Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2000 Apr;78(4):2116–2126. doi: 10.1016/S0006-3495(00)76758-0

Effects of temperature on calcium-sensitive fluorescent probes.

A E Oliver 1, G A Baker 1, R D Fugate 1, F Tablin 1, J H Crowe 1
PMCID: PMC1300803  PMID: 10733989

Abstract

The effect of temperature on the binding equilibria of calcium-sensing dyes has been extensively studied, but there are also important temperature-related changes in the photophysics of the dyes that have been largely ignored. We conducted a systematic study of thermal effects on five calcium-sensing dyes under calcium-saturated and calcium-free conditions. Quin-2, chlortetracycline, calcium green dextran, Indo-1, and Fura-2 all show temperature-dependent effects on fluorescence in all or part of the range tested (5-40 degrees C). Specifically, the intensity of the single-wavelength dyes increased at low temperature. The ratiometric dyes, because of variable effects at the two wavelengths, showed, in general, a reduction in the fluorescence ratio as temperature decreased. Changes in viscosity, pH, oxygen quenching, or fluorescence maxima could not fully explain the effects of temperature on fluorescence. The excited-state lifetimes of the dyes were determined, in both the presence and absence of calcium, using multifrequency phase-modulation fluorimetry. In most cases, low temperature led to prolonged fluorescence lifetimes. The increase in lifetimes at reduced temperature is probably largely responsible for the effects of temperature on the physical properties of the calcium-sensing dyes. Clearly, these temperature effects can influence reported calcium concentrations and must therefore be taken into consideration during any investigation involving variable temperatures.

Full Text

The Full Text of this article is available as a PDF (142.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Becker P. L., Fay F. S. Photobleaching of fura-2 and its effect on determination of calcium concentrations. Am J Physiol. 1987 Oct;253(4 Pt 1):C613–C618. doi: 10.1152/ajpcell.1987.253.4.C613. [DOI] [PubMed] [Google Scholar]
  2. Bhattacharya J., Chakrabarti M. K. Rise of intracellular free calcium levels with activation of inositol triphosphate in a human colonic carcinoma cell line (COLO 205) by heat-stable enterotoxin of Escherichia coli. Biochim Biophys Acta. 1998 May 27;1403(1):1–4. doi: 10.1016/s0167-4889(98)00026-3. [DOI] [PubMed] [Google Scholar]
  3. Bi G. Q., Alderton J. M., Steinhardt R. A. Calcium-regulated exocytosis is required for cell membrane resealing. J Cell Biol. 1995 Dec;131(6 Pt 2):1747–1758. doi: 10.1083/jcb.131.6.1747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brancaleon L., Crippa P. R., Diemmi D. Anomalous temperature fluorescence quenching of N-Trp terminal peptides. Biopolymers. 1995 Dec;36(6):723–733. doi: 10.1002/bip.360360606. [DOI] [PubMed] [Google Scholar]
  5. Brancaleon L., Gasparini G., Manfredi M., Mazzini A. A model for the explanation of the thermally induced increase of the overall fluorescence in tryptophan-X peptides. Arch Biochem Biophys. 1997 Dec 1;348(1):125–133. doi: 10.1006/abbi.1997.0368. [DOI] [PubMed] [Google Scholar]
  6. Busa W. B. Spectral characterization of the effect of viscosity on Fura-2 fluorescence: excitation wavelength optimization abolishes the viscosity artifact. Cell Calcium. 1992 May;13(5):313–319. doi: 10.1016/0143-4160(92)90066-2. [DOI] [PubMed] [Google Scholar]
  7. Carroll J., Swann K., Whittingham D., Whitaker M. Spatiotemporal dynamics of intracellular [Ca2+]i oscillations during the growth and meiotic maturation of mouse oocytes. Development. 1994 Dec;120(12):3507–3517. doi: 10.1242/dev.120.12.3507. [DOI] [PubMed] [Google Scholar]
  8. Chen S. J., Wang M. H., Chen I. J. Antiplatelet and calcium inhibitory properties of eugenol and sodium eugenol acetate. Gen Pharmacol. 1996 Jun;27(4):629–633. doi: 10.1016/0306-3623(95)02089-6. [DOI] [PubMed] [Google Scholar]
  9. Deber C. M., Hsu L. C. Calcium transport by ionophorous peptides in dog and human lymphocytes detected by quin-2 fluorescence. Biochem Biophys Res Commun. 1986 Jan 29;134(2):731–735. doi: 10.1016/s0006-291x(86)80481-8. [DOI] [PubMed] [Google Scholar]
  10. Demchenko A. P., Ladokhin A. S. Temperature-dependent shift of fluorescence spectra without conformational changes in protein; studies of dipole relaxation in the melittin molecule. Biochim Biophys Acta. 1988 Aug 10;955(3):352–360. doi: 10.1016/0167-4838(88)90215-4. [DOI] [PubMed] [Google Scholar]
  11. Eastman J. W., Rosa E. J. The fluorescence of adenine. The effects of solvent and temperature on the quantum yield. Photochem Photobiol. 1968 Feb;7(2):189–201. doi: 10.1111/j.1751-1097.1968.tb08005.x. [DOI] [PubMed] [Google Scholar]
  12. Fetcho J. R., O'Malley D. M. Visualization of active neural circuitry in the spinal cord of intact zebrafish. J Neurophysiol. 1995 Jan;73(1):399–406. doi: 10.1152/jn.1995.73.1.399. [DOI] [PubMed] [Google Scholar]
  13. Gambassi G., Cerbai E., Pahor M., Capogrossi M. C., Carbonin P., Mugelli A. Temperature modulates calcium homeostasis and ventricular arrhythmias in myocardial preparations. Cardiovasc Res. 1994 Mar;28(3):391–399. doi: 10.1093/cvr/28.3.391. [DOI] [PubMed] [Google Scholar]
  14. Greco N. J., Tandon N. N., Jones G. D., Kornhauser R., Jackson B., Yamamoto N., Tanoue K., Jamieson G. A. Contributions of glycoprotein Ib and the seven transmembrane domain receptor to increases in platelet cytoplasmic [Ca2+] induced by alpha-thrombin. Biochemistry. 1996 Jan 23;35(3):906–914. doi: 10.1021/bi951503y. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Harris P. J. Caffeine-induced calcium release in sea urchin eggs and the effect of continuous versus pulsed application on the mitotic apparatus. Dev Biol. 1994 Feb;161(2):370–378. doi: 10.1006/dbio.1994.1038. [DOI] [PubMed] [Google Scholar]
  17. Harrison S. M., Bers D. M. Correction of proton and Ca association constants of EGTA for temperature and ionic strength. Am J Physiol. 1989 Jun;256(6 Pt 1):C1250–C1256. doi: 10.1152/ajpcell.1989.256.6.C1250. [DOI] [PubMed] [Google Scholar]
  18. Harrison S. M., Bers D. M. The effect of temperature and ionic strength on the apparent Ca-affinity of EGTA and the analogous Ca-chelators BAPTA and dibromo-BAPTA. Biochim Biophys Acta. 1987 Aug 13;925(2):133–143. doi: 10.1016/0304-4165(87)90102-4. [DOI] [PubMed] [Google Scholar]
  19. Howarth F. C., Singh J., Waring J. J., Hustler B. I., Bailey M. Effects of monovalent cations, pH and temperature on the dissociation constant (KD) for the fluorescent indicator mag-fura-2 at different excitation wavelengths. Magnes Res. 1995 Dec;8(4):299–306. [PubMed] [Google Scholar]
  20. Jacob R., Murphy E., Lieberman M. Free calcium in isolated chick embryo heart cells measured using quin2 and fura-2. Am J Physiol. 1987 Aug;253(2 Pt 1):C337–C342. doi: 10.1152/ajpcell.1987.253.2.C337. [DOI] [PubMed] [Google Scholar]
  21. Jacquemond V., Allard B. Activation of Ca2+-activated K+ channels by an increase in intracellular Ca2+ induced by depolarization of mouse skeletal muscle fibres. J Physiol. 1998 May 15;509(Pt 1):93–102. doi: 10.1111/j.1469-7793.1998.093bo.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ju Y. K., Allen D. G. Intracellular calcium and Na+-Ca2+ exchange current in isolated toad pacemaker cells. J Physiol. 1998 Apr 1;508(Pt 1):153–166. doi: 10.1111/j.1469-7793.1998.153br.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. June C. H., Rabinovitch P. S. Intracellular ionized calcium. Methods Cell Biol. 1994;41:149–174. [PubMed] [Google Scholar]
  24. Jy W., Haynes D. H. Calcium uptake and release characteristics of the dense tubules of digitonin-permeabilized human platelets. Biochim Biophys Acta. 1988 Oct 20;944(3):374–382. doi: 10.1016/0005-2736(88)90508-1. [DOI] [PubMed] [Google Scholar]
  25. Jy W., Haynes D. H. Intracellular calcium storage and release in the human platelet. Chlorotetracycline as a continuous monitor. Circ Res. 1984 Nov;55(5):595–608. doi: 10.1161/01.res.55.5.595. [DOI] [PubMed] [Google Scholar]
  26. Jy W., Haynes D. H. Thrombin-induced calcium movements in platelet activation. Biochim Biophys Acta. 1987 Jun 15;929(1):88–102. doi: 10.1016/0167-4889(87)90244-8. [DOI] [PubMed] [Google Scholar]
  27. Kao J. P., Tsien R. Y. Ca2+ binding kinetics of fura-2 and azo-1 from temperature-jump relaxation measurements. Biophys J. 1988 Apr;53(4):635–639. doi: 10.1016/S0006-3495(88)83142-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kenyon J. L., Goff H. R. Temperature dependencies of Ca2+ current, Ca(2+)-activated Cl- current and Ca2+ transients in sensory neurones. Cell Calcium. 1998 Jul;24(1):35–48. doi: 10.1016/s0143-4160(98)90087-2. [DOI] [PubMed] [Google Scholar]
  29. Kono T., Jones K. T., Bos-Mikich A., Whittingham D. G., Carroll J. A cell cycle-associated change in Ca2+ releasing activity leads to the generation of Ca2+ transients in mouse embryos during the first mitotic division. J Cell Biol. 1996 Mar;132(5):915–923. doi: 10.1083/jcb.132.5.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kumke M. U., Shu L., McGown L. B., Walker G. T., Pitner J. B., Linn C. P. Temperature and quenching studies of fluorescence polarization detection of DNA hybridization. Anal Chem. 1997 Feb 1;69(3):500–506. doi: 10.1021/ac9608230. [DOI] [PubMed] [Google Scholar]
  31. Lakowicz J. R., Szmacinski H., Nowaczyk K., Johnson M. L. Fluorescence lifetime imaging of calcium using Quin-2. Cell Calcium. 1992 Mar;13(3):131–147. doi: 10.1016/0143-4160(92)90041-p. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lakowicz J. R., Szmacinski H., Nowaczyk K., Lederer W. J., Kirby M. S., Johnson M. L. Fluorescence lifetime imaging of intracellular calcium in COS cells using Quin-2. Cell Calcium. 1994 Jan;15(1):7–27. doi: 10.1016/0143-4160(94)90100-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lattanzio F. A., Jr, Bartschat D. K. The effect of pH on rate constants, ion selectivity and thermodynamic properties of fluorescent calcium and magnesium indicators. Biochem Biophys Res Commun. 1991 May 31;177(1):184–191. doi: 10.1016/0006-291x(91)91966-g. [DOI] [PubMed] [Google Scholar]
  34. Lattanzio F. A., Jr The effects of pH and temperature on fluorescent calcium indicators as determined with Chelex-100 and EDTA buffer systems. Biochem Biophys Res Commun. 1990 Aug 31;171(1):102–108. doi: 10.1016/0006-291x(90)91362-v. [DOI] [PubMed] [Google Scholar]
  35. Liu B., Wang L. C., Belke D. D. Effect of low temperature on the cytosolic free Ca2+ in rat ventricular myocytes. Cell Calcium. 1991 Jan;12(1):11–18. doi: 10.1016/0143-4160(91)90080-x. [DOI] [PubMed] [Google Scholar]
  36. Luby-Phelps K., Mujumdar S., Mujumdar R. B., Ernst L. A., Galbraith W., Waggoner A. S. A novel fluorescence ratiometric method confirms the low solvent viscosity of the cytoplasm. Biophys J. 1993 Jul;65(1):236–242. doi: 10.1016/S0006-3495(93)81075-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Martin G. R., Jain R. K. Noninvasive measurement of interstitial pH profiles in normal and neoplastic tissue using fluorescence ratio imaging microscopy. Cancer Res. 1994 Nov 1;54(21):5670–5674. [PubMed] [Google Scholar]
  38. McClellan A. D., McPherson D., O'Donovan M. J. Combined retrograde labeling and calcium imaging in spinal cord and brainstem neurons of the lamprey. Brain Res. 1994 Nov 7;663(1):61–68. doi: 10.1016/0006-8993(94)90462-6. [DOI] [PubMed] [Google Scholar]
  39. Miao N., Nagao K., Lynch C., 3rd Thiopental and methohexital depress Ca2+ entry into and glutamate release from cultured neurons. Anesthesiology. 1998 Jun;88(6):1643–1653. doi: 10.1097/00000542-199806000-00029. [DOI] [PubMed] [Google Scholar]
  40. Miyoshi N., Hara K., Kimura S., Nakanishi K., Fukuda M. A new method of determining intracellular free Ca2+ concentration using Quin2-fluorescence. Photochem Photobiol. 1991 Mar;53(3):415–418. doi: 10.1111/j.1751-1097.1991.tb03650.x. [DOI] [PubMed] [Google Scholar]
  41. Oliver A. E., Tablin F., Walker N. J., Crowe J. H. The internal calcium concentration of human platelets increases during chilling. Biochim Biophys Acta. 1999 Jan 12;1416(1-2):349–360. doi: 10.1016/s0005-2736(98)00239-9. [DOI] [PubMed] [Google Scholar]
  42. Paltauf-Doburzynska J., Graier W. F. Temperature dependence of agonist-stimulated Ca2+ signaling in cultured endothelial cells. Cell Calcium. 1997 Jan;21(1):43–51. doi: 10.1016/s0143-4160(97)90095-6. [DOI] [PubMed] [Google Scholar]
  43. Poenie M. Alteration of intracellular Fura-2 fluorescence by viscosity: a simple correction. Cell Calcium. 1990 Feb-Mar;11(2-3):85–91. doi: 10.1016/0143-4160(90)90062-y. [DOI] [PubMed] [Google Scholar]
  44. Puglisi J. L., Bassani R. A., Bassani J. W., Amin J. N., Bers D. M. Temperature and relative contributions of Ca transport systems in cardiac myocyte relaxation. Am J Physiol. 1996 May;270(5 Pt 2):H1772–H1778. doi: 10.1152/ajpheart.1996.270.5.H1772. [DOI] [PubMed] [Google Scholar]
  45. Rink T. J., Smith S. W., Tsien R. Y. Cytoplasmic free Ca2+ in human platelets: Ca2+ thresholds and Ca-independent activation for shape-change and secretion. FEBS Lett. 1982 Nov 1;148(1):21–26. doi: 10.1016/0014-5793(82)81234-9. [DOI] [PubMed] [Google Scholar]
  46. Rink T. J., Tsien R. Y., Pozzan T. Cytoplasmic pH and free Mg2+ in lymphocytes. J Cell Biol. 1982 Oct;95(1):189–196. doi: 10.1083/jcb.95.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ritucci N. A., Erlichman J. S., Dean J. B., Putnam R. W. A fluorescence technique to measure intracellular pH of single neurons in brainstem slices. J Neurosci Methods. 1996 Oct;68(2):149–163. doi: 10.1016/0165-0270(96)00051-9. [DOI] [PubMed] [Google Scholar]
  48. Sebille S., Pereira M., Millot J. M., Jacquot J., Delabroise A. M., Arnaud M., Manfait M. Extracellular Mg2+ inhibits both histamine-stimulated Ca(2+)-signaling and exocytosis in human tracheal secretory gland cells. Biochem Biophys Res Commun. 1998 May 8;246(1):111–116. doi: 10.1006/bbrc.1998.8494. [DOI] [PubMed] [Google Scholar]
  49. Shuttleworth T. J., Thompson J. L. Effect of temperature on receptor-activated changes in [Ca2+]i and their determination using fluorescent probes. J Biol Chem. 1991 Jan 25;266(3):1410–1414. [PubMed] [Google Scholar]
  50. Sipido K. R., Callewaert G. How to measure intracellular [Ca2+] in single cardiac cells with fura-2 or indo-1. Cardiovasc Res. 1995 May;29(5):717–726. [PubMed] [Google Scholar]
  51. Somogyi B., Punyiczki M., Hedstrom J., Norman J. A., Prendergast F. G., Rosenberg A. Coupling between external viscosity and the intramolecular dynamics of ribonuclease T1: a two-phase model for the quenching of protein fluorescence. Biochim Biophys Acta. 1994 Nov 16;1209(1):61–68. doi: 10.1016/0167-4838(94)90137-6. [DOI] [PubMed] [Google Scholar]
  52. Song P. S., Chae Q., Briggs W. R. Temperature dependence of the fluorescence quantum yield of phytochrome. Photochem Photobiol. 1975 Jul-Aug;22(1-2):75–76. doi: 10.1111/j.1751-1097.1975.tb06726.x. [DOI] [PubMed] [Google Scholar]
  53. Suelter C. H. Effects of temperature and activating cations on the fluorescence of pyruvate kinase. Biochemistry. 1967 Feb;6(2):418–423. doi: 10.1021/bi00854a008. [DOI] [PubMed] [Google Scholar]
  54. Swaminathan R., Hoang C. P., Verkman A. S. Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion. Biophys J. 1997 Apr;72(4):1900–1907. doi: 10.1016/S0006-3495(97)78835-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Szmacinski H., Gryczynski I., Lakowicz J. R. Calcium-dependent fluorescence lifetimes of Indo-1 for one- and two-photon excitation of fluorescence. Photochem Photobiol. 1993 Sep;58(3):341–345. doi: 10.1111/j.1751-1097.1993.tb09571.x. [DOI] [PubMed] [Google Scholar]
  56. Szmacinski H., Lakowicz J. R. Optical measurements of pH using fluorescence lifetimes and phase-modulation fluorometry. Anal Chem. 1993 Jul 1;65(13):1668–1674. doi: 10.1021/ac00061a007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Tamm S. L., Terasaki M. Visualization of calcium transients controlling orientation of ciliary beat. J Cell Biol. 1994 Jun;125(5):1127–1135. doi: 10.1083/jcb.125.5.1127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Tao J., Johansson J. S., Haynes D. H. Protein kinase C stimulates dense tubular Ca2+ uptake in the intact human platelet by increasing the Vm of the Ca(2+)-ATPase pump: stimulation by phorbol ester, inhibition by calphostin C. Biochim Biophys Acta. 1992 Jun 30;1107(2):213–222. doi: 10.1016/0005-2736(92)90407-d. [DOI] [PubMed] [Google Scholar]
  59. Tsien R., Pozzan T. Measurement of cytosolic free Ca2+ with quin2. Methods Enzymol. 1989;172:230–262. doi: 10.1016/s0076-6879(89)72017-6. [DOI] [PubMed] [Google Scholar]
  60. Vijayaraghavan S., Hoskins D. Quantitation of bovine sperm cytoplasmic calcium with Quin-2 and Fura-2: evidence that external calcium does not have direct access to the sperm cytoplasm. Cell Calcium. 1989 May-Jun;10(4):241–253. doi: 10.1016/0143-4160(89)90007-9. [DOI] [PubMed] [Google Scholar]
  61. Wang R., Sun S., Bekos E. J., Bright F. V. Dynamics surrounding Cys-34 in native, chemically denatured, and silica-adsorbed bovine serum albumin. Anal Chem. 1995 Jan 1;67(1):149–159. doi: 10.1021/ac00097a024. [DOI] [PubMed] [Google Scholar]
  62. Waris R., Acree W. E., Jr, Street K. W., Jr Py and BPe solvent polarity scales: effect of temperature on pyrene and benzo[ghi]perylene fluorescence spectra. Analyst. 1988 Sep;113(9):1465–1467. doi: 10.1039/an9881301465. [DOI] [PubMed] [Google Scholar]
  63. Xu Y. J., Shao Q., Dhalla N. S. Fura-2 fluorescent technique for the assessment of Ca2+ homeostasis in cardiomyocytes. Mol Cell Biochem. 1997 Jul;172(1-2):149–157. [PubMed] [Google Scholar]
  64. Zhao Y., Buhr M. M. Cryopreservation extenders affect calcium flux in bovine spermatozoa during a temperature challenge. J Androl. 1995 May-Jun;16(3):278–285. [PubMed] [Google Scholar]
  65. Zicha J., Kunes J., David-Dufilho M., Pernollet M. G., Devynck M. A. Cell calcium handling and intracellular pH regulation in hereditary hypertriglyceridemic rats: reduced platelet response to thrombin stimulation. Life Sci. 1996;59(10):803–813. doi: 10.1016/0024-3205(96)00371-2. [DOI] [PubMed] [Google Scholar]
  66. van den Zegel M., Boens N., de Schryver F. C. Fluorescence decay of 1-methylpyrene in small unilamellar l-alpha-dimyristoylphosphatidylcholine vesicles. A temperature and concentration dependence study. Biophys Chem. 1984 Nov;20(4):333–345. doi: 10.1016/0301-4622(84)80023-x. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES