decreased 53 ± 18% in 120 s, whereas the
emission at wavelengths of 663-685 (red) decreased 41 ± 16%
(P = 0.011, for red vs. green, paired 2-tailed
t-test). The ratio of the two emission signals (green/red) decreased only 18 ± 13% (P < 0.015 for value
vs. decrease in red or green emission, paired 2-tailed
t-test).
The signals obtained with ratiometry may depend on the choice of
emission wavelengths that are collected. To understand this, we
examined the emission spectrum and its change during an action potential or potassium bolus. Figure 7
shows the results for an action potential. The diastolic emission
spectrum is shown (Fig. 7A). Also, the changes in emission
(emission after the phase-zero depolarization minus emission before it)
for each diode are shown as a fraction of both the diastolic emission
(Fig. 7B) and RMS noise [i.e., signal-to-noise ratio
(signal/noise), Fig. 7C]. The emission of green
light increased while emission of red light decreased, which is
consistent with action potentials in previous figures.
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Ratiometric measurement of action potential repolarization.
The ability of the ratio to indicate action potential repolarization
was tested in six hearts with simultaneous microelectrode and optical
recordings during perfusion with warm solution containing 15 or 20 mM
diacetyl monoxime. Because multiple trials were performed at some laser
spots, the durations of recordings were shortened to 2-4 s to
lessen photobleaching. Figure 10 shows
recordings of the ratio and transmembrane voltage during pacing at
intervals of 150-1,250 ms in one heart. Ratio and microelectrode
signals indicate similar action potential contours and durations for a given pacing interval. Even though activation time at the laser recording spot differed from that at the microelectrode tip by 3.8 ± 1 ms as expected due to their spatial separation, the plots of ratio
versus transmembrane voltage indicate a linear relationship between the
two signals during most of the action potential duration.
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0.05 for ratiometric or microelectrode measurements at 500 ms vs.
those at 150 ms, paired 2-tail t-tests).
The effect of a potassium bolus on the action potential duration was
recorded with the two-photomultiplier tube system in two hearts
perfused with warm solution containing 15 mM diacetyl monoxime. The
APD75 measured ratiometrically at a single laser spot or
with microelectrodes changed from 159 ± 25 ms to 102 ± 8 ms
when potassium was introduced (P = 0.005, paired
2-tailed t-test, n = 4 hearts × recording methods). Two-way analysis of variance of the
APD75 indicated P = 0.022 for an effect of
the potassium and P = 0.65 for an effect of the
recording method (i.e., microelectrode vs. ratiometry).
Calibration of ratiometric measurements.
The calibration factor between the ratio and the transmembrane voltage
during perfusion with solution containing normal potassium was
determined in the six hearts in which microelectrode recordings were
obtained (of which 2 were studied with the 2-photomultiplier tube
system and 4 were studied with the spectrofluorometer). For spectrofluorometric measurements, emission wavelength bands of 510-576 nm and 598-751 nm were used for the numerator and
denominator, respectively, which were wavelengths that produced large
fractional changes and low noise in the ratio described. When all
recordings were included, the calibration slope depended on the order
of the recording in each heart (P = 0.03, analysis of
variance, n = 30). A linear regression of the
calibration slope versus the order of the recording indicated that the
calibration slope decreased at an average rate of 0.0058 per recording.
This decrease may be due to dye internalization or photobleaching
(14). To estimate the calibration slope when these effects
are minimized, only a single ratiometric recording from each heart was
included. The criterion for selection was that the recording had the
largest Ratio/Ratio found in the heart, which consistently occurred
for recordings obtained within 20 min after staining the heart with d-4-ANEPPS.
85 ± 6 mV and 18 ± 7 mV
(P > 0.6 for hearts studied with photomultiplier tubes
versus hearts studied with spectrofluorometer, nonpaired 2-tail
t-test). In the two hearts studied with photomultiplier
tubes for which gains were initially set to produce a ratio of unity,
ratios before and after phase-zero depolarization were 0.98 ± 0.03 and 1.05 ± 0.02, and the calibration slope was (0.071 ± 0.017)/100 mV. In the four hearts studied with the
spectrofluorometer, ratios before and after phase-zero depolarization
were 0.21 ± 0.03 and 0.23 ± 0.04, and the calibration slope
was (0.017 ± 0.002)/100 mV.
Reduction of effects of laser noise.
The ability of emission ratiometry to reduce common mode noise due to
fluctuations of the excitation light intensity was studied by disabling
a regulator in the laser power supply that controlled laser light
intensity. Under this condition, the peak-to-peak magnitude of periodic
laser intensity fluctuations at a rate of 60 Hz was 1.7% of the
average laser intensity. For green and red emission signals, 60-Hz
fluctuations were approximately half as large as the deflections
produced during the phase-zero depolarization, as shown in Fig.
12, A and B.
However, for the ratio of these signals (Fig. 12C), any
60-Hz fluctuations were much smaller than the phase-zero depolarization.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Emission ratiometry for transmembrane voltage measurements in hearts. The results indicate that emission ratiometry reduces drift and effects of epicardial motion, which is indicated in individual recordings (Figs. 4-6 and 12), in the measurements of drift over a 2-min period and in the decreased coefficient of variation. The ratiometry allows optical recordings of cardiac repolarization without requiring electromechanical uncoupling drugs. This is a potentially important advantage because the drugs can have undesired effects in myocardium (3, 16, 25, 26). Also ratiometry reduces drift, which affects long-duration recordings. Whereas slow changes in transmembrane potential that occur over a longer time period than that studied here may still be obscured by drift, our results show that ratiometry can reveal changes in transmembrane potential that occur during a potassium bolus.
Photobleaching-induced drift was only partly canceled by ratiometry as evident from drift in the ratio of green light to red light (Fig. 5). The spectrofluorometric results indicated that there was a greater decrease in recorded green emission compared with red. This is not expected if photobleaching only destroys fluorophore molecules, in which case the fractional decrease in emission should be the same for all fluorescence wavelengths. A greater decrease in green emission may result from a different emission spectrum of the remaining fluorophore, possibly due to the environment of the fluorophore, or from a phosphorescence emission at the longer wavelengths. (11, 24) Also, dye internalization may be enhanced with intense laser illumination. If the emission spectrum and photobleaching decay rates differ in different membranes within the cells, this would produce drift of the ratio during photobleaching when dye has internalized, as proposed by Kao, WY, Davis C, Kim YI, and Beach JM (unpublished obervations). Also a change in the membrane environment, if it occurs intracellularly, may alter spectral properties of internalized dye because of changes in the membrane dipole potential (14). The emission ratiometry has potential advantages for studies in which changes in absorption due to chemical effects unrelated to transmembrane voltage may interfere with transmembrane voltage measurements. The ratio is expected to reject effects of changes in absorption because they are common to both green and red emissions. The finding that ratiometry detected similar transmembrane voltage-dependent shifts of emission wavelengths for the cardiac action potential and hyperkalemia indicates that any absorption changes due to potassium were rejected. Regarding the possibility of improved calibration with ratiometry, Bullen and Saggau (5) found that slopes of ratio versus transmembrane voltage were approximately constant among cells; however, variable offset existed in the ratio that required subtraction. They also found that when offset was subtracted, slopes indicated a conversion factor between transmembrane voltage and ratio of 0.015/100 mV, which is similar to our mean slope of 0.017/100 mV found with the spectrofluorometer. The calibration slope may depend on detector sensitivities and optical bandwidths. Whereas gains and bandwidths were constant among all diodes in our spectrofluorometer, they were different in our photomultiplier tubes, which produced a mean slope of 0.071/100 mV when the gains were adjusted to produce an initial ratio of unity. Had our spectrofluorometer diode gains been adjusted to produce an initial ratio of unity (which would require increasing the gains of diodes sensing green light by a factor of ~4.5), then a slope of 0.077/100 mV can be calculated, which is close to the value obtained with our two photomultiplier tubes. Despite similarities in our mean slopes obtained with the spectrofluorometer compared with that reported in neurons by Bullen and Saggau, the variation of slopes among hearts was larger than the variation among neurons. This may be due to nonspecific fluorescence in the heart in which various types of tissue are present, as discussed by Bullen and Saggau. The signal/noise obtained ratiometrically (Fig. 9) is optimal at numerator wavelengths slightly longer than those that give the largest fractional ratio change. This may be due to larger fluorescence emission at the longer numerator wavelengths (Fig. 7), which increases DC level and lessens effects of shot noise. In systems that use two detectors for which the optical bandwidths of numerator and denominator signals can be selected a priori, the shot noise should decrease when a wider optical bandwidth is used. This is expected to increase signal/noise if the additional wavelengths add to the change in ratio. For the denominator, wavelengths will add to the signal in a wide band of approximately >600 nm (Fig. 8). For the numerator, the widest possible band is bounded by the excitation wavelength and ~580 nm, above which wavelengths add no fractional change in the ratio. However, a narrower numerator band gives a larger fractional change and signal/ noise by excluding wavelengths that have a small change (Figs. 8 and 9). Also, neither band needs to be wide if there is sufficient light intensity to overcome shot noise, because the largest signal/noise was obtained with narrow numerator and denominator bands near 550 and 680 nm. Another issue impacting on the choice of bands is that nonspecific fluorescence from surrounding cellular structures or dye bound in nonspecific orientation may be rejected when optical bandwidth is small (5). Motion artifacts in optical action potential recordings may have multiple causes that include possible changes in light transport properties (reflectance, scattering, or absorption of light) at the excitation and emission wavelengths due to stretching of the tissue and translational movement of the tissue when the light transport properties or dye distribution among voltage-sensing and nonspecific environments are heterogeneous. Ratiometry may eliminate distortion of the action potential due to changes in light transport properties provided that identical changes occur for the two emission wavelength bands used for the numerator and denominator. Even when ratiometry eliminates visible distortion in the optically recorded action potential, translational movement would cause different phases of an action potential to be recorded from different groups of cells. This limitation is potentially important in cases of heterogeneous repolarization and resting potential. In these cases, ratiometry may be advantageous with endocardially prefrozen or mechanically stabilized hearts that already have motion lessened by contact with adjacent underlying necrotic tissue or external stabilizers.Laser scanner method for ratiometry.
Recording systems in which spatial resolution of the recording is
determined by a narrow excitation laser beam have potential advantages
for emission ratiometry in hearts as well as microscopy (5,
7). The fact that localization of a recording is controlled by
the excitation beam ensures that both red and green emission signals
are collocal. For systems in which broad-field excitation is used and
spatial resolution is determined by locations of detectors in image
planes, collocal ratiometric recordings depend on exact alignment of
detectors (23). Effects of laser intensity fluctuations, which have been a limitation of laser scanner systems and required additional methods for cancellation (15, 19), are reduced by emission ratiometry because the effect is common to numerator and
denominator signals (Fig. 12). Also, the use of a narrow laser excitation bandwidth (~10
5 nm) allows short emission
wavelengths (e.g., 500 nm, Fig. 8) to be included in the numerator if necessary.
The emission spectrum in hearts. The emission spectrum and its change during depolarization in hearts are qualitatively similar to previous measurements of di-4-ANEPPS emission in other preparations. For Retzius cells from the leech, Fromherz and Lambacher (11) found an emission peak at 604 nm and emission crossover at 575 nm for excitation at 482 nm with a positive change of ~6% in emission at 533 nm and a negative change of 5% at 636 nm for 100 mV of depolarization. For hemispherical bilayers, Fluhler et al. (10) found an emission peak at 640 nm and emission crossover at 620 nm with a positive change of 7.6% at 540 nm and a negative change of ~2.2% at 680 nm for 100 mV of depolarization. Our emission peak (Fig. 7) is similar to that of Fluhler et al. (10), whereas our emission crossover for the action potential is similar to that of Fromherz and Lambacher (11). Both of these reports agree with our findings in hearts in which positive changes in green emission and negative changes in red emission occur during membrane depolarization.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institutes of Health Grant HL-52003 and American Heart Association Grant 9740173N. S. B. Knisley is an Established Investigator Awardee of the American Heart Association.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. B. Knisley, The Univ. of Alabama at Birmingham, B122 Volker Hall, 1670 University Blvd., Birmingham, AL 35294-0019 (E-mail: sbk{at}crml.uab.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 8 November 1999; accepted in final form 8 March 2000.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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