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. 2001 Jan;80(1):549–561. doi: 10.1016/S0006-3495(01)76037-7

Calcium measurements in perfused mouse heart: quantitating fluorescence and absorbance of Rhod-2 by application of photon migration theory.

C Du 1, G A MacGowan 1, D L Farkas 1, A P Koretsky 1
PMCID: PMC1301256  PMID: 11159425

Abstract

Both theoretical and experimental results are presented for the quantitative detection of calcium transients in the perfused mouse heart loaded with the calcium-sensitive fluorescent dye Rhod-2. Analytical models are proposed to calculate both the reflected absorbance and fluorescence spectra detected from the mouse heart. These models allow correlation of the measured spectral intensities with the relative quantity of Rhod-2 in the heart and measurement of the changes in quantum yield of Rhod-2 upon binding calcium in the heart in which multiple scattering effects are predominant. Theoretical modeling and experimental results demonstrate that both reflected absorbance and fluorescence emission are attenuated linearly with Rhod-2 washout. According to this relation, a ratiometric method using fluorescence and absorbance is validated as a measure of the quantum yield of calcium-dependent fluorescence, enabling determination of the dynamics of cytosolic calcium in the perfused mouse heart. The feasibility of this approach is confirmed by experiments quantifying calcium transients in the perfused mouse heart stimulated at 8 Hz. The calculated cytosolic calcium concentrations are 368 +/- 68 nM and 654 +/- 164 nM in diastole and systole, respectively. Spectral distortions induced by tissue scattering and absorption and errors induced by the geometry of the detection optics in the calcium quantification are shown to be eliminated by using the ratio method. Methods to effectively minimize motion-induced artifacts and to monitor the oxygenation status of the whole perfused heart are also discussed.

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Selected References

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  1. Brandes R., Figueredo V. M., Camacho S. A., Baker A. J., Weiner M. W. Quantitation of cytosolic [Ca2+] in whole perfused rat hearts using Indo-1 fluorometry. Biophys J. 1993 Nov;65(5):1973–1982. doi: 10.1016/S0006-3495(93)81274-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brandes R., Figueredo V. M., Camacho S. A., Massie B. M., Weiner M. W. Suppression of motion artifacts in fluorescence spectroscopy of perfused hearts. Am J Physiol. 1992 Sep;263(3 Pt 2):H972–H980. doi: 10.1152/ajpheart.1992.263.3.H972. [DOI] [PubMed] [Google Scholar]
  3. Brandes R., Figueredo V. M., Camacho S. A., Weiner M. W. Compensation for changes in tissue light absorption in fluorometry of hypoxic perfused rat hearts. Am J Physiol. 1994 Jun;266(6 Pt 2):H2554–H2567. doi: 10.1152/ajpheart.1994.266.6.H2554. [DOI] [PubMed] [Google Scholar]
  4. Chance B., Liu H., Kitai T., Zhang Y. Effects of solutes on optical properties of biological materials: models, cells, and tissues. Anal Biochem. 1995 May 20;227(2):351–362. doi: 10.1006/abio.1995.1291. [DOI] [PubMed] [Google Scholar]
  5. Del Nido P. J., Glynn P., Buenaventura P., Salama G., Koretsky A. P. Fluorescence measurement of calcium transients in perfused rabbit heart using rhod 2. Am J Physiol. 1998 Feb;274(2 Pt 2):H728–H741. doi: 10.1152/ajpheart.1998.274.2.H728. [DOI] [PubMed] [Google Scholar]
  6. Efimov I. R., Ermentrout B., Huang D. T., Salama G. Activation and repolarization patterns are governed by different structural characteristics of ventricular myocardium: experimental study with voltage-sensitive dyes and numerical simulations. J Cardiovasc Electrophysiol. 1996 Jun;7(6):512–530. doi: 10.1111/j.1540-8167.1996.tb00558.x. [DOI] [PubMed] [Google Scholar]
  7. Farrell T. J., Patterson M. S., Wilson B. A diffusion theory model of spatially resolved, steady-state diffuse reflectance for the noninvasive determination of tissue optical properties in vivo. Med Phys. 1992 Jul-Aug;19(4):879–888. doi: 10.1118/1.596777. [DOI] [PubMed] [Google Scholar]
  8. Field M. L., Azzawi A., Styles P., Henderson C., Seymour A. M., Radda G. K. Intracellular Ca2+ transients in isolated perfused rat heart: measurement using the fluorescent indicator Fura-2/AM. Cell Calcium. 1994 Aug;16(2):87–100. doi: 10.1016/0143-4160(94)90004-3. [DOI] [PubMed] [Google Scholar]
  9. Flock S. T., Wilson B. C., Patterson M. S. Total attenuation coefficients and scattering phase functions of tissues and phantom materials at 633 nm. Med Phys. 1987 Sep-Oct;14(5):835–841. doi: 10.1118/1.596010. [DOI] [PubMed] [Google Scholar]
  10. Fralix T. A., Heineman F. W., Balaban R. S. Effects of tissue absorbance on NAD(P)H and Indo-1 fluorescence from perfused rabbit hearts. FEBS Lett. 1990 Mar 26;262(2):287–292. doi: 10.1016/0014-5793(90)80212-2. [DOI] [PubMed] [Google Scholar]
  11. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  12. Hampton T. G., Amende I., Travers K. E., Morgan J. P. Intracellular calcium dynamics in mouse model of myocardial stunning. Am J Physiol. 1998 May;274(5 Pt 2):H1821–H1827. doi: 10.1152/ajpheart.1998.274.5.H1821. [DOI] [PubMed] [Google Scholar]
  13. Haworth R. A., Redon D. Calibration of intracellular Ca transients of isolated adult heart cells labelled with fura-2 by acetoxymethyl ester loading. Cell Calcium. 1998 Oct;24(4):263–273. doi: 10.1016/s0143-4160(98)90050-1. [DOI] [PubMed] [Google Scholar]
  14. Koretsky A. P., Katz L. A., Balaban R. S. Determination of pyridine nucleotide fluorescence from the perfused heart using an internal standard. Am J Physiol. 1987 Oct;253(4 Pt 2):H856–H862. doi: 10.1152/ajpheart.1987.253.4.H856. [DOI] [PubMed] [Google Scholar]
  15. Miyata H., Silverman H. S., Sollott S. J., Lakatta E. G., Stern M. D., Hansford R. G. Measurement of mitochondrial free Ca2+ concentration in living single rat cardiac myocytes. Am J Physiol. 1991 Oct;261(4 Pt 2):H1123–H1134. doi: 10.1152/ajpheart.1991.261.4.H1123. [DOI] [PubMed] [Google Scholar]
  16. Parrish J. A. New concepts in therapeutic photomedicine: photochemistry, optical targeting and the therapeutic window. J Invest Dermatol. 1981 Jul;77(1):45–50. doi: 10.1111/1523-1747.ep12479235. [DOI] [PubMed] [Google Scholar]
  17. Potter W. R., Mang T. S. Photofrin II levels by in vivo fluorescence photometry. Prog Clin Biol Res. 1984;170:177–186. [PubMed] [Google Scholar]
  18. Richards-Kortum R., Rava R. P., Fitzmaurice M., Tong L. L., Ratliff N. B., Kramer J. R., Feld M. S. A one-layer model of laser-induced fluorescence for diagnosis of disease in human tissue: applications to atherosclerosis. IEEE Trans Biomed Eng. 1989 Dec;36(12):1222–1232. doi: 10.1109/10.42117. [DOI] [PubMed] [Google Scholar]
  19. Salama G., Lombardi R., Elson J. Maps of optical action potentials and NADH fluorescence in intact working hearts. Am J Physiol. 1987 Feb;252(2 Pt 2):H384–H394. doi: 10.1152/ajpheart.1987.252.2.H384. [DOI] [PubMed] [Google Scholar]
  20. Trollinger D. R., Cascio W. E., Lemasters J. J. Selective loading of Rhod 2 into mitochondria shows mitochondrial Ca2+ transients during the contractile cycle in adult rabbit cardiac myocytes. Biochem Biophys Res Commun. 1997 Jul 30;236(3):738–742. doi: 10.1006/bbrc.1997.7042. [DOI] [PubMed] [Google Scholar]
  21. Wilson B. C., Patterson M. S. The physics of photodynamic therapy. Phys Med Biol. 1986 Apr;31(4):327–360. doi: 10.1088/0031-9155/31/4/001. [DOI] [PubMed] [Google Scholar]
  22. van der Putten W. J., van Gemert M. J. A modelling approach to the detection of subcutaneous tumours by haematoporphyrin-derivative fluorescence. Phys Med Biol. 1983 Jun;28(6):639–645. doi: 10.1088/0031-9155/28/6/004. [DOI] [PubMed] [Google Scholar]

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