Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1999 Oct;77(4):2226–2236. doi: 10.1016/S0006-3495(99)77063-3

Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage.

H J Koester 1, D Baur 1, R Uhl 1, S W Hell 1
PMCID: PMC1300503  PMID: 10512842

Abstract

The signal and limitations of calcium florescence imaging using nonresonant multiphoton absorption of near-infrared femto- and picosecond laser pulses were examined. The fluorescence changes of various Ca(2+)-indicators induced by transient increases of the intradendritic calcium concentration were evaluated by evoking physiological activity in neocortical neurons in rat brain slices. Photodamage was noticeable as irreversible changes in the parameters describing the calcium fluorescence transients. At higher two-photon excitation rates, a great variety of irregular functional and structural alterations occurred. Thus, signal and observation time were limited by phototoxic effects. At lower excitation rates, photodamage accumulated linearly with exposure time. Femtosecond and picosecond laser pulses were directly compared with respect to this cumulative photodamage. The variation of the pulse length at a constant two-photon excitation rate indicated that a two-photon excitation mechanism is mainly responsible for the cumulative photodamage within the investigated window of 75 fs to 3.2 ps. As a direct consequence, at low excitation rates, the same image quality is achieved irrespective of whether two-photon Ca(2+)-imaging is carried out with femto- or picosecond laser pulses.

Full Text

The Full Text of this article is available as a PDF (320.7 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. Booth M. J., Hell S. W. Continuous wave excitation two-photon fluorescence microscopy exemplified with the 647-nm ArKr laser line. J Microsc. 1998 Jun;190(Pt 3):298–304. doi: 10.1046/j.1365-2818.1998.00375.x. [DOI] [PubMed] [Google Scholar]
  3. Denk W., Holt J. R., Shepherd G. M., Corey D. P. Calcium imaging of single stereocilia in hair cells: localization of transduction channels at both ends of tip links. Neuron. 1995 Dec;15(6):1311–1321. doi: 10.1016/0896-6273(95)90010-1. [DOI] [PubMed] [Google Scholar]
  4. Denk W., Strickler J. H., Webb W. W. Two-photon laser scanning fluorescence microscopy. Science. 1990 Apr 6;248(4951):73–76. doi: 10.1126/science.2321027. [DOI] [PubMed] [Google Scholar]
  5. Denk W., Yuste R., Svoboda K., Tank D. W. Imaging calcium dynamics in dendritic spines. Curr Opin Neurobiol. 1996 Jun;6(3):372–378. doi: 10.1016/s0959-4388(96)80122-x. [DOI] [PubMed] [Google Scholar]
  6. Dodt H. U., Frick A., Kampe K., Zieglgänsberger W. NMDA and AMPA receptors on neocortical neurons are differentially distributed. Eur J Neurosci. 1998 Nov;10(11):3351–3357. doi: 10.1046/j.1460-9568.1998.00338.x. [DOI] [PubMed] [Google Scholar]
  7. Helmchen F., Imoto K., Sakmann B. Ca2+ buffering and action potential-evoked Ca2+ signaling in dendrites of pyramidal neurons. Biophys J. 1996 Feb;70(2):1069–1081. doi: 10.1016/S0006-3495(96)79653-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jenei A., Kirsch A. K., Subramaniam V., Arndt-Jovin D. J., Jovin T. M. Picosecond multiphoton scanning near-field optical microscopy. Biophys J. 1999 Feb;76(2):1092–1100. doi: 10.1016/S0006-3495(99)77274-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Koester H. J., Sakmann B. Calcium dynamics in single spines during coincident pre- and postsynaptic activity depend on relative timing of back-propagating action potentials and subthreshold excitatory postsynaptic potentials. Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9596–9601. doi: 10.1073/pnas.95.16.9596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Maiti S., Shear J. B., Williams R. M., Zipfel W. R., Webb W. W. Measuring serotonin distribution in live cells with three-photon excitation. Science. 1997 Jan 24;275(5299):530–532. doi: 10.1126/science.275.5299.530. [DOI] [PubMed] [Google Scholar]
  11. Markram H., Lübke J., Frotscher M., Roth A., Sakmann B. Physiology and anatomy of synaptic connections between thick tufted pyramidal neurones in the developing rat neocortex. J Physiol. 1997 Apr 15;500(Pt 2):409–440. doi: 10.1113/jphysiol.1997.sp022031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Sako Y., Sekihata A., Yanagisawa Y., Yamamoto M., Shimada Y., Ozaki K., Kusumi A. Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1. J Microsc. 1997 Jan;185(Pt 1):9–20. doi: 10.1046/j.1365-2818.1997.1480707.x. [DOI] [PubMed] [Google Scholar]
  13. Schiller J., Schiller Y., Clapham D. E. NMDA receptors amplify calcium influx into dendritic spines during associative pre- and postsynaptic activation. Nat Neurosci. 1998 Jun;1(2):114–118. doi: 10.1038/363. [DOI] [PubMed] [Google Scholar]
  14. Soeller C., Cannell M. B. Construction of a two-photon microscope and optimisation of illumination pulse duration. Pflugers Arch. 1996 Jul;432(3):555–561. doi: 10.1007/s004240050169. [DOI] [PubMed] [Google Scholar]
  15. Svaasand L. O., Ellingsen R. Optical properties of human brain. Photochem Photobiol. 1983 Sep;38(3):293–299. doi: 10.1111/j.1751-1097.1983.tb02674.x. [DOI] [PubMed] [Google Scholar]
  16. Svoboda K., Tank D. W., Denk W. Direct measurement of coupling between dendritic spines and shafts. Science. 1996 May 3;272(5262):716–719. doi: 10.1126/science.272.5262.716. [DOI] [PubMed] [Google Scholar]
  17. Yuste R., Denk W. Dendritic spines as basic functional units of neuronal integration. Nature. 1995 Jun 22;375(6533):682–684. doi: 10.1038/375682a0. [DOI] [PubMed] [Google Scholar]
  18. Yuste R., Majewska A., Cash S. S., Denk W. Mechanisms of calcium influx into hippocampal spines: heterogeneity among spines, coincidence detection by NMDA receptors, and optical quantal analysis. J Neurosci. 1999 Mar 15;19(6):1976–1987. doi: 10.1523/JNEUROSCI.19-06-01976.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES