Technical features of a CCD video camera system to record cardiac fluorescence data
A charge-coupled device (CCD) camera was used to acquie movies of transmembrane activity from thin slices of sheep ventricular epicardial muscle stained with a voltage-sensitive dye. Compared with photodiodes, CCDs have high spatial resolution, but low temporal resolution. Spatial resolution in our system ranged from 0.04 to 0.14 mm/pixel; the acquisition rate was 60, 120, or 240 frames/sec. Propagating waves were readily visualized after subtraction of a background image. The optical signal had an amplitude of 1 to 6 gray levels, with signal-to-noise ratios between 1.5 and 4.4. Because CCD cameras in-tegrate light over the frame interval, moving objects, including propagating waves, are blurred in the resulting movies. A computer model of such an integrating imaging system was developed to study the effects of blur, noise, filtering, and quantization on the ability to measure conduction velocity and action potential duration (APD). The model indicated that blurring, filtering, and quantization do not affect the ability to localize wave fronts in the optical data (i.e., no systematic error in determining spatial position), but noise does increase the uncertainty of the measurements. The model also showed that the low frame rates of the CCD camera introduced a systematic error in the calculation of APD: for cutoff levels >50%, the APD was erroneusly long. Both noise and quantization increased the uncertainty in the APD measurements. The optical measures of conduction velocity were not significantly different from those measured simultaneously with microelectrodes. Optical APDs, however, were longer than the electrically recorded APDs. This APD error could be reduced by using the 50% cutoff level and the fastest frame rate possible.
KeywordsOptical mapping Voltage-sensitive dyes Electro-physiology Conduction velocity Action potential duration
Baxter, W. T., J. M. Davidenko, C. Cabo, and J. Jalife. Video imaging of cardiac transmembrane activity.SPIE Proc. Clin. Appl. Modern Imaging Technol.
2132:357–366, 1994.Google Scholar
Blasdel, G. G., and G. Salama. Voltage-sensitive dyes reveal a modular organization in monkey striate cortex.Nature
321:579–585, 1986.PubMedCrossRefGoogle Scholar
Cabo, C., A. M. Pertsov, W. T. Baxter, J. M. Davidenko, R. A. Gray, and J. Jalife. Wave-front curvature as a cause of slow conduction and block in isolated cardiac muscle.Circ. Res.
75:1014–1028, 1994.PubMedGoogle Scholar
Cohen, L. B., B. M. Salzberg, H. V. Davila, W. N. Ross, D. Landowne, A. S. Waggoner, and C. H. Wang. Changes in axon fluorescence during activity: molecular probes of membrane potential.J. Membr. Biol.
19:1–36, 1974.PubMedCrossRefGoogle Scholar
Davidenko, J. M., A. V. Pertsov, R. Salomonsz, W. Baxter, and J. Jalife. Stationary and drifting spiral waves of excitation in isolated cardiac muscle.Nature
355:349–351, 1992.PubMedCrossRefGoogle Scholar
Dillon, S. M. Use of voltage sensitive dyes to record, map and image cardiac electrical activation. In: Imaging analysis and simulation of the cardiac system, edited by S. Sideman and R. Beyar. London: Freund Publishing, 1990, pp. 739–766.Google Scholar
Dillon, S., and M. Morad. A new laser scanning system for measuring action potential propagation in the heart.Science
214:453–456, 1981.PubMedCrossRefGoogle Scholar
Dillon, S. M., M. A. Allessie, P. C. Ursell, and A. L. Wit. Influence of anisotropic tissue on reentrant circuit in the epicardial border zone of subacute canine infarcts.Circ. Res.
63:182–206, 1988.PubMedGoogle Scholar
Effimov, I. R., D. T. Huang, J. M. Rendt, and G. Salama. Optical mapping of repolarization and refractoriness from intact hearts.Circulation
90:1469–1480, 1994.Google Scholar
Falk, C. X., J. Y. Wu, L. B. Cohen, and A. K. Tang. Non-uniform expression of habituation in the activity of distinct classes of neurons in theAplysia
abdominal ganglion.J. Neurosci.
13:4072–4081, 1993.PubMedGoogle Scholar
Fast, V. G., and A. G. Kléber. Microscopic conduction in cultured strands of neonatal rat heart cells measured with voltage-sensitive-dyes.Circ. Res.
73:914–925, 1993.PubMedGoogle Scholar
Frazier, D. W., P. D. Wolf, J. M. Wharton, A. S. L. Tang, W. M. Smith, and R. E. Ideker Stimulus-induced critical point: mechanism for initiation of reentry in normal canine myocardium.J. Clin. Invest.
83:1039–1052, 1989.PubMedGoogle Scholar
Girouard, S. D., K. R. Laurita, and D. S. Rosenbaum. Unique characteristics of optically recorded action potentials.J. Cardiovasc. Electrophysiol.
7:1024–1038, 1996.PubMedCrossRefGoogle Scholar
Gray, R. A., J. Jalife, A. Panfilov, W. T. Baxter, C. Cabo, J. Davidenko, and A. M. Pertsov. Nonstationary vortexlike reentrant activity as a mechanism of polymorphic ventricular tachycardia in the isolated rabbit heart.Circulation
91:2454–2469, 1995.PubMedGoogle Scholar
Grinvald, A. Real-time optical mapping of neuronal activity: from single growth cones to the intact mammalian brain.Ann. Rev. Neurosci.
8:263–305, 1985.PubMedCrossRefGoogle Scholar
Hirota, A, K. Sato, Y. Momose-Sato, T. Sakai, and K. Kamino. A new simultaneous 1020-site optical recording system for monitoring neural activity using voltage-sensitive dyes.J. Neurosci. Methods
56:187–194, 1995.PubMedCrossRefGoogle Scholar
Janesick, J. R., T. Elliott S. Collins, M. M. Blouke, and J. Freeman. Scientific charge-coupled devices.Opt. Eng.
26: 692–714, 1987.Google Scholar
Kauer, J. S.: Real-time imaging of evoked activity in local circuits of the salamander olfactory bulb.Nature
331:166–168, 1988.PubMedCrossRefGoogle Scholar
Lasser-Ross, N., H. Miyakawa, V. Lev-Ram, S. R. Young, and W. N. Ross. High time resolution fluorescence imaging with a CCD camera.J. Neurosci. Methods
36:253–261, 1991.PubMedCrossRefGoogle Scholar
Liu, Y., C. Cabo, R. Salomonsz, M. Delmar J. Davidenko, and J. Jalife. Effects of diacetyl monoxime on the electrical properties of sheep and guinea pig ventricular muscle.Cardiovasc. Res.
27:1991–1997, 1993.PubMedCrossRefGoogle Scholar
Loew, L. M., L. B. Cohen, J. Dix, E. N. Fluhler, V. Montana, G. Salama, and J. Y. Wu. A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue, cell, and model membrane preparations.J. Membr. Biol.
130:1–10, 1992.PubMedGoogle Scholar
MacKay, C.D. Fast optical imaging techniques. In: Fluorescence spectroscopy, edited by O. S. Wolfbeis. New York: Springer-Verlag, 1993, pp. 25–30.Google Scholar
Pertsov, A. M., J. M. Davidenko, R. Salomonsz, W. T. Baxter, and J. Jalife. Spiral waves of excitation underlie reentrant activity in isolated cardiac muscle.Circ. Res.
72:631–650, 1993.PubMedGoogle Scholar
Ratzlaff, E. H., and A. Grinvald. A tandem-lens epifluorescence macroscope: hundred-fold brightness advantage for wide-field imaging.J. Neurosci. Methods
36:127–137, 1991.PubMedCrossRefGoogle Scholar
Rohr, S., and B. M. Salzberg. Multiple site optical recording of transmembrane voltage (MSORTV) in patterned growth heart cell cultures: assessing electrical behavior, with microsecond resolution, on a cellular and subcellular seale.Biophys. J.
67:1301–1315, 1994.PubMedCrossRefGoogle Scholar
Russ, J. C.. The Image Processing Handbook, Boca Raton, FL: CRC Press, 1992, pp. 1–445.Google Scholar
Salama, G. Optical measurements of transmembrane potential in heart. In: Spectrosopic membrane probes, vol. 3, edited by L. M. Loew. Boca Raton, FL: CRC Press, 1988, pp. 137–199.Google Scholar
Salama, G., and M. Morad. Merocyanine 540 as an optical probe of transmembrane electrical activity in the heart.Science
191:485–487, 1976.PubMedCrossRefGoogle Scholar
© Biomedical Engineering Society 1997