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Optical Studies of Excitation‐Contraction Coupling Using Voltage‐Sensitive and Calcium‐Sensitive Probes

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Abstract

The sections in this article are:

1 Optical Signals Sensitive to Changes in Surface Potential and T‐System Potential
1.1 Intrinsic Birefringence Signal: First Component
1.2 Signals From Impermeant Potentiometric Dyes
2 Optical Signals Possibly Sensitive to Changes in Sr Potential
2.1 Intrinsic Birefringence Signal: Second Component
2.2 Signals From Membrane‐Permeant Dyes
2.3 Simultaneous Comparisons of Signals From Birefringence and Membrane‐Permeant Dyes
2.4 Vesicular Preparations
2.5 Use of Impermeant Dyes to Possibly Stain SR Membranes
2.6 Voltage‐Clamp Studies
3 Optical Signals Sensitive to Changes in Myoplasmic Calcium
3.1 Luminescence Response From Aequorin
3.2 Arsenazo III Signals in Intact Fibers
3.3 Charge‐Movement Currents and Antipyrylazo III Signals in Cut Fibers
3.4 Comparison of Signals From Calcium‐Indicator Dyes on the Same Preparation
4 Conclusions
Figure 1. Figure 1.

Kinetics of Ca2+‐murexide complex in toad muscle after action potential stimulation. An increase in transmission is in the upward direction. Vertical lines provide a time scale at every 25 ms. Effects of 4 series of 10 contractions. A: time relations between tension development (upper trace), light scattering (middle trace), and formation of Ca2+‐murexide complex (lower trace). Formation was approximated by subtracting transmission changes at 440 and 505 nm, wavelengths relatively insensitive to Ca, from transmission change at 470 nm, a wavelength corresponding to a peak in Ca2+‐murexide difference spectrum. The subtraction procedure was designed to minimize interference from nonspecific effects. Stimulus delayed 12 ms after start of sweep; 12°C. B: time relation of optical response at 2 wavelengths on opposite sides of isosbestic point for Ca2+‐murexide reaction. Upper trace, 540 nm (relative to 505 and 580 nm); lower trace, 470 nm (relative to 440 and 505 nm). Stimulus delayed 25 ms from start of sweep. Results on 2 preparations from different toads; 9°C–10°C. [From Jöbsis and O'Connor .]

Figure 2. Figure 2.

Simultaneous recordings of action potential (less‐noisy trace) and optical change from squid giant axon. Arrow calibrates fractional change in light intensity, ΔI/I. A: intrinsic birefringence change, i.e., signal detected with axon positioned between crossed polarizers oriented at ±45° to fiber axis. Time constant for optical recording, 24 μs; number of sweeps averaged, 2,030; 14°C. B: extrinsic absorbance change after staining with WW375, a merocyanine‐rhodanine dye. Time constant for optical recording, 5 μs; number of sweeps averaged, 32; room temperature (21°C–23°C); dye exposure, 10 min with a concentration of 0.2 mg/ml. [A from Cohen et al. ; B from Ross et al. .]

Figure 3. Figure 3.

Top: schematic diagram (top and side views) of recording chamber and optical arrangement used for detection of changes in scattering, birefringence, and fluorescence of skeletal muscle fibers of Rana pipiens after electric stimulation. M, muscle fibers; S, stimulating electrodes; R, recording electrodes; W and W, windows for illumination and detection; L, light source; F1, interference filter; F2, sharp‐cut filter; P and A, polarizer and analyzer, respectively; and D (or D′), photomultiplier at 0° (or 90°). Bottom: records of changes in light intensity (lower traces) coincident with externally recorded action potentials (upper traces). Optical signals represent changes in light scattering (A) recorded with photodetector in position D′, changes in birefringence (B) recorded with photodetector in position D and with polarizer and analyzer in place and oriented at ±45° with respect to the fiber axis, and changes in fluorescence of muscle fibers stained with pyronine B (C) recorded with photodetector in position D′ and with F1 and F2 in place (F1 selects dye excitation wavelengths near 550 nm, and F2 selects emission wavelengths beyond 610 nm). Different muscle preparations were used in each record. Upward deflection of lower trace represents an increase in light intensity. CAT computer was used to record both the action potentials and the optical signals. Vertical bars represent an increase of 2 × 10−4 (A and B) and 10−4 (C) times the resting level of illumination. Dye staining with pyronine B involved a 10‐min exposure using a concentration of 0.05 mg/ml. Normal Ringer's solution; 22°C; signal‐averaging was used. [From Camay and Barry . Copyright 1969 by the American Association for the Advancement of Science.]

Figure 4. Figure 4.

Intrinsic birefringence signal from intact single fibers of Rana temporaria. Room temperature (19°C–23°C). A: separation of 1st and 2nd components. Optical recordings of fractional intensity changes from the same fiber in Ringer's solutions of various tonicities superimposed by lining up the stimulus artifact (arrow): normal tonicity (1T), 120 mM NaCl; 2.2T, 270 mM NaCl; 2.6T, 320 mM NaCl; 3T, 370 mM NaCl Ringer's solution. In 1T Ringer's solution the 2 components cannot be distinguished temporally; in 3T Ringer's solution the 1st component, with a time to peak of about 2 ms after stimulation, is kinetically distinct from the second component, which is still rising at end of record. Each trace is the average of 64 sweeps. Optical recording was a 500‐μm field, 1.5 mm from stimulus cathode. B: optical (o) and intracellular voltage (v) recordings taken simultaneously in 3T Ringer's solution (370 mM NaCl). Optical trace is inverted to facilitate comparison with action potential. Upper vertical calibration refers to the optical trace, and the lower to the potential trace, each of which is an average of 300 sweeps. Optical recording was 7 mm from stimulus cathode. [From Baylor and Oetliker .]

Figure 5. Figure 5.

A: simultaneous recordings of optical signals (upper trace) and intracellularly recorded action potentials (lower trace) in a single muscle fiber of Xenopus laevis stained with WW375 (100 μg/ml) for 15 min. Optical signal was signal‐averaged by an averager, and action potential represents the average of the photographs of the 4 spikes. Optical calibrations refer to ΔI/I; the wavelength was 702 nm; slit size = fiber diameter × 500 μm. Arrows indicate start of massive stimulation. Fiber diameter, 108 μm; sarcomere length, 2.6 μm; room temperature (25°C). Response time constant of optical system was 25 μs and that of the electrical system was 30 μs. B: estimation of radial conduction velocity of tubular action potential by analysis of simultaneously recorded optical and electrical signals. Continuous curve is the best‐fit optical signal calculated from the experimentally determined electrical signal (action potential) obtained by adjusting values of access delay and radial conduction velocity. Filled circles, recorded optical signal. Normalizing factors were introduced to the continuous curve to make height approximately the same as optical signal. Data are from experiment illustrated in A. [From Nakajima and Gilai .]

Figure 6. Figure 6.

Wavelength and polarization dependence of the WW375 transmission change from a single fiber of Rana temporaria after action potential stimulation in 3T D2O Ringer's solution. A: original records of fractional changes in light intensity. 0° indicates that light polarized at 0° to fiber's axis was used, and 90° indicates that light polarized at 90° to fiber's axis was used. Wavelength is indicated alongside the 0° traces. M is tension response. Optical traces obtained with a 27 × 300‐μn slit and traces signal‐averaged as follows: 570 nm, 0°, 64 times; 690 nm, 0°, 32 times; 750 nm, 0°, 8 times; 570 nm, 90°, 32 times; 690 nm, 90°, 16 times; 750 nm, 90°, 4 times. B: results of fitting 570‐nm, 0° waveform by scaling the other optical traces shown in A. The least‐squares fitting constants are shown alongside each optical trace. The 570‐nm, 90° trace and the 690‐nm, 90° trace are noisy traces. The 570‐nm, 0° trace is not well fitted by their scaled versions, indicating more than a single temporal process. [From Baylor et al. .]

Figure 7. Figure 7.

Simultaneous recordings of optical signals and action potentials after staining of cut single fibers of Rana catesbeiana with impermeant potentiometric dyes. In both parts of the figure the noisier records are optical traces. A: absorbance change from WW375 (1 mg/ml) obtained with 750‐nm light at 10°C. B: fluorescence change from WW781 (approx. 0.5 mg/ml). Average of 4 sweeps. Excitation was with 625‐nm light; fluorescence was measured with a 665‐nm, cut‐on filter at 10°C. [From Vergara and Bezanilla .]

Figure 8. Figure 8.

Nile blue fluorescence change from whole muscle of Rana pipiens after action potential stimulation. A: external action currents (upper trace) and fluorescence signal (lower trace) in response to one supramaximal stimulus. Time calibration bar is 50 ms; fluorescence calibration bar is 2 mV. Resting fluorescence, 0.78 V. Temperature, 17.9°C. B: relation of intracellular action potential to fluorescence signal. Upper trace, external 0‐potential reference; middle trace, fluorescence signal; lower trace, internal potential. Time calibration bar is 2 ms. Calibration for internal potential is 25 mV and for fluorescence is 2 mV. Resting fluorescence, 0.9 V; 25°C. C: time relation of fluorescence to mechanical response. Upper trace, fluorescence signal in response to single stimulus; lower trace, tension response. Time calibration bar is 10 ms for both traces; tension calibration bar is 28 mg; fluorescence calibration bar is 10 mV; 23.5°C. [From Bezanilla and Horowicz .]

Figure 9. Figure 9.

Temporal comparison of intrinsic birefringence signal and fluorescence signals from single muscle fibers of Rana temporaria stained with membrane‐permeant dyes. In both experiments action potentials were elicited by an external shock occurring at 0 ms on the horizontal time calibration. In both parts of figure, ΔI/I calibration applies to birefringence and ΔF/F calibration applies to fluorescence trace. A: upper traces show birefringence and Nile blue signals. Birefringence signal peaks earlier than Nile blue signal. Birefringence recorded in Ringer's solution before dye; fluorescence recorded after exposing fiber to Ringer's solution containing 0.5 μg/ml Nile blue. Birefringence was obtained with white light and signal‐averaged twice. Nile blue was a single sweep, obtained with a 570‐nm primary filter, 90° polarized light, and a 645‐nm secondary filter. Lower traces show tension records; larger response was recorded after dye and shows that Nue blue caused a small potentiation of the twitch. B: upper traces show birefringence and downward cyanine signals; lower traces show tension. Birefringence signal decays more rapidly than cyanine signal. Birefringence recorded in Ringer's solution before and fluorescence recorded after adding 1 μg/ml of cyanine dye. Birefringence trace was signal‐averaged twice and obtained with a 570‐nm filter. Cyanine signal, a single sweep, was obtained with a 570‐nm primary filter, unpolarized light, and 695‐nm secondary filter. [From Baylor et al. .]

Figure 10. Figure 10.

Upper traces, temporal comparison of intrinsic birefringence signal and fluorescence signal due to the injection of impermeant dye WW781 (intact single fiber from Rana temporaria) Birefringence obtained after WW781 had been iontophoresed into myoplasm. Birefringence trace (single sweep) was obtained with white light and is plotted upside down; fluorescence trace (signal‐averaged 31 times) obtained with 630‐nm primary filter, unpolarized light, and 695‐nm secondary filter. Lower trace, tension. Field of optical recording, 300 μm; fiber diameter, 80 μm; sarcomere spacing, 4.5 μn; normal Ringer's solution; 19°C. [From Baylor et al. .]

Figure 11. Figure 11.

Absorbance change from a skinned fiber of Xenopus laevis stained with impermeant dye NK2367 (0.2 mg/ml) as a function of changes in bath K+ concentration after exposure of fiber to valinomycin. Scale on abscissa has been converted to mV by Nernst relationship, under the assumption that the SR membrane potential behaves as a potassium electrode. A straight‐line relationship has been fit to the data by method of least squares. [From Best et al. .]

Figure 12. Figure 12.

Changes in intrinsic transmission from a cut fiber of Rana pipiens as a function of pulse amplitude under voltage‐clamp conditions. Fiber was exposed to tetrodotoxin (TTX) and tetraethylammonium (TEA) at 3.9°C. A: ΔI/I records for 60‐ms pulses to the following values of membrane potential (mV): a, −165; b, −44; c, −35; d, −25; e, −16; f, −7; g, +3. Each record is a single sweep. Horizontal calibration bar represents 60 ms; vertical calibration bar represents 4 × 10−4. B: maximum ΔI/I as a function of membrane potential during pulse for records in A. [From Kovacs and Schneider .]

Figure 13. Figure 13.

Fluorescence signals from a cut fiber of Rana catesbeiana stained with Nile blue (0.5–2 μg/ml) as a function of pulse amplitude under voltage‐clamp conditions. A: 1st trace, sample voltage pulse of 125 mV; 2nd trace, sample current for 125‐mV pulse, 3rd–8th traces, fluorescence signals associated with depolarizing voltage steps of the magnitudes indicated from a holding potential of −100 mV. Fiber was exposed to a Na‐free, 115 mM tetramethylammonium solution; 15°C. B: peak amplitude of fluorescence change in A as a function of membrane potential during pulse. Circles are experimental peak values of ΔF/F plotted as a function of absolute membrane potential; dashed line corresponds to ΔF/F = 0.0205/[1 + exp([V + 35]/9)]; continuous curve corresponds to ΔF/F = 0.0462/[1 + 1.2(1 + exp[−(V + 40)/12])2], where V = absolute membrane potential (mV). [From Vergara et al. .]

Figure 14. Figure 14.

A: birefringence signals near contractile threshold in a voltage‐clamp experiment on a highly stretched single fiber of Rana temporaria exposed to TTX and TEA. Upper traces show 2 voltage pulses—100 ms and 60 ms in duration; middle traces show birefringence records; lower traces show superimposed tension records. All traces are single sweeps. Dashed vertical lines mark beginning and end of the pulses. Sarcomere spacing was 3.9 μ at site of optical recording in the middle of the fiber. Illumination was with a narrow transverse slit (about 60 μm wide) of white light positioned about 50 μm from voltage‐sensing microelectrode. Resting potential of fiber was −90 mV; holding potential was −100 mV. Numbers to the left of optical traces indicate membrane potential (mV) during pulse; 10°C. B: logarithmic plot of maximum rate of change of birefringence signal vs. membrane potential during pulse. Solid circles, from records in A; open circles, from a bracketing record during run. Straight line was drawn by eye; its slope corresponds to an e‐fold change per 2.5 mV. Scale for ordinate has been arbitrarily shifted. [From Baylor and Chandler .]

Figure 15. Figure 15.

Luminescent and mechanical responses in isometric twitches from a single fiber of Rana temporaria injected with aequorin. Striation spacing, 2.3 μm; temperature, 10°C. A: records of light (noisy trace) and force from a single rested‐state contraction. B: 7 such twitches have been averaged to reduce photomultiplier shot noise. Time of stimulus is indicated by the vertical mark below the base line. (Note: recent improvements in techniques of light collection now allow a single‐sweep signal‐to‐noise ratio for aequorin response comparable to that seen for averaged response in B—see, e.g., ref. ) [From Blinks et al. .]

Figure 16. Figure 16.

Absorbance changes from a costocutaneous muscle fiber of Rana temporaria injected with arsenazo III. A: action potential conditions; 7.5°C. Upper trace is (minus) the absorbance record at 532 nm. Middle trace is the absorbance change at 602 nm minus the change at 532 nm, with the difference normalized by the resting absorbance at 532 nm. Subtraction procedure is designed so nonspecific changes tend to cancel, whereas changes on opposite sides of the isosbestic wavelength (approx. 570 nm) will summate. Lower trace, intracellularly recorded action potential. B: analysis of voltage‐clamp records; 7°C. Relation between membrane potential and amplitude of the differentially recorded (602 nm relative to 532 nm) and normalized optical signal for a depolarizing pulse of 10‐ms duration to the level indicated. Holding potential, −75 mV. [From Miledi et al. .]

Figure 17. Figure 17.

Changes in transmission recorded from highly stretched intact single fibers of Rana temporaria injected with relatively high concentrations of arsenazo III. A: action potential conditions. Upper traces show superimposed optical records taken at wavelengths indicated with light polarized transversely (90°) to the fiber axis. Lower trace is the residual tension response. External shock occurred at 0 ms on the horizontal time axis. Sarcomere spacing, 4.0 μm; 16 °C; normal Ringer's solution; 0.6–0.8 mM arsenazo III. B: voltage‐clamp conditions. Upper trace shows a sample 200‐ms voltage pulse. Middle trace is the transmission change at 720 nm, which is representative of non‐dye‐related effects. Lower traces are superimposed transmission changes at 660 nm for steps of potential to the levels indicated. Holding potential, −100 mV; sarcomere spacing, 3.9 μm; 16°C; D2O Ringer's solution; 0.8 mM arsenazo III; 90° polarized light. [From Baylor et al. .]

Figure 18. Figure 18.

Voltage dependence of Ca transients and intramembrane charge movement for a cut fiber of Rana pipiens exposed to TTX and TEA. A: superimposed ΔA signals at 720 nm for 100‐ms depolarizing pulses to indicated values of membrane potential (mV). Each record is average of 6 determinations with no correction for fiber movement. Calibration bar corresponds to ΔA720/A550 of 1.4 × 10−2. Pulse is diagrammed below records. B: records of intramembrane charge‐movement currents for same pulses as in A. Vertical calibration denotes 3.2 μA/μF, and the horizontal calibration denotes 30 ms; holding potential, −100 mV; sarcomere spacing, 3.5 μm; 3°C. Concentration of antipyrylazo III was 1 mM. [From Kovacs et al. .]

Figure 19. Figure 19.

Absorbance signals from cut fibers of Rana temporaria and Rana catesbeiana exposed to Ca‐indicator dyes. A: action potential conditions with arsenazo III; 21°C. Superimposed traces show the electrically recorded action potential (earliest deflection, left calibration) and absorbance records at indicated wavelengths (later deflections, right calibration). B: voltage‐clamp conditions with 1 mM antipyrylazo III; 10°C. Ringer's solution without TTX. Horizontal calibration is 50 ms; vertical calibration is 0.02 units of the absorbance change at 710 nm minus the fractional transmission change at 790 nm and normalized by the resting absorbance at 550 nm. C: voltage‐clamp conditions with arsenazo III; 10°C. Ringer's solution without TTX. Horizontal calibration is 50 ms; vertical calibration is 0.2 units of the absorbance change at 660 nm minus the fractional transmission change at 740 nm and normalized by the resting absorbance at 532 nm. [From Palade and Vergara .]



Figure 1.

Kinetics of Ca2+‐murexide complex in toad muscle after action potential stimulation. An increase in transmission is in the upward direction. Vertical lines provide a time scale at every 25 ms. Effects of 4 series of 10 contractions. A: time relations between tension development (upper trace), light scattering (middle trace), and formation of Ca2+‐murexide complex (lower trace). Formation was approximated by subtracting transmission changes at 440 and 505 nm, wavelengths relatively insensitive to Ca, from transmission change at 470 nm, a wavelength corresponding to a peak in Ca2+‐murexide difference spectrum. The subtraction procedure was designed to minimize interference from nonspecific effects. Stimulus delayed 12 ms after start of sweep; 12°C. B: time relation of optical response at 2 wavelengths on opposite sides of isosbestic point for Ca2+‐murexide reaction. Upper trace, 540 nm (relative to 505 and 580 nm); lower trace, 470 nm (relative to 440 and 505 nm). Stimulus delayed 25 ms from start of sweep. Results on 2 preparations from different toads; 9°C–10°C. [From Jöbsis and O'Connor .]



Figure 2.

Simultaneous recordings of action potential (less‐noisy trace) and optical change from squid giant axon. Arrow calibrates fractional change in light intensity, ΔI/I. A: intrinsic birefringence change, i.e., signal detected with axon positioned between crossed polarizers oriented at ±45° to fiber axis. Time constant for optical recording, 24 μs; number of sweeps averaged, 2,030; 14°C. B: extrinsic absorbance change after staining with WW375, a merocyanine‐rhodanine dye. Time constant for optical recording, 5 μs; number of sweeps averaged, 32; room temperature (21°C–23°C); dye exposure, 10 min with a concentration of 0.2 mg/ml. [A from Cohen et al. ; B from Ross et al. .]



Figure 3.

Top: schematic diagram (top and side views) of recording chamber and optical arrangement used for detection of changes in scattering, birefringence, and fluorescence of skeletal muscle fibers of Rana pipiens after electric stimulation. M, muscle fibers; S, stimulating electrodes; R, recording electrodes; W and W, windows for illumination and detection; L, light source; F1, interference filter; F2, sharp‐cut filter; P and A, polarizer and analyzer, respectively; and D (or D′), photomultiplier at 0° (or 90°). Bottom: records of changes in light intensity (lower traces) coincident with externally recorded action potentials (upper traces). Optical signals represent changes in light scattering (A) recorded with photodetector in position D′, changes in birefringence (B) recorded with photodetector in position D and with polarizer and analyzer in place and oriented at ±45° with respect to the fiber axis, and changes in fluorescence of muscle fibers stained with pyronine B (C) recorded with photodetector in position D′ and with F1 and F2 in place (F1 selects dye excitation wavelengths near 550 nm, and F2 selects emission wavelengths beyond 610 nm). Different muscle preparations were used in each record. Upward deflection of lower trace represents an increase in light intensity. CAT computer was used to record both the action potentials and the optical signals. Vertical bars represent an increase of 2 × 10−4 (A and B) and 10−4 (C) times the resting level of illumination. Dye staining with pyronine B involved a 10‐min exposure using a concentration of 0.05 mg/ml. Normal Ringer's solution; 22°C; signal‐averaging was used. [From Camay and Barry . Copyright 1969 by the American Association for the Advancement of Science.]



Figure 4.

Intrinsic birefringence signal from intact single fibers of Rana temporaria. Room temperature (19°C–23°C). A: separation of 1st and 2nd components. Optical recordings of fractional intensity changes from the same fiber in Ringer's solutions of various tonicities superimposed by lining up the stimulus artifact (arrow): normal tonicity (1T), 120 mM NaCl; 2.2T, 270 mM NaCl; 2.6T, 320 mM NaCl; 3T, 370 mM NaCl Ringer's solution. In 1T Ringer's solution the 2 components cannot be distinguished temporally; in 3T Ringer's solution the 1st component, with a time to peak of about 2 ms after stimulation, is kinetically distinct from the second component, which is still rising at end of record. Each trace is the average of 64 sweeps. Optical recording was a 500‐μm field, 1.5 mm from stimulus cathode. B: optical (o) and intracellular voltage (v) recordings taken simultaneously in 3T Ringer's solution (370 mM NaCl). Optical trace is inverted to facilitate comparison with action potential. Upper vertical calibration refers to the optical trace, and the lower to the potential trace, each of which is an average of 300 sweeps. Optical recording was 7 mm from stimulus cathode. [From Baylor and Oetliker .]



Figure 5.

A: simultaneous recordings of optical signals (upper trace) and intracellularly recorded action potentials (lower trace) in a single muscle fiber of Xenopus laevis stained with WW375 (100 μg/ml) for 15 min. Optical signal was signal‐averaged by an averager, and action potential represents the average of the photographs of the 4 spikes. Optical calibrations refer to ΔI/I; the wavelength was 702 nm; slit size = fiber diameter × 500 μm. Arrows indicate start of massive stimulation. Fiber diameter, 108 μm; sarcomere length, 2.6 μm; room temperature (25°C). Response time constant of optical system was 25 μs and that of the electrical system was 30 μs. B: estimation of radial conduction velocity of tubular action potential by analysis of simultaneously recorded optical and electrical signals. Continuous curve is the best‐fit optical signal calculated from the experimentally determined electrical signal (action potential) obtained by adjusting values of access delay and radial conduction velocity. Filled circles, recorded optical signal. Normalizing factors were introduced to the continuous curve to make height approximately the same as optical signal. Data are from experiment illustrated in A. [From Nakajima and Gilai .]



Figure 6.

Wavelength and polarization dependence of the WW375 transmission change from a single fiber of Rana temporaria after action potential stimulation in 3T D2O Ringer's solution. A: original records of fractional changes in light intensity. 0° indicates that light polarized at 0° to fiber's axis was used, and 90° indicates that light polarized at 90° to fiber's axis was used. Wavelength is indicated alongside the 0° traces. M is tension response. Optical traces obtained with a 27 × 300‐μn slit and traces signal‐averaged as follows: 570 nm, 0°, 64 times; 690 nm, 0°, 32 times; 750 nm, 0°, 8 times; 570 nm, 90°, 32 times; 690 nm, 90°, 16 times; 750 nm, 90°, 4 times. B: results of fitting 570‐nm, 0° waveform by scaling the other optical traces shown in A. The least‐squares fitting constants are shown alongside each optical trace. The 570‐nm, 90° trace and the 690‐nm, 90° trace are noisy traces. The 570‐nm, 0° trace is not well fitted by their scaled versions, indicating more than a single temporal process. [From Baylor et al. .]



Figure 7.

Simultaneous recordings of optical signals and action potentials after staining of cut single fibers of Rana catesbeiana with impermeant potentiometric dyes. In both parts of the figure the noisier records are optical traces. A: absorbance change from WW375 (1 mg/ml) obtained with 750‐nm light at 10°C. B: fluorescence change from WW781 (approx. 0.5 mg/ml). Average of 4 sweeps. Excitation was with 625‐nm light; fluorescence was measured with a 665‐nm, cut‐on filter at 10°C. [From Vergara and Bezanilla .]



Figure 8.

Nile blue fluorescence change from whole muscle of Rana pipiens after action potential stimulation. A: external action currents (upper trace) and fluorescence signal (lower trace) in response to one supramaximal stimulus. Time calibration bar is 50 ms; fluorescence calibration bar is 2 mV. Resting fluorescence, 0.78 V. Temperature, 17.9°C. B: relation of intracellular action potential to fluorescence signal. Upper trace, external 0‐potential reference; middle trace, fluorescence signal; lower trace, internal potential. Time calibration bar is 2 ms. Calibration for internal potential is 25 mV and for fluorescence is 2 mV. Resting fluorescence, 0.9 V; 25°C. C: time relation of fluorescence to mechanical response. Upper trace, fluorescence signal in response to single stimulus; lower trace, tension response. Time calibration bar is 10 ms for both traces; tension calibration bar is 28 mg; fluorescence calibration bar is 10 mV; 23.5°C. [From Bezanilla and Horowicz .]



Figure 9.

Temporal comparison of intrinsic birefringence signal and fluorescence signals from single muscle fibers of Rana temporaria stained with membrane‐permeant dyes. In both experiments action potentials were elicited by an external shock occurring at 0 ms on the horizontal time calibration. In both parts of figure, ΔI/I calibration applies to birefringence and ΔF/F calibration applies to fluorescence trace. A: upper traces show birefringence and Nile blue signals. Birefringence signal peaks earlier than Nile blue signal. Birefringence recorded in Ringer's solution before dye; fluorescence recorded after exposing fiber to Ringer's solution containing 0.5 μg/ml Nile blue. Birefringence was obtained with white light and signal‐averaged twice. Nile blue was a single sweep, obtained with a 570‐nm primary filter, 90° polarized light, and a 645‐nm secondary filter. Lower traces show tension records; larger response was recorded after dye and shows that Nue blue caused a small potentiation of the twitch. B: upper traces show birefringence and downward cyanine signals; lower traces show tension. Birefringence signal decays more rapidly than cyanine signal. Birefringence recorded in Ringer's solution before and fluorescence recorded after adding 1 μg/ml of cyanine dye. Birefringence trace was signal‐averaged twice and obtained with a 570‐nm filter. Cyanine signal, a single sweep, was obtained with a 570‐nm primary filter, unpolarized light, and 695‐nm secondary filter. [From Baylor et al. .]



Figure 10.

Upper traces, temporal comparison of intrinsic birefringence signal and fluorescence signal due to the injection of impermeant dye WW781 (intact single fiber from Rana temporaria) Birefringence obtained after WW781 had been iontophoresed into myoplasm. Birefringence trace (single sweep) was obtained with white light and is plotted upside down; fluorescence trace (signal‐averaged 31 times) obtained with 630‐nm primary filter, unpolarized light, and 695‐nm secondary filter. Lower trace, tension. Field of optical recording, 300 μm; fiber diameter, 80 μm; sarcomere spacing, 4.5 μn; normal Ringer's solution; 19°C. [From Baylor et al. .]



Figure 11.

Absorbance change from a skinned fiber of Xenopus laevis stained with impermeant dye NK2367 (0.2 mg/ml) as a function of changes in bath K+ concentration after exposure of fiber to valinomycin. Scale on abscissa has been converted to mV by Nernst relationship, under the assumption that the SR membrane potential behaves as a potassium electrode. A straight‐line relationship has been fit to the data by method of least squares. [From Best et al. .]



Figure 12.

Changes in intrinsic transmission from a cut fiber of Rana pipiens as a function of pulse amplitude under voltage‐clamp conditions. Fiber was exposed to tetrodotoxin (TTX) and tetraethylammonium (TEA) at 3.9°C. A: ΔI/I records for 60‐ms pulses to the following values of membrane potential (mV): a, −165; b, −44; c, −35; d, −25; e, −16; f, −7; g, +3. Each record is a single sweep. Horizontal calibration bar represents 60 ms; vertical calibration bar represents 4 × 10−4. B: maximum ΔI/I as a function of membrane potential during pulse for records in A. [From Kovacs and Schneider .]



Figure 13.

Fluorescence signals from a cut fiber of Rana catesbeiana stained with Nile blue (0.5–2 μg/ml) as a function of pulse amplitude under voltage‐clamp conditions. A: 1st trace, sample voltage pulse of 125 mV; 2nd trace, sample current for 125‐mV pulse, 3rd–8th traces, fluorescence signals associated with depolarizing voltage steps of the magnitudes indicated from a holding potential of −100 mV. Fiber was exposed to a Na‐free, 115 mM tetramethylammonium solution; 15°C. B: peak amplitude of fluorescence change in A as a function of membrane potential during pulse. Circles are experimental peak values of ΔF/F plotted as a function of absolute membrane potential; dashed line corresponds to ΔF/F = 0.0205/[1 + exp([V + 35]/9)]; continuous curve corresponds to ΔF/F = 0.0462/[1 + 1.2(1 + exp[−(V + 40)/12])2], where V = absolute membrane potential (mV). [From Vergara et al. .]



Figure 14.

A: birefringence signals near contractile threshold in a voltage‐clamp experiment on a highly stretched single fiber of Rana temporaria exposed to TTX and TEA. Upper traces show 2 voltage pulses—100 ms and 60 ms in duration; middle traces show birefringence records; lower traces show superimposed tension records. All traces are single sweeps. Dashed vertical lines mark beginning and end of the pulses. Sarcomere spacing was 3.9 μ at site of optical recording in the middle of the fiber. Illumination was with a narrow transverse slit (about 60 μm wide) of white light positioned about 50 μm from voltage‐sensing microelectrode. Resting potential of fiber was −90 mV; holding potential was −100 mV. Numbers to the left of optical traces indicate membrane potential (mV) during pulse; 10°C. B: logarithmic plot of maximum rate of change of birefringence signal vs. membrane potential during pulse. Solid circles, from records in A; open circles, from a bracketing record during run. Straight line was drawn by eye; its slope corresponds to an e‐fold change per 2.5 mV. Scale for ordinate has been arbitrarily shifted. [From Baylor and Chandler .]



Figure 15.

Luminescent and mechanical responses in isometric twitches from a single fiber of Rana temporaria injected with aequorin. Striation spacing, 2.3 μm; temperature, 10°C. A: records of light (noisy trace) and force from a single rested‐state contraction. B: 7 such twitches have been averaged to reduce photomultiplier shot noise. Time of stimulus is indicated by the vertical mark below the base line. (Note: recent improvements in techniques of light collection now allow a single‐sweep signal‐to‐noise ratio for aequorin response comparable to that seen for averaged response in B—see, e.g., ref. ) [From Blinks et al. .]



Figure 16.

Absorbance changes from a costocutaneous muscle fiber of Rana temporaria injected with arsenazo III. A: action potential conditions; 7.5°C. Upper trace is (minus) the absorbance record at 532 nm. Middle trace is the absorbance change at 602 nm minus the change at 532 nm, with the difference normalized by the resting absorbance at 532 nm. Subtraction procedure is designed so nonspecific changes tend to cancel, whereas changes on opposite sides of the isosbestic wavelength (approx. 570 nm) will summate. Lower trace, intracellularly recorded action potential. B: analysis of voltage‐clamp records; 7°C. Relation between membrane potential and amplitude of the differentially recorded (602 nm relative to 532 nm) and normalized optical signal for a depolarizing pulse of 10‐ms duration to the level indicated. Holding potential, −75 mV. [From Miledi et al. .]



Figure 17.

Changes in transmission recorded from highly stretched intact single fibers of Rana temporaria injected with relatively high concentrations of arsenazo III. A: action potential conditions. Upper traces show superimposed optical records taken at wavelengths indicated with light polarized transversely (90°) to the fiber axis. Lower trace is the residual tension response. External shock occurred at 0 ms on the horizontal time axis. Sarcomere spacing, 4.0 μm; 16 °C; normal Ringer's solution; 0.6–0.8 mM arsenazo III. B: voltage‐clamp conditions. Upper trace shows a sample 200‐ms voltage pulse. Middle trace is the transmission change at 720 nm, which is representative of non‐dye‐related effects. Lower traces are superimposed transmission changes at 660 nm for steps of potential to the levels indicated. Holding potential, −100 mV; sarcomere spacing, 3.9 μm; 16°C; D2O Ringer's solution; 0.8 mM arsenazo III; 90° polarized light. [From Baylor et al. .]



Figure 18.

Voltage dependence of Ca transients and intramembrane charge movement for a cut fiber of Rana pipiens exposed to TTX and TEA. A: superimposed ΔA signals at 720 nm for 100‐ms depolarizing pulses to indicated values of membrane potential (mV). Each record is average of 6 determinations with no correction for fiber movement. Calibration bar corresponds to ΔA720/A550 of 1.4 × 10−2. Pulse is diagrammed below records. B: records of intramembrane charge‐movement currents for same pulses as in A. Vertical calibration denotes 3.2 μA/μF, and the horizontal calibration denotes 30 ms; holding potential, −100 mV; sarcomere spacing, 3.5 μm; 3°C. Concentration of antipyrylazo III was 1 mM. [From Kovacs et al. .]



Figure 19.

Absorbance signals from cut fibers of Rana temporaria and Rana catesbeiana exposed to Ca‐indicator dyes. A: action potential conditions with arsenazo III; 21°C. Superimposed traces show the electrically recorded action potential (earliest deflection, left calibration) and absorbance records at indicated wavelengths (later deflections, right calibration). B: voltage‐clamp conditions with 1 mM antipyrylazo III; 10°C. Ringer's solution without TTX. Horizontal calibration is 50 ms; vertical calibration is 0.02 units of the absorbance change at 710 nm minus the fractional transmission change at 790 nm and normalized by the resting absorbance at 550 nm. C: voltage‐clamp conditions with arsenazo III; 10°C. Ringer's solution without TTX. Horizontal calibration is 50 ms; vertical calibration is 0.2 units of the absorbance change at 660 nm minus the fractional transmission change at 740 nm and normalized by the resting absorbance at 532 nm. [From Palade and Vergara .]

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Stephen M. Baylor. Optical Studies of Excitation‐Contraction Coupling Using Voltage‐Sensitive and Calcium‐Sensitive Probes. Compr Physiol 2011, Supplement 27: Handbook of Physiology, Skeletal Muscle: 355-379. First published in print 1983. doi: 10.1002/cphy.cp100113