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. 1999 Jan;76(1 Pt 1):489–499. doi: 10.1016/S0006-3495(99)77217-6

Photolysis of caged calcium in femtoliter volumes using two-photon excitation.

E B Brown 1, J B Shear 1, S R Adams 1, R Y Tsien 1, W W Webb 1
PMCID: PMC1302539  PMID: 9876162

Abstract

A new technique for the determination of the two-photon uncaging action cross section (deltau) of photolyzable calcium cages is described. This technique is potentially applicable to other caged species that can be chelated by a fluorescent indicator dye, as well as caged fluorescent compounds. The two-photon action cross sections of three calcium cages, DM-nitrophen, NP-EGTA, and azid-1, are studied in the range of excitation wavelengths between 700 and 800 nm. Azid-1 has a maximum deltau of approximately 1.4 GM at 700 nm, DM-nitrophen has a maximum deltau of approximately 0.013 GM at 730 nm, and NP-EGTA has no measurable uncaging yield. The equations necessary to predict the amount of cage photolyzed and the temporal behavior of the liberated calcium distribution under a variety of conditions are derived. These equations predict that by using 700-nm light from a Ti:sapphire laser focused with a 1.3-NA objective, essentially all of the azid-1 within the two-photon focal volume would be photolyzed with a 10-micros pulse train of approximately 7 mW average power. The initially localized distributions of free calcium will dissipate rapidly because of diffusion of free calcium and uptake by buffers. In buffer-free cytoplasm, the elevation of the calcium concentration at the center of the focal volume is expected to last for approximately 165 micros.

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Selected References

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  1. Adams S. R., Lev-Ram V., Tsien R. Y. A new caged Ca2+, azid-1, is far more photosensitive than nitrobenzyl-based chelators. Chem Biol. 1997 Nov;4(11):867–878. doi: 10.1016/s1074-5521(97)90119-8. [DOI] [PubMed] [Google Scholar]
  2. Allbritton N. L., Meyer T., Stryer L. Range of messenger action of calcium ion and inositol 1,4,5-trisphosphate. Science. 1992 Dec 11;258(5089):1812–1815. doi: 10.1126/science.1465619. [DOI] [PubMed] [Google Scholar]
  3. Augustine G. J., Neher E. Neuronal Ca2+ signalling takes the local route. Curr Opin Neurobiol. 1992 Jun;2(3):302–307. doi: 10.1016/0959-4388(92)90119-6. [DOI] [PubMed] [Google Scholar]
  4. Cheng H., Lederer W. J., Cannell M. B. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science. 1993 Oct 29;262(5134):740–744. doi: 10.1126/science.8235594. [DOI] [PubMed] [Google Scholar]
  5. Denk W. Two-photon scanning photochemical microscopy: mapping ligand-gated ion channel distributions. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6629–6633. doi: 10.1073/pnas.91.14.6629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ellis-Davies G. C., Kaplan J. H., Barsotti R. J. Laser photolysis of caged calcium: rates of calcium release by nitrophenyl-EGTA and DM-nitrophen. Biophys J. 1996 Feb;70(2):1006–1016. doi: 10.1016/S0006-3495(96)79644-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ellis-Davies G. C., Kaplan J. H. Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):187–191. doi: 10.1073/pnas.91.1.187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Escobar A. L., Velez P., Kim A. M., Cifuentes F., Fill M., Vergara J. L. Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis. Pflugers Arch. 1997 Sep;434(5):615–631. doi: 10.1007/s004240050444. [DOI] [PubMed] [Google Scholar]
  9. Forsén S., Linse S., Thulin E., Lindegård B., Martin S. R., Bayley P. M., Brodin P., Grundström T. Kinetics of calcium binding to calbindin mutants. Eur J Biochem. 1988 Oct 15;177(1):47–52. doi: 10.1111/j.1432-1033.1988.tb14343.x. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Kaplan J. H., Ellis-Davies G. C. Photolabile chelators for the rapid photorelease of divalent cations. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6571–6575. doi: 10.1073/pnas.85.17.6571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kaplan J. H. Photochemical manipulation of divalent cation levels. Annu Rev Physiol. 1990;52:897–914. doi: 10.1146/annurev.ph.52.030190.004225. [DOI] [PubMed] [Google Scholar]
  13. Klein M. G., Cheng H., Santana L. F., Jiang Y. H., Lederer W. J., Schneider M. F. Two mechanisms of quantized calcium release in skeletal muscle. Nature. 1996 Feb 1;379(6564):455–458. doi: 10.1038/379455a0. [DOI] [PubMed] [Google Scholar]
  14. Klingauf J., Neher E. Modeling buffered Ca2+ diffusion near the membrane: implications for secretion in neuroendocrine cells. Biophys J. 1997 Feb;72(2 Pt 1):674–690. doi: 10.1016/s0006-3495(97)78704-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lipp P., Lüscher C., Niggli E. Photolysis of caged compounds characterized by ratiometric confocal microscopy: a new approach to homogeneously control and measure the calcium concentration in cardiac myocytes. Cell Calcium. 1996 Mar;19(3):255–266. doi: 10.1016/s0143-4160(96)90026-3. [DOI] [PubMed] [Google Scholar]
  16. Lipp P., Niggli E. Fundamental calcium release events revealed by two-photon excitation photolysis of caged calcium in Guinea-pig cardiac myocytes. J Physiol. 1998 May 1;508(Pt 3):801–809. doi: 10.1111/j.1469-7793.1998.801bp.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Martin S. R., Linse S., Johansson C., Bayley P. M., Forsén S. Protein surface charges and Ca2+ binding to individual sites in calbindin D9k: stopped-flow studies. Biochemistry. 1990 May 1;29(17):4188–4193. doi: 10.1021/bi00469a023. [DOI] [PubMed] [Google Scholar]
  18. McCray J. A., Fidler-Lim N., Ellis-Davies G. C., Kaplan J. H. Rate of release of Ca2+ following laser photolysis of the DM-nitrophen-Ca2+ complex. Biochemistry. 1992 Sep 22;31(37):8856–8861. doi: 10.1021/bi00152a023. [DOI] [PubMed] [Google Scholar]
  19. McCray J. A., Trentham D. R. Properties and uses of photoreactive caged compounds. Annu Rev Biophys Biophys Chem. 1989;18:239–270. doi: 10.1146/annurev.bb.18.060189.001323. [DOI] [PubMed] [Google Scholar]
  20. Naraghi M. T-jump study of calcium binding kinetics of calcium chelators. Cell Calcium. 1997 Oct;22(4):255–268. doi: 10.1016/s0143-4160(97)90064-6. [DOI] [PubMed] [Google Scholar]
  21. Nelson M. T., Cheng H., Rubart M., Santana L. F., Bonev A. D., Knot H. J., Lederer W. J. Relaxation of arterial smooth muscle by calcium sparks. Science. 1995 Oct 27;270(5236):633–637. doi: 10.1126/science.270.5236.633. [DOI] [PubMed] [Google Scholar]
  22. Stern M. D. Buffering of calcium in the vicinity of a channel pore. Cell Calcium. 1992 Mar;13(3):183–192. doi: 10.1016/0143-4160(92)90046-u. [DOI] [PubMed] [Google Scholar]
  23. Xu T., Naraghi M., Kang H., Neher E. Kinetic studies of Ca2+ binding and Ca2+ clearance in the cytosol of adrenal chromaffin cells. Biophys J. 1997 Jul;73(1):532–545. doi: 10.1016/S0006-3495(97)78091-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Zhou Z., Neher E. Mobile and immobile calcium buffers in bovine adrenal chromaffin cells. J Physiol. 1993 Sep;469:245–273. doi: 10.1113/jphysiol.1993.sp019813. [DOI] [PMC free article] [PubMed] [Google Scholar]

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