Skip to main content
SpringerLink
Log in

A confocal scanning laser microscope for quantitative ratiometric 3D measurements of [Ca2+] and Ca2+ diffusions in living cells stained with Fura-2

  • Original Article
  • Molecular and Cellular Physiology
  • Published:
Pflügers Archiv Aims and scope Submit manuscript

Abstract

A confocal scanning laser microscope (CSLM) for observation and quantitative ratiometric measurements of the intracellular dynamics of Ca2+ ions in living neurons has been developed. The instrument consists of a UV-enhanced CSLM, an optical arrangement providing simultaneous excitation at two wavelengths, an electronic arrangement for processing the simultaneous fluorescence response, and software for computing the absolute Ca2+ concentrations, ([Ca2+]). The instrument can be used for any excitation ratiometric measurements, provided that the dye substance used is excitable by wavelengths between 334 nm and 750 nm (such as, e.g. Fura-2). The spatial resolution of the CSLM, as well as a temporal resolution of 20 ms per line (maximum sampling rate) for dynamic measurements are provided by the instrument. Using Fura-2 in calibrated Ca2+ buffer solutions, the instrument measures [Ca2+] between 0 and 1.35 μmol·1−1 with an error of less than 1%. The capability of the instrument to measure absolute [Ca2+] was verified by recording fluorescence images of test solutions with well defined [Ca2+] values (Molecular Probes, Eugene, Ore., USA, C-3009 calibration solutions). In order to verify the dynamic capability of the instrument in real biological specimens, fluorescence changes of Fura-2 that were due to an intracellular flux of Ca2+ ions, and to an increase of [Ca2+]i (the intracellular Ca2+ concentration) have been recorded in Fura-2-loaded cultured cells of the line TE 671.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Almers W, Neher E (1985) The calcium signal from Fura-2 loaded mast cells depends strongly on the method of dyeloading. FEBS Lett 192:13–18

    Google Scholar 

  2. Becker PL, Fay FS (1987) Photobleaching of Fura-2 and its effect on determination of calcium concentrations. Am J Physiol 253:C613-C618

    Google Scholar 

  3. Blatter LA, Wier WG (1990) Intracellular diffusion, binding, and compartmentalization of the fluorescent calcium indicators Indo-1 and Fura-2. Biophys J 58:1491–1499

    Google Scholar 

  4. Bioprobes (1993) Molecular Probes, Eugene, Ore., USA 13:4

    Google Scholar 

  5. Bioprobes (1992) Molecular Probes, Eugene, Ore., USA 15:5–6

    Google Scholar 

  6. Bioprobes (1993) Molecular Probes, Eugene, Ore., USA 18:18

    Google Scholar 

  7. Burch CR (1947) Reflecting microscopes Proc Phys Soc (Lond) 59:41–49

    Google Scholar 

  8. Cannon J, Armas M (1993) Ultraviolet lasers expand uses of confocal microscopes. Laser Focus World 29:99–104

    Google Scholar 

  9. Carlsson K, Liljeborg A (1989) A confocal laser microscope scanner for digital recording of optical serial sections. J Microsc 153:171–180

    Google Scholar 

  10. Carlsson C, Mossberg K, Helm PJ, Philip J (1992) Use of UV-excitation in confocal laser scanning fluorescence microscopy. Micron Microscopica Acta 23:413–428

    Google Scholar 

  11. Williams DA, Fay FS (eds) (1990) Imaging of cell calcium — collected papers and reviews, Cell Calcium 11

  12. Cuthbertson KSR, Cobbold PH (eds) (1991) Oscillations in cell calcium — collected papers and reviews. Cell Calcium 12

  13. Diliberto PA, Herman B (1993) Quantitative estimation of PDGF-induced nuclear free calcium oscillations in single cells performed by confocal microscopy with Fluo-3 and Fura-Red (abstract). Biophys J 64:130

    Google Scholar 

  14. Dodt HU, Pawelzik H, Zieglgänsberger W (1989) Infrared DIC-videomicroscopy of living brain slices. Soc Neurosci Abstr 15:280

    Google Scholar 

  15. Dodt HU, Zieglgänsberger W (1990) Visualizing unstained neurons in living brain slices by infrared DIC-videomicroscopy. Brain Res 537:333–336

    Google Scholar 

  16. Grey DS (1950) A family of catadioptric microscope objectives. Proceedings of the London Conference of Optical Instrumentation Chapman Hall, London pp 65–76

    Google Scholar 

  17. Grey DS (1949) A new series of microscope objectives: II. Preliminary investigation of catadioptric Schwarzschild systems. J Opt Soc Am 39:723–728

    Google Scholar 

  18. Grey DS (1950) A new series of microscope objectives: III. Ultraviolet objectives of intermediate numerical aperture. J Opt Soc Am 40:283–290

    Google Scholar 

  19. Grey DS, Lee PH (1949) A new series of microscope objectives: I. Catadioptric Newtonian systems, J Opt Soc Am 39:719–723

    Google Scholar 

  20. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ -indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450

    Google Scholar 

  21. Haugland RP, Larison K (ed) (1992) Handbook of fluorescent probes and research chemicals. Molecular Probes, Eugene, Ore., pp 113–128

    Google Scholar 

  22. Kuba K, Hua SY, Nohmi M (1991) Spatial and dynamic changes in intracellular Ca2+ measured by confocal laser scanning microscopy in bullfrog sympathetic ganglion. Neurosci Res 10:245–259

    Google Scholar 

  23. Kuba K, Hua SY, Nohmi M, Hayashi T (1992) Ultraviolet laser-scanning confocal microscopy for studying dynamic functions of neurons. New Trends on Scanning Optical Microscopy, Second International OITDA-Forum, 8–10 January 1992, Naha, Okinawa, Japan, pp 25–30

    Google Scholar 

  24. Kurebayashi N (1992) Use of Fura-Red as a myoplasmic Ca2+-indicator in intact frog skeletal muscle fibers (abstract). Biophys J 61:A160

    Google Scholar 

  25. De Marinis RM, Katerinopoulos HE, Muirhead KA (1989) United States Patent 4,849,362

  26. Mc Allister RM, Isaacs H, Rongey R, Peer M, Au W, Soukup SW, Gardner MB (1977) Establishment of a human medulloblastoma cell line. Int J Cancer 20:206–212

    Google Scholar 

  27. McGuigan JAS, Lüthi D, Buri A (1991) Calcium buffer solutions and how to make them: a do it yourself guide. Can J Physiol Pharmacol 69:1733–1749

    Google Scholar 

  28. Miller RJ (1988) Neuronal Ca2+: getting it up and keeping it up. Trends Neurosci 15:317–319

    Google Scholar 

  29. Minamikawa T, Takamatsu T, Fujita S (1993) Dynamic imaging of cytosolic calcium using UV-laser confocal fluorescence microscope. The 1993 International Conference on Confocal Microscopy and 3D Image Processing, Sidney, Australia, 8–11 February 1993, Program and Book of Abstracts, p 56

  30. Minamikawa T, Takamatsu T, Kashima S, Fushiki S, Fujita S (1993) Confocal calcium imaging with ultraviolet laser scanning microscopy and Indo-1. Micron 24:551–556

    Google Scholar 

  31. Minta A, Kao JPY, Tsien RY (1989) Fluorescent indicators for cytosolic calcium based on rhodamine and fluoresceine chromophores. J Biol Chem 264:8171–8178

    Google Scholar 

  32. Moore CE (1971), Atomic energy levels, National Standards Reference Data Series National Bureau of Standards (USA), 35/V.I, December 1971, p 247

  33. Moore EDW, Becker PL, Fogarty KE, Williams DA, Fay FS (1990) Ca2+-imaging in single living cells: theoretical and practical issues Cell Calcium 11:157–179

    Google Scholar 

  34. Niggli E, Piston DW, Kirby MS, Cheng H, Sandison DR, Webb WW, Lederer WJ (1994) A confocal laser scanning microscope designed for indicators with ultraviolet excitation wavelengths. Am J Physiol 266:C303-C310

    Google Scholar 

  35. Olympus catalogue “LSM-GB200 UV”, 1993

  36. Poenie M (1990) Alteration of intracellular Fura-2 fluorescence by viscosity: a simple correction. Cell Calcium 11:85–91

    Google Scholar 

  37. Stratton MR, Darling J, Pilkington GJ, Lantos PL, Reeves BR, Cooper CS (1989) Carcinogenesis 10:899–905

    Google Scholar 

  38. Miller RJ (1988) Calcium signalling in neurons. Trends Neurosci 11:415–418

    Google Scholar 

  39. Tsien RY (1988) Fluorescence measurement and photochemical manipulation of cytosolic free calcium. Trends Neurosci 11:419–424

    Google Scholar 

  40. Campbell KP, Leung AT, Sharp AH (1988) The biochemistry and molecular biology of the dihydropyridine-sensitive calcium channel. Trends Neurosci 11:425–430

    Google Scholar 

  41. Tsien RW, Lipscombe D, Madison DV, Bley KR, Fox AP (1988) Multiple types of neuronal calcium channels and their selective modulation. Trends Neurosci 11:431–437

    Google Scholar 

  42. Blaustein MP (1988) Calcium transport and buffering in neurons. Trends Neurosci 11:438–443

    Google Scholar 

  43. Nahorski SR (1988) Inositol polyphosphates and neuronal calcium homeostasis. Trends Neurosci 11:444–448

    Google Scholar 

  44. Meldolesi J, Volpe P, Pozzan T (1988) The intracellular distribution of calcium homeostasis. Trends Neurosci 11:449–452

    Google Scholar 

  45. Fill M, Coronado R (1988) Ryanodine receptor channel of sarcoplasmic reticulum. Trends Neurosci 11:453–457

    Google Scholar 

  46. Smith SJ, Augustine GJ (1988) Calcium ions, active zones and synaptic transmitter release. Trends Neurosci 11:458–464

    Google Scholar 

  47. Choi DW (1988) Calcium-medicated neurotoxicity; relationship to specific channel types and role in ischemic damage. Trends Neurosci 11:465–468

    Google Scholar 

  48. Ulfhake B, Carlsson K, Mossberg K, Arvidsson U, Helm PJ (1991) Imaging of fluorescent neurons labelled with fluoro-gold and fluorescent axon-terminals labelled with AMCA (7-amino-4-methylcoumarine-3-acetic-acid) conjugated antiserum using a UV-laser confocal scanning microscope J Neurosci Methods 40:39–48

    Google Scholar 

  49. Uto A, Arai H, Ogawa Y (1991) Reassessment of Fura-2 and the ratio-method for determination of intracellular Ca2+ concentrations. Cell Calcium 12:29–37

    Google Scholar 

  50. Whitaker JE, Haugland RP, Prendergast FG (1991) Spectral and photophysical studies of benzo[c]xanthene dyes: dual emission pH sensors. Anal Biochem 194:330–334

    Google Scholar 

  51. Wilson T (ed) (1990) Confocal microscopy. Academic, London

    Google Scholar 

  52. Åslund N. Carlsson K (1993) Confocal scanning microfluorometry of dual-labelled specimens using two excitation wavelengths and lock-in detection technique. Micron 24:603–609

    Google Scholar 

  53. Åslund N, Carlsson K, Liljeborg A, Majlöf L (1983) PHOIBOS, a microscope scanner designed for microfluorometric applications, using laser induced fluorescence. Proceedings of the third Scandinavian conference on image analysis, pp 338–343

Download references

Author information

Authors and Affiliations

  1. Department of Cell Physiology, The Max Planck Institute for Medical Research, P.O. Box 103820, D-69028, Heidelberg, Germany

    Paul Johannes Helm

  2. Physics 4, The Royal Institute of Technology, S-100 44, Stockholm, Sweden

    Paul Johannes Helm, Olof Franksson & Kjell Carlsson

Authors
  1. Paul Johannes Helm
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Olof Franksson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kjell Carlsson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Paul Johannes Helm.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Helm, P.J., Franksson, O. & Carlsson, K. A confocal scanning laser microscope for quantitative ratiometric 3D measurements of [Ca2+] and Ca2+ diffusions in living cells stained with Fura-2. Pflügers Arch. 429, 672–681 (1995). https://doi.org/10.1007/BF00373988

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00373988

Key words

Search

Navigation