Abstract

We have investigated the possibility that variations in the level of intracellular Ca2+ in excitable cells might be induced as an artifact of the incoherent illumination that is being used to monitor transient responses. In order to avoid the fluctuations in power of an arc lamp source, a microscope using a light emitting diode that was calibrated accurately at low power levels, was constructed to provide good control over the dose of light applied to the biological specimen. We report here that higher powers of illumination increased the probability of occurrence of Ca2+ transients even in the sub-mW range normally used to measure such transients in epi-fluorescence work, suggesting that caution should be exercised when designing experiments and interpreting data.

© 2012 OSA

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2010 (1)

V. Ntziachristos, “Going deeper than microscopy: the optical imaging frontier in biology,” Nat. Methods7(8), 603–614 (2010).
[CrossRef] [PubMed]

2008 (2)

R. W. Cole and J. N. Turner, “Light-emitting diodes are better illumination sources for biological microscopy than conventional sources,” Microsc. Microanal.14(03), 243–250 (2008).
[CrossRef] [PubMed]

D. F. Albeanu, E. Soucy, T. F. Sato, M. Meister, and V. N. Murthy, “LED arrays as cost effective and efficient light sources for widefield microscopy,” PLoS ONE3(5), e2146 (2008).
[CrossRef] [PubMed]

2006 (2)

N. I. Smith, S. Iwanaga, T. Beppu, K. Fujita, O. Nakamura, and S. Kawata, “Photostimulation of two types of Ca2+ waves in rat pheochromocytoma PC12 cells by ultrashort pulsed near-infrared laser irradiation,” Laser Phys. Lett.3(3), 154–161 (2006).
[CrossRef]

S. Iwanaga, N. I. Smith, K. Fujita, and S. Kawata, “Slow Ca(2+) wave stimulation using low repetition rate femtosecond pulsed irradiation,” Opt. Express14(2), 717–725 (2006).
[CrossRef] [PubMed]

2005 (1)

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, “A guide to choosing fluorescent proteins,” Nat. Methods2(12), 905–909 (2005).
[CrossRef] [PubMed]

2003 (3)

M. M. Knight, S. R. Roberts, D. A. Lee, and D. L. Bader, “Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death,” Am. J. Physiol. Cell Physiol.284(4), C1083–C1089 (2003).
[PubMed]

R. Dixit and R. Cyr, “Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy,” Plant J.36(2), 280–290 (2003).
[CrossRef] [PubMed]

R. Lavi, A. Shainberg, H. Friedmann, V. Shneyvays, O. Rickover, M. Eichler, D. Kaplan, and R. Lubart, “Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells,” J. Biol. Chem.278(42), 40917–40922 (2003).
[CrossRef] [PubMed]

2002 (3)

N. I. Kiskin, R. Chillingworth, J. A. McCray, D. Piston, and D. Ogden, “The efficiency of two-photon photolysis of a “caged” fluorophore, o-1-(2-nitrophenyl)ethylpyranine, in relation to photodamage of synaptic terminals,” Eur. Biophys. J.30(8), 588–604 (2002).
[CrossRef] [PubMed]

J. Dempster, D. Wokosin, K. McCloskey, J. Girkin, and A. Gurney, “WinFluor-an integrated system for the simultaneous recording of cell fluorescence images and electrophysiological signals on a single computer system,” Br. J. Pharmacol.137(Suppl. S), 146P (2002).

J. Zhang, R. E. Campbell, A. Y. Ting, and R. Y. Tsien, “Creating new fluorescent probes for cell biology,” Nat. Rev. Mol. Cell Biol.3(12), 906–918 (2002).
[CrossRef] [PubMed]

2001 (1)

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, “Studying protein dynamics in living cells,” Nat. Rev. Mol. Cell Biol.2(6), 444–456 (2001).
[CrossRef] [PubMed]

2000 (1)

R. Lubart, H. Friedmann, and R. Lavie, “Photobiostimulation as a function of different wavelengths,” Laser Therapy12(1), 38–41 (2000).
[CrossRef]

1999 (3)

Y. J. Suzuki and G. D. Ford, “Redox regulation of signal transduction in cardiac and smooth muscle,” J. Mol. Cell. Cardiol.31(2), 345–353 (1999).
[CrossRef] [PubMed]

J. G. McCarron and T. C. Muir, “Mitochondrial regulation of the cytosolic Ca2+ concentration and the InsP3-sensitive Ca2+ store in guinea-pig colonic smooth muscle,” J. Physiol.516(1), 149–161 (1999).
[CrossRef] [PubMed]

J. White and E. Stelzer, “Photobleaching GFP reveals protein dynamics inside live cells,” Trends Cell Biol.9(2), 61–65 (1999).
[CrossRef] [PubMed]

1998 (1)

Albeanu, D. F.

D. F. Albeanu, E. Soucy, T. F. Sato, M. Meister, and V. N. Murthy, “LED arrays as cost effective and efficient light sources for widefield microscopy,” PLoS ONE3(5), e2146 (2008).
[CrossRef] [PubMed]

Bader, D. L.

M. M. Knight, S. R. Roberts, D. A. Lee, and D. L. Bader, “Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death,” Am. J. Physiol. Cell Physiol.284(4), C1083–C1089 (2003).
[PubMed]

Beppu, T.

N. I. Smith, S. Iwanaga, T. Beppu, K. Fujita, O. Nakamura, and S. Kawata, “Photostimulation of two types of Ca2+ waves in rat pheochromocytoma PC12 cells by ultrashort pulsed near-infrared laser irradiation,” Laser Phys. Lett.3(3), 154–161 (2006).
[CrossRef]

Campbell, R. E.

J. Zhang, R. E. Campbell, A. Y. Ting, and R. Y. Tsien, “Creating new fluorescent probes for cell biology,” Nat. Rev. Mol. Cell Biol.3(12), 906–918 (2002).
[CrossRef] [PubMed]

Chillingworth, R.

N. I. Kiskin, R. Chillingworth, J. A. McCray, D. Piston, and D. Ogden, “The efficiency of two-photon photolysis of a “caged” fluorophore, o-1-(2-nitrophenyl)ethylpyranine, in relation to photodamage of synaptic terminals,” Eur. Biophys. J.30(8), 588–604 (2002).
[CrossRef] [PubMed]

Cole, R. W.

R. W. Cole and J. N. Turner, “Light-emitting diodes are better illumination sources for biological microscopy than conventional sources,” Microsc. Microanal.14(03), 243–250 (2008).
[CrossRef] [PubMed]

Cyr, R.

R. Dixit and R. Cyr, “Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy,” Plant J.36(2), 280–290 (2003).
[CrossRef] [PubMed]

Dempster, J.

J. Dempster, D. Wokosin, K. McCloskey, J. Girkin, and A. Gurney, “WinFluor-an integrated system for the simultaneous recording of cell fluorescence images and electrophysiological signals on a single computer system,” Br. J. Pharmacol.137(Suppl. S), 146P (2002).

Dixit, R.

R. Dixit and R. Cyr, “Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy,” Plant J.36(2), 280–290 (2003).
[CrossRef] [PubMed]

Eichler, M.

R. Lavi, A. Shainberg, H. Friedmann, V. Shneyvays, O. Rickover, M. Eichler, D. Kaplan, and R. Lubart, “Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells,” J. Biol. Chem.278(42), 40917–40922 (2003).
[CrossRef] [PubMed]

Ford, G. D.

Y. J. Suzuki and G. D. Ford, “Redox regulation of signal transduction in cardiac and smooth muscle,” J. Mol. Cell. Cardiol.31(2), 345–353 (1999).
[CrossRef] [PubMed]

Friedmann, H.

R. Lavi, A. Shainberg, H. Friedmann, V. Shneyvays, O. Rickover, M. Eichler, D. Kaplan, and R. Lubart, “Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells,” J. Biol. Chem.278(42), 40917–40922 (2003).
[CrossRef] [PubMed]

R. Lubart, H. Friedmann, and R. Lavie, “Photobiostimulation as a function of different wavelengths,” Laser Therapy12(1), 38–41 (2000).
[CrossRef]

Fujita, K.

N. I. Smith, S. Iwanaga, T. Beppu, K. Fujita, O. Nakamura, and S. Kawata, “Photostimulation of two types of Ca2+ waves in rat pheochromocytoma PC12 cells by ultrashort pulsed near-infrared laser irradiation,” Laser Phys. Lett.3(3), 154–161 (2006).
[CrossRef]

S. Iwanaga, N. I. Smith, K. Fujita, and S. Kawata, “Slow Ca(2+) wave stimulation using low repetition rate femtosecond pulsed irradiation,” Opt. Express14(2), 717–725 (2006).
[CrossRef] [PubMed]

Girkin, J.

J. Dempster, D. Wokosin, K. McCloskey, J. Girkin, and A. Gurney, “WinFluor-an integrated system for the simultaneous recording of cell fluorescence images and electrophysiological signals on a single computer system,” Br. J. Pharmacol.137(Suppl. S), 146P (2002).

Gurney, A.

J. Dempster, D. Wokosin, K. McCloskey, J. Girkin, and A. Gurney, “WinFluor-an integrated system for the simultaneous recording of cell fluorescence images and electrophysiological signals on a single computer system,” Br. J. Pharmacol.137(Suppl. S), 146P (2002).

Hell, S. W.

Iwanaga, S.

N. I. Smith, S. Iwanaga, T. Beppu, K. Fujita, O. Nakamura, and S. Kawata, “Photostimulation of two types of Ca2+ waves in rat pheochromocytoma PC12 cells by ultrashort pulsed near-infrared laser irradiation,” Laser Phys. Lett.3(3), 154–161 (2006).
[CrossRef]

S. Iwanaga, N. I. Smith, K. Fujita, and S. Kawata, “Slow Ca(2+) wave stimulation using low repetition rate femtosecond pulsed irradiation,” Opt. Express14(2), 717–725 (2006).
[CrossRef] [PubMed]

Kaplan, D.

R. Lavi, A. Shainberg, H. Friedmann, V. Shneyvays, O. Rickover, M. Eichler, D. Kaplan, and R. Lubart, “Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells,” J. Biol. Chem.278(42), 40917–40922 (2003).
[CrossRef] [PubMed]

Kawata, S.

S. Iwanaga, N. I. Smith, K. Fujita, and S. Kawata, “Slow Ca(2+) wave stimulation using low repetition rate femtosecond pulsed irradiation,” Opt. Express14(2), 717–725 (2006).
[CrossRef] [PubMed]

N. I. Smith, S. Iwanaga, T. Beppu, K. Fujita, O. Nakamura, and S. Kawata, “Photostimulation of two types of Ca2+ waves in rat pheochromocytoma PC12 cells by ultrashort pulsed near-infrared laser irradiation,” Laser Phys. Lett.3(3), 154–161 (2006).
[CrossRef]

Kenworthy, A.

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, “Studying protein dynamics in living cells,” Nat. Rev. Mol. Cell Biol.2(6), 444–456 (2001).
[CrossRef] [PubMed]

Kiskin, N. I.

N. I. Kiskin, R. Chillingworth, J. A. McCray, D. Piston, and D. Ogden, “The efficiency of two-photon photolysis of a “caged” fluorophore, o-1-(2-nitrophenyl)ethylpyranine, in relation to photodamage of synaptic terminals,” Eur. Biophys. J.30(8), 588–604 (2002).
[CrossRef] [PubMed]

Knight, M. M.

M. M. Knight, S. R. Roberts, D. A. Lee, and D. L. Bader, “Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death,” Am. J. Physiol. Cell Physiol.284(4), C1083–C1089 (2003).
[PubMed]

Lavi, R.

R. Lavi, A. Shainberg, H. Friedmann, V. Shneyvays, O. Rickover, M. Eichler, D. Kaplan, and R. Lubart, “Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells,” J. Biol. Chem.278(42), 40917–40922 (2003).
[CrossRef] [PubMed]

Lavie, R.

R. Lubart, H. Friedmann, and R. Lavie, “Photobiostimulation as a function of different wavelengths,” Laser Therapy12(1), 38–41 (2000).
[CrossRef]

Lee, D. A.

M. M. Knight, S. R. Roberts, D. A. Lee, and D. L. Bader, “Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death,” Am. J. Physiol. Cell Physiol.284(4), C1083–C1089 (2003).
[PubMed]

Lippincott-Schwartz, J.

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, “Studying protein dynamics in living cells,” Nat. Rev. Mol. Cell Biol.2(6), 444–456 (2001).
[CrossRef] [PubMed]

Lubart, R.

R. Lavi, A. Shainberg, H. Friedmann, V. Shneyvays, O. Rickover, M. Eichler, D. Kaplan, and R. Lubart, “Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells,” J. Biol. Chem.278(42), 40917–40922 (2003).
[CrossRef] [PubMed]

R. Lubart, H. Friedmann, and R. Lavie, “Photobiostimulation as a function of different wavelengths,” Laser Therapy12(1), 38–41 (2000).
[CrossRef]

McCarron, J. G.

J. G. McCarron and T. C. Muir, “Mitochondrial regulation of the cytosolic Ca2+ concentration and the InsP3-sensitive Ca2+ store in guinea-pig colonic smooth muscle,” J. Physiol.516(1), 149–161 (1999).
[CrossRef] [PubMed]

McCloskey, K.

J. Dempster, D. Wokosin, K. McCloskey, J. Girkin, and A. Gurney, “WinFluor-an integrated system for the simultaneous recording of cell fluorescence images and electrophysiological signals on a single computer system,” Br. J. Pharmacol.137(Suppl. S), 146P (2002).

McCray, J. A.

N. I. Kiskin, R. Chillingworth, J. A. McCray, D. Piston, and D. Ogden, “The efficiency of two-photon photolysis of a “caged” fluorophore, o-1-(2-nitrophenyl)ethylpyranine, in relation to photodamage of synaptic terminals,” Eur. Biophys. J.30(8), 588–604 (2002).
[CrossRef] [PubMed]

Meister, M.

D. F. Albeanu, E. Soucy, T. F. Sato, M. Meister, and V. N. Murthy, “LED arrays as cost effective and efficient light sources for widefield microscopy,” PLoS ONE3(5), e2146 (2008).
[CrossRef] [PubMed]

Muir, T. C.

J. G. McCarron and T. C. Muir, “Mitochondrial regulation of the cytosolic Ca2+ concentration and the InsP3-sensitive Ca2+ store in guinea-pig colonic smooth muscle,” J. Physiol.516(1), 149–161 (1999).
[CrossRef] [PubMed]

Murthy, V. N.

D. F. Albeanu, E. Soucy, T. F. Sato, M. Meister, and V. N. Murthy, “LED arrays as cost effective and efficient light sources for widefield microscopy,” PLoS ONE3(5), e2146 (2008).
[CrossRef] [PubMed]

Nakamura, O.

N. I. Smith, S. Iwanaga, T. Beppu, K. Fujita, O. Nakamura, and S. Kawata, “Photostimulation of two types of Ca2+ waves in rat pheochromocytoma PC12 cells by ultrashort pulsed near-infrared laser irradiation,” Laser Phys. Lett.3(3), 154–161 (2006).
[CrossRef]

Ntziachristos, V.

V. Ntziachristos, “Going deeper than microscopy: the optical imaging frontier in biology,” Nat. Methods7(8), 603–614 (2010).
[CrossRef] [PubMed]

Ogden, D.

N. I. Kiskin, R. Chillingworth, J. A. McCray, D. Piston, and D. Ogden, “The efficiency of two-photon photolysis of a “caged” fluorophore, o-1-(2-nitrophenyl)ethylpyranine, in relation to photodamage of synaptic terminals,” Eur. Biophys. J.30(8), 588–604 (2002).
[CrossRef] [PubMed]

Piston, D.

N. I. Kiskin, R. Chillingworth, J. A. McCray, D. Piston, and D. Ogden, “The efficiency of two-photon photolysis of a “caged” fluorophore, o-1-(2-nitrophenyl)ethylpyranine, in relation to photodamage of synaptic terminals,” Eur. Biophys. J.30(8), 588–604 (2002).
[CrossRef] [PubMed]

Rickover, O.

R. Lavi, A. Shainberg, H. Friedmann, V. Shneyvays, O. Rickover, M. Eichler, D. Kaplan, and R. Lubart, “Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells,” J. Biol. Chem.278(42), 40917–40922 (2003).
[CrossRef] [PubMed]

Roberts, S. R.

M. M. Knight, S. R. Roberts, D. A. Lee, and D. L. Bader, “Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death,” Am. J. Physiol. Cell Physiol.284(4), C1083–C1089 (2003).
[PubMed]

Sato, T. F.

D. F. Albeanu, E. Soucy, T. F. Sato, M. Meister, and V. N. Murthy, “LED arrays as cost effective and efficient light sources for widefield microscopy,” PLoS ONE3(5), e2146 (2008).
[CrossRef] [PubMed]

Schönle, A.

Shainberg, A.

R. Lavi, A. Shainberg, H. Friedmann, V. Shneyvays, O. Rickover, M. Eichler, D. Kaplan, and R. Lubart, “Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells,” J. Biol. Chem.278(42), 40917–40922 (2003).
[CrossRef] [PubMed]

Shaner, N. C.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, “A guide to choosing fluorescent proteins,” Nat. Methods2(12), 905–909 (2005).
[CrossRef] [PubMed]

Shneyvays, V.

R. Lavi, A. Shainberg, H. Friedmann, V. Shneyvays, O. Rickover, M. Eichler, D. Kaplan, and R. Lubart, “Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells,” J. Biol. Chem.278(42), 40917–40922 (2003).
[CrossRef] [PubMed]

Smith, N. I.

S. Iwanaga, N. I. Smith, K. Fujita, and S. Kawata, “Slow Ca(2+) wave stimulation using low repetition rate femtosecond pulsed irradiation,” Opt. Express14(2), 717–725 (2006).
[CrossRef] [PubMed]

N. I. Smith, S. Iwanaga, T. Beppu, K. Fujita, O. Nakamura, and S. Kawata, “Photostimulation of two types of Ca2+ waves in rat pheochromocytoma PC12 cells by ultrashort pulsed near-infrared laser irradiation,” Laser Phys. Lett.3(3), 154–161 (2006).
[CrossRef]

Snapp, E.

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, “Studying protein dynamics in living cells,” Nat. Rev. Mol. Cell Biol.2(6), 444–456 (2001).
[CrossRef] [PubMed]

Soucy, E.

D. F. Albeanu, E. Soucy, T. F. Sato, M. Meister, and V. N. Murthy, “LED arrays as cost effective and efficient light sources for widefield microscopy,” PLoS ONE3(5), e2146 (2008).
[CrossRef] [PubMed]

Steinbach, P. A.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, “A guide to choosing fluorescent proteins,” Nat. Methods2(12), 905–909 (2005).
[CrossRef] [PubMed]

Stelzer, E.

J. White and E. Stelzer, “Photobleaching GFP reveals protein dynamics inside live cells,” Trends Cell Biol.9(2), 61–65 (1999).
[CrossRef] [PubMed]

Suzuki, Y. J.

Y. J. Suzuki and G. D. Ford, “Redox regulation of signal transduction in cardiac and smooth muscle,” J. Mol. Cell. Cardiol.31(2), 345–353 (1999).
[CrossRef] [PubMed]

Ting, A. Y.

J. Zhang, R. E. Campbell, A. Y. Ting, and R. Y. Tsien, “Creating new fluorescent probes for cell biology,” Nat. Rev. Mol. Cell Biol.3(12), 906–918 (2002).
[CrossRef] [PubMed]

Tsien, R. Y.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, “A guide to choosing fluorescent proteins,” Nat. Methods2(12), 905–909 (2005).
[CrossRef] [PubMed]

J. Zhang, R. E. Campbell, A. Y. Ting, and R. Y. Tsien, “Creating new fluorescent probes for cell biology,” Nat. Rev. Mol. Cell Biol.3(12), 906–918 (2002).
[CrossRef] [PubMed]

Turner, J. N.

R. W. Cole and J. N. Turner, “Light-emitting diodes are better illumination sources for biological microscopy than conventional sources,” Microsc. Microanal.14(03), 243–250 (2008).
[CrossRef] [PubMed]

White, J.

J. White and E. Stelzer, “Photobleaching GFP reveals protein dynamics inside live cells,” Trends Cell Biol.9(2), 61–65 (1999).
[CrossRef] [PubMed]

Wokosin, D.

J. Dempster, D. Wokosin, K. McCloskey, J. Girkin, and A. Gurney, “WinFluor-an integrated system for the simultaneous recording of cell fluorescence images and electrophysiological signals on a single computer system,” Br. J. Pharmacol.137(Suppl. S), 146P (2002).

Zhang, J.

J. Zhang, R. E. Campbell, A. Y. Ting, and R. Y. Tsien, “Creating new fluorescent probes for cell biology,” Nat. Rev. Mol. Cell Biol.3(12), 906–918 (2002).
[CrossRef] [PubMed]

Am. J. Physiol. Cell Physiol. (1)

M. M. Knight, S. R. Roberts, D. A. Lee, and D. L. Bader, “Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death,” Am. J. Physiol. Cell Physiol.284(4), C1083–C1089 (2003).
[PubMed]

Br. J. Pharmacol. (1)

J. Dempster, D. Wokosin, K. McCloskey, J. Girkin, and A. Gurney, “WinFluor-an integrated system for the simultaneous recording of cell fluorescence images and electrophysiological signals on a single computer system,” Br. J. Pharmacol.137(Suppl. S), 146P (2002).

Eur. Biophys. J. (1)

N. I. Kiskin, R. Chillingworth, J. A. McCray, D. Piston, and D. Ogden, “The efficiency of two-photon photolysis of a “caged” fluorophore, o-1-(2-nitrophenyl)ethylpyranine, in relation to photodamage of synaptic terminals,” Eur. Biophys. J.30(8), 588–604 (2002).
[CrossRef] [PubMed]

J. Biol. Chem. (1)

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Supplementary Material (1)

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Figures (5)

Fig. 1
Fig. 1

Schematic of widefield epi-fluorescence and brightfield transmission microscope system. Brightfield transmission was collected by the condenser lens and viewed through the eyepiece. White light illumination from the lamp was computer controlled via a flip mirror. The 488 nm LED excitation source was lensed and filtered externally from the inverted microscope to uniformly fill the field of view. Fluorescence was detected using a highly sensitive fast CCD camera with a 520nm LP filter in the detection path.

Fig. 2
Fig. 2

The number of cells exhibiting Ca2+ responses for n = 30 cells are shown for each optical power setting. The responses are categorized as global or localized response. The data is expressed with a 5% error in detection for optical power.

Fig. 3
Fig. 3

(a) Extracted frames from a movie of an isolated smooth muscle cell with 150 μW of 488 nm irradiation. The progression of a localized Ca2+ transient is shown at time points i) 14.5 seconds, ii) 16.4 seconds, iii) 17.2 seconds and iv) 20.8 seconds. The region of interest is indicated by the broken circle. (b) Extracted frames of the same cell shown in (a) at a later time point. The progression of a global Ca2+ transient is shown over the following time points i) 35.6 seconds, ii) 35.9 seconds, iii) 36.8 seconds and iv) 50.1 seconds. (c) Corresponding fluorescence signal intensity profiles over the course of the acquisition period shown in (a) (b). 3(c)i shows the intensity in the region circled in (a) over time, and 3(c)ii shows the intensity of the full cell over time. Ca2+ images (a) and (b), are taken from the time points indicated in (ci) and (cii), respectively.

Fig. 4
Fig. 4

A global Ca2+ transient was observed after a step-wise increase in optical power (Media 1). The specimen was initially irradiated from t = 0-80 seconds at 30μW (not shown). The 30 μW irradiation continued from t = 80 seconds until t = 164 seconds, when the average power was increased to 70 μW. The specimen was illuminated from t = 164 seconds to t = 271.2 seconds, and then the power was incremented to 110 μW for the remainder of the experiment. A global Ca2+ transient is observed with illumination of 110 μW average power at t = 307.6 seconds. The selected frame shows the recovered cell at t = 400.00 seconds. The cross and ‘1’ indicates a highlighted single point from which the fluorescence intensity over time data was plotted.

Fig. 5
Fig. 5

A global Ca2+ rise resulting in contraction of a single smooth muscle cell under epi-fluorescence excitation. Frames show (a) an unresponsive cell under 70 μW illumination at t = 309.10 seconds, (b) a global Ca2+ rise under 110 μW illumination at t = 318.40 seconds, (c) subsequent cell contraction at t = 322.90 seconds followed by (d) recovery at t = 381.40 seconds. The cross (+) and ‘1’ indicates a highlighted single point from which it was possible to plot fluorescence intensity over time data (not shown).

Tables (1)

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Table 1 LED current correlated with power at sample, with scale reading uncertainty

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