Abstract

This paper discusses the task of true physical quantity reconstruction from experimental data on the basis of fluorescence microscopy images when a specific ion indicator is used for an investigation into ion concentration. Brain cell functional activity in the form of calcium oscillation may be processed as quantitation on the basis of measurements of single-wavelength fluorescence intensity using data about dye loading protocol, excitation, and detection conditions without calibration of every experiment.

© 2012 Optical Society of America

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References

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  1. J. B. Pawley, ed., Handbook of Biological Confocal Microscopy (Springer, 2006).
  2. M. Zaidi, A. S. M Towhidul Alam, Ch. Bax, V. Shankar, P. F. R. Bevis, Ch. L. H. Huang, M. Pazianas, and B. S. Moonga, “Cytosolic free calcium measurements in single cell using calcium-sensitive fluorochromes,” in Methods in Molecular Biology, J. Graham and J. A. Higgins, eds. (Humana, 1994), pp. 279–293.
  3. R. P. Haugland and M. T. Z. Spence, eds., Handbook of Fluorescent Probes and Research Products, 9th ed. (Molecular Probes, 2002).
  4. G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260, 3440–3450 (1985).
  5. I. V. Mukhina, V. B. Kazantsev, L. G. Khaspekov, Yu. N. Zackharov, M. V. Vedunova, E. V. Mitroshina, S. A. Korotchenko, and E. A. Koryagina, “Multielectrode arrays—new possibilities in investigation of the neuronal network plasticity,” Mod. Tech. Med.1, 8–15 (2009).
  6. BioProbes, “Fluorescent calcium indicators for in vivoimaging of neuronal networks” (Invitrogen, 2007), Vol. 54, pp. 26–27.
  7. S. Namiki, T. Sasaki, N. Matsuski, and Y. Ikegaya, “Regional difference in stainability with calcium-sensitive acetoxymetyl-ester probes in mice brain slices,” Int. J. Neurosci. 119, 214–216 (2008).

2008

S. Namiki, T. Sasaki, N. Matsuski, and Y. Ikegaya, “Regional difference in stainability with calcium-sensitive acetoxymetyl-ester probes in mice brain slices,” Int. J. Neurosci. 119, 214–216 (2008).

1985

G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260, 3440–3450 (1985).

Bax, Ch.

M. Zaidi, A. S. M Towhidul Alam, Ch. Bax, V. Shankar, P. F. R. Bevis, Ch. L. H. Huang, M. Pazianas, and B. S. Moonga, “Cytosolic free calcium measurements in single cell using calcium-sensitive fluorochromes,” in Methods in Molecular Biology, J. Graham and J. A. Higgins, eds. (Humana, 1994), pp. 279–293.

Bevis, P. F. R.

M. Zaidi, A. S. M Towhidul Alam, Ch. Bax, V. Shankar, P. F. R. Bevis, Ch. L. H. Huang, M. Pazianas, and B. S. Moonga, “Cytosolic free calcium measurements in single cell using calcium-sensitive fluorochromes,” in Methods in Molecular Biology, J. Graham and J. A. Higgins, eds. (Humana, 1994), pp. 279–293.

BioProbes,

BioProbes, “Fluorescent calcium indicators for in vivoimaging of neuronal networks” (Invitrogen, 2007), Vol. 54, pp. 26–27.

Grynkiewicz, G.

G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260, 3440–3450 (1985).

Huang, Ch. L. H.

M. Zaidi, A. S. M Towhidul Alam, Ch. Bax, V. Shankar, P. F. R. Bevis, Ch. L. H. Huang, M. Pazianas, and B. S. Moonga, “Cytosolic free calcium measurements in single cell using calcium-sensitive fluorochromes,” in Methods in Molecular Biology, J. Graham and J. A. Higgins, eds. (Humana, 1994), pp. 279–293.

Ikegaya, Y.

S. Namiki, T. Sasaki, N. Matsuski, and Y. Ikegaya, “Regional difference in stainability with calcium-sensitive acetoxymetyl-ester probes in mice brain slices,” Int. J. Neurosci. 119, 214–216 (2008).

Kazantsev, V. B.

I. V. Mukhina, V. B. Kazantsev, L. G. Khaspekov, Yu. N. Zackharov, M. V. Vedunova, E. V. Mitroshina, S. A. Korotchenko, and E. A. Koryagina, “Multielectrode arrays—new possibilities in investigation of the neuronal network plasticity,” Mod. Tech. Med.1, 8–15 (2009).

Khaspekov, L. G.

I. V. Mukhina, V. B. Kazantsev, L. G. Khaspekov, Yu. N. Zackharov, M. V. Vedunova, E. V. Mitroshina, S. A. Korotchenko, and E. A. Koryagina, “Multielectrode arrays—new possibilities in investigation of the neuronal network plasticity,” Mod. Tech. Med.1, 8–15 (2009).

Korotchenko, S. A.

I. V. Mukhina, V. B. Kazantsev, L. G. Khaspekov, Yu. N. Zackharov, M. V. Vedunova, E. V. Mitroshina, S. A. Korotchenko, and E. A. Koryagina, “Multielectrode arrays—new possibilities in investigation of the neuronal network plasticity,” Mod. Tech. Med.1, 8–15 (2009).

Koryagina, E. A.

I. V. Mukhina, V. B. Kazantsev, L. G. Khaspekov, Yu. N. Zackharov, M. V. Vedunova, E. V. Mitroshina, S. A. Korotchenko, and E. A. Koryagina, “Multielectrode arrays—new possibilities in investigation of the neuronal network plasticity,” Mod. Tech. Med.1, 8–15 (2009).

Matsuski, N.

S. Namiki, T. Sasaki, N. Matsuski, and Y. Ikegaya, “Regional difference in stainability with calcium-sensitive acetoxymetyl-ester probes in mice brain slices,” Int. J. Neurosci. 119, 214–216 (2008).

Mitroshina, E. V.

I. V. Mukhina, V. B. Kazantsev, L. G. Khaspekov, Yu. N. Zackharov, M. V. Vedunova, E. V. Mitroshina, S. A. Korotchenko, and E. A. Koryagina, “Multielectrode arrays—new possibilities in investigation of the neuronal network plasticity,” Mod. Tech. Med.1, 8–15 (2009).

Moonga, B. S.

M. Zaidi, A. S. M Towhidul Alam, Ch. Bax, V. Shankar, P. F. R. Bevis, Ch. L. H. Huang, M. Pazianas, and B. S. Moonga, “Cytosolic free calcium measurements in single cell using calcium-sensitive fluorochromes,” in Methods in Molecular Biology, J. Graham and J. A. Higgins, eds. (Humana, 1994), pp. 279–293.

Mukhina, I. V.

I. V. Mukhina, V. B. Kazantsev, L. G. Khaspekov, Yu. N. Zackharov, M. V. Vedunova, E. V. Mitroshina, S. A. Korotchenko, and E. A. Koryagina, “Multielectrode arrays—new possibilities in investigation of the neuronal network plasticity,” Mod. Tech. Med.1, 8–15 (2009).

Namiki, S.

S. Namiki, T. Sasaki, N. Matsuski, and Y. Ikegaya, “Regional difference in stainability with calcium-sensitive acetoxymetyl-ester probes in mice brain slices,” Int. J. Neurosci. 119, 214–216 (2008).

Pazianas, M.

M. Zaidi, A. S. M Towhidul Alam, Ch. Bax, V. Shankar, P. F. R. Bevis, Ch. L. H. Huang, M. Pazianas, and B. S. Moonga, “Cytosolic free calcium measurements in single cell using calcium-sensitive fluorochromes,” in Methods in Molecular Biology, J. Graham and J. A. Higgins, eds. (Humana, 1994), pp. 279–293.

Poenie, M.

G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260, 3440–3450 (1985).

Sasaki, T.

S. Namiki, T. Sasaki, N. Matsuski, and Y. Ikegaya, “Regional difference in stainability with calcium-sensitive acetoxymetyl-ester probes in mice brain slices,” Int. J. Neurosci. 119, 214–216 (2008).

Shankar, V.

M. Zaidi, A. S. M Towhidul Alam, Ch. Bax, V. Shankar, P. F. R. Bevis, Ch. L. H. Huang, M. Pazianas, and B. S. Moonga, “Cytosolic free calcium measurements in single cell using calcium-sensitive fluorochromes,” in Methods in Molecular Biology, J. Graham and J. A. Higgins, eds. (Humana, 1994), pp. 279–293.

Towhidul Alam, A. S. M

M. Zaidi, A. S. M Towhidul Alam, Ch. Bax, V. Shankar, P. F. R. Bevis, Ch. L. H. Huang, M. Pazianas, and B. S. Moonga, “Cytosolic free calcium measurements in single cell using calcium-sensitive fluorochromes,” in Methods in Molecular Biology, J. Graham and J. A. Higgins, eds. (Humana, 1994), pp. 279–293.

Tsien, R. Y.

G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260, 3440–3450 (1985).

Vedunova, M. V.

I. V. Mukhina, V. B. Kazantsev, L. G. Khaspekov, Yu. N. Zackharov, M. V. Vedunova, E. V. Mitroshina, S. A. Korotchenko, and E. A. Koryagina, “Multielectrode arrays—new possibilities in investigation of the neuronal network plasticity,” Mod. Tech. Med.1, 8–15 (2009).

Zackharov, Yu. N.

I. V. Mukhina, V. B. Kazantsev, L. G. Khaspekov, Yu. N. Zackharov, M. V. Vedunova, E. V. Mitroshina, S. A. Korotchenko, and E. A. Koryagina, “Multielectrode arrays—new possibilities in investigation of the neuronal network plasticity,” Mod. Tech. Med.1, 8–15 (2009).

Zaidi, M.

M. Zaidi, A. S. M Towhidul Alam, Ch. Bax, V. Shankar, P. F. R. Bevis, Ch. L. H. Huang, M. Pazianas, and B. S. Moonga, “Cytosolic free calcium measurements in single cell using calcium-sensitive fluorochromes,” in Methods in Molecular Biology, J. Graham and J. A. Higgins, eds. (Humana, 1994), pp. 279–293.

Int. J. Neurosci.

S. Namiki, T. Sasaki, N. Matsuski, and Y. Ikegaya, “Regional difference in stainability with calcium-sensitive acetoxymetyl-ester probes in mice brain slices,” Int. J. Neurosci. 119, 214–216 (2008).

J. Biol. Chem.

G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260, 3440–3450 (1985).

Other

I. V. Mukhina, V. B. Kazantsev, L. G. Khaspekov, Yu. N. Zackharov, M. V. Vedunova, E. V. Mitroshina, S. A. Korotchenko, and E. A. Koryagina, “Multielectrode arrays—new possibilities in investigation of the neuronal network plasticity,” Mod. Tech. Med.1, 8–15 (2009).

BioProbes, “Fluorescent calcium indicators for in vivoimaging of neuronal networks” (Invitrogen, 2007), Vol. 54, pp. 26–27.

J. B. Pawley, ed., Handbook of Biological Confocal Microscopy (Springer, 2006).

M. Zaidi, A. S. M Towhidul Alam, Ch. Bax, V. Shankar, P. F. R. Bevis, Ch. L. H. Huang, M. Pazianas, and B. S. Moonga, “Cytosolic free calcium measurements in single cell using calcium-sensitive fluorochromes,” in Methods in Molecular Biology, J. Graham and J. A. Higgins, eds. (Humana, 1994), pp. 279–293.

R. P. Haugland and M. T. Z. Spence, eds., Handbook of Fluorescent Probes and Research Products, 9th ed. (Molecular Probes, 2002).

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Figures (5)

Fig. 1.
Fig. 1.

Microscopic fluorescent image of cell culture, loaded by specific calcium dye.

Fig. 2.
Fig. 2.

Fluorescence intensity (RUs) of glial cell image ROI 1.

Fig. 3.
Fig. 3.

Fluorescence intensity (RUs) of neuron image ROI 2.

Fig. 4.
Fig. 4.

Time dependence of [CA2+] in glial cell (ROI 1).

Fig. 5.
Fig. 5.

Time dependence of [CA2+] in neuron (ROI 2).

Equations (13)

Equations on this page are rendered with MathJax. Learn more.

[Ca]=KdFFminFmaxF,
F=ΦQDQFαnI0.
S=ΦQDQFαI0
F=Sfnf+Sbnb,
nbnf=[Ion]fKd.
nΣ=nf+nb,
[Ion]f=KdFSfnΣSbnΣF.
[Ion]total=KdFSfnΣSbnΣF+FSfnΣSbSf.
0.1Kd<[Ion]f<10Kd,
Iabs=I0(1exp{αnl}),
IabsI0αnl,
I0=P0Sfocal=166Wmm2,
[Ca2+]i=KdFSfnΣSbnΣF.

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