Abstract

We report the design, development, and implementation of an improved instrumentation approach for frequency-domain fluorescence lifetime (FDFL) optrodic sensing without a concurrent reference LED. FDFL traditionally uses a reference LED, at approximately the same wavelength as the sensor fluorophore emission, to measure phase shifts associated with changes in the fluorescence lifetime of fluorophore. For this work we used an oxygen optrode to design, develop, and test the reference-LED-free FDFL approach. Electronics and optics were optimized, and key system parameters, such as inherent system phase shifts, were determined to insure best performance. In our tests with the oxygen optrode, we observed that several key performance characteristics were improved by the implementation of the reference-LED-free instrumentation platform. This system can potentially be adapted to other analyte-selective fluorophores, which will enable scientists and researchers to expand the application of optrodic sensors as basic research tools in biology, medicine, and agriculture.

© 2009 Optical Society of America

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References

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  1. S. M. Borisov and O. S. Wolfbeis, “Optical biosensors,” Chem. Rev. 108, 423-461 (2008).
    [CrossRef]
  2. O. S. Wolfbeis, “Fiber optic chemical sensors and biosensors,” Anal. Chem. 76, 3269-3284 (2004).
    [CrossRef]
  3. A. ul Haque, M. R. Chatni, G. Li, and D. M. Porterfield, “Biochips and other microtechnologies for physiomics,” Expert Rev. Proteomics 4, 553-563 (2007).
    [CrossRef]
  4. J. R. Lackowicz, Principles of Fluorescence Spectroscopy (Springer, 2006).
  5. K. Carlsson and A. Liljeborg, “Simultaneous confocal lifetime imaging of multiple imaging of fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique,” J. Microsc. 191, 119-127 (1998).
    [CrossRef]
  6. B. Chance, M. Cope, E. Gratton, N. Ramanujam, and B. Tromberg, “Phase measurement of light absorption and scatter in human tissue,” Rev. Sci. Instrum. 69, 3457-3481(1998).
    [CrossRef]
  7. J. E. Cantle, Atomic Absorption Spectrometry (Elsevier, 1982).
  8. D. M. Porterfield, J. L. Rickus, and R. Kopelmann, “Non-invasive approaches to measuring respiratory patterns using a PtTFPP based phase-lifetime self-referencing oxygen optrode,” Proc. SPIE 6380, 63800S (2006).
    [CrossRef]
  9. M. R. Chatni, D. E. Maier, and D. M. Porterfield, “Evaluation of microparticle materials for enhancing the performance of fluorescence lifetime based optrodes,” Sens. Actuators B 141, 471-477 (2009).
    [CrossRef]
  10. M. J. O'Brien, E. Rabinovich, S. R. J. Brueck, and G. P. Lopez “Technique for detecting changes in fluorescence lifetime by means of optoelectronic circuit auto-oscillation,” Opt. Lett. 26, 1256-1258 (2001).
    [CrossRef]
  11. O. S. Knudsen, Biological Membranes: Theory of Transport Potentials and Electric Impulses (Cambridge U. Press, 2003).
  12. T. Mock, G. S. Dieckmann, C. Haas, A. Krell, J. L. Tison, A. L. Belem, S. Papadimitriou, and D. N. Thomas, “Micro-optrodes in sea-ice: a new approach to investigate oxygen dynamics during sea ice formation,” Aquat. Microb. Ecol. 29, 297-306 (2002).
    [CrossRef]
  13. B. C. Sanchez, H. Ochoa-Acuna, D. M. Porterfield, and M. S. Sepulvada, “Oxygen flux as an indicator of physiological stress in fathead minnow embryos: a real-time biomonitoring system of water quality,” Environ. Sci. Technol. 42, 7010-7017(2008).
    [CrossRef]
  14. K. Kolasinski, Surface Science: Foundations of Catalysis and Nanoscience (Wiley, 2002).
  15. J. N. Demas, B. A. DeGraff, and W. Xu, “Modeling of luminescence quenching-based sensors: comparison of multi-site and non-linear gas solubility models,” Anal. Chem. 67, 1377-1380(1995).
    [CrossRef]
  16. V. I. Ogurtsov and D. B. Papkovsky, “Approximation of calibration for phase-fluorometric oxygen sensors on the basis of physical models,” Sens. Actuators B 88, 89-100(2003).
    [CrossRef]
  17. K. A. Kneas, J. N. Demas, B. A. DeGraff, and A. Periasamy, “Fluorescence microscopy study of heterogeneity in polymer-supported luminescence-based oxygen sensors,” Microsc. Microanal. 6, 551-561 (2000).
  18. E. R. Carraway, J. N. Demas, B. A. DeGraff, and R. Bacon, “Photophysics and oxygen quenching of transition-metal complexes on fumed silica,” Langmuir 7, 2991-2998 (1991).
    [CrossRef]

2009 (1)

M. R. Chatni, D. E. Maier, and D. M. Porterfield, “Evaluation of microparticle materials for enhancing the performance of fluorescence lifetime based optrodes,” Sens. Actuators B 141, 471-477 (2009).
[CrossRef]

2008 (2)

S. M. Borisov and O. S. Wolfbeis, “Optical biosensors,” Chem. Rev. 108, 423-461 (2008).
[CrossRef]

B. C. Sanchez, H. Ochoa-Acuna, D. M. Porterfield, and M. S. Sepulvada, “Oxygen flux as an indicator of physiological stress in fathead minnow embryos: a real-time biomonitoring system of water quality,” Environ. Sci. Technol. 42, 7010-7017(2008).
[CrossRef]

2007 (1)

A. ul Haque, M. R. Chatni, G. Li, and D. M. Porterfield, “Biochips and other microtechnologies for physiomics,” Expert Rev. Proteomics 4, 553-563 (2007).
[CrossRef]

2006 (1)

D. M. Porterfield, J. L. Rickus, and R. Kopelmann, “Non-invasive approaches to measuring respiratory patterns using a PtTFPP based phase-lifetime self-referencing oxygen optrode,” Proc. SPIE 6380, 63800S (2006).
[CrossRef]

2004 (1)

O. S. Wolfbeis, “Fiber optic chemical sensors and biosensors,” Anal. Chem. 76, 3269-3284 (2004).
[CrossRef]

2003 (1)

V. I. Ogurtsov and D. B. Papkovsky, “Approximation of calibration for phase-fluorometric oxygen sensors on the basis of physical models,” Sens. Actuators B 88, 89-100(2003).
[CrossRef]

2002 (1)

T. Mock, G. S. Dieckmann, C. Haas, A. Krell, J. L. Tison, A. L. Belem, S. Papadimitriou, and D. N. Thomas, “Micro-optrodes in sea-ice: a new approach to investigate oxygen dynamics during sea ice formation,” Aquat. Microb. Ecol. 29, 297-306 (2002).
[CrossRef]

2001 (1)

2000 (1)

K. A. Kneas, J. N. Demas, B. A. DeGraff, and A. Periasamy, “Fluorescence microscopy study of heterogeneity in polymer-supported luminescence-based oxygen sensors,” Microsc. Microanal. 6, 551-561 (2000).

1998 (2)

K. Carlsson and A. Liljeborg, “Simultaneous confocal lifetime imaging of multiple imaging of fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique,” J. Microsc. 191, 119-127 (1998).
[CrossRef]

B. Chance, M. Cope, E. Gratton, N. Ramanujam, and B. Tromberg, “Phase measurement of light absorption and scatter in human tissue,” Rev. Sci. Instrum. 69, 3457-3481(1998).
[CrossRef]

1995 (1)

J. N. Demas, B. A. DeGraff, and W. Xu, “Modeling of luminescence quenching-based sensors: comparison of multi-site and non-linear gas solubility models,” Anal. Chem. 67, 1377-1380(1995).
[CrossRef]

1991 (1)

E. R. Carraway, J. N. Demas, B. A. DeGraff, and R. Bacon, “Photophysics and oxygen quenching of transition-metal complexes on fumed silica,” Langmuir 7, 2991-2998 (1991).
[CrossRef]

Bacon, R.

E. R. Carraway, J. N. Demas, B. A. DeGraff, and R. Bacon, “Photophysics and oxygen quenching of transition-metal complexes on fumed silica,” Langmuir 7, 2991-2998 (1991).
[CrossRef]

Belem, A. L.

T. Mock, G. S. Dieckmann, C. Haas, A. Krell, J. L. Tison, A. L. Belem, S. Papadimitriou, and D. N. Thomas, “Micro-optrodes in sea-ice: a new approach to investigate oxygen dynamics during sea ice formation,” Aquat. Microb. Ecol. 29, 297-306 (2002).
[CrossRef]

Borisov, S. M.

S. M. Borisov and O. S. Wolfbeis, “Optical biosensors,” Chem. Rev. 108, 423-461 (2008).
[CrossRef]

Brueck, S. R. J.

Cantle, J. E.

J. E. Cantle, Atomic Absorption Spectrometry (Elsevier, 1982).

Carlsson, K.

K. Carlsson and A. Liljeborg, “Simultaneous confocal lifetime imaging of multiple imaging of fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique,” J. Microsc. 191, 119-127 (1998).
[CrossRef]

Carraway, E. R.

E. R. Carraway, J. N. Demas, B. A. DeGraff, and R. Bacon, “Photophysics and oxygen quenching of transition-metal complexes on fumed silica,” Langmuir 7, 2991-2998 (1991).
[CrossRef]

Chance, B.

B. Chance, M. Cope, E. Gratton, N. Ramanujam, and B. Tromberg, “Phase measurement of light absorption and scatter in human tissue,” Rev. Sci. Instrum. 69, 3457-3481(1998).
[CrossRef]

Chatni, M. R.

M. R. Chatni, D. E. Maier, and D. M. Porterfield, “Evaluation of microparticle materials for enhancing the performance of fluorescence lifetime based optrodes,” Sens. Actuators B 141, 471-477 (2009).
[CrossRef]

A. ul Haque, M. R. Chatni, G. Li, and D. M. Porterfield, “Biochips and other microtechnologies for physiomics,” Expert Rev. Proteomics 4, 553-563 (2007).
[CrossRef]

Cope, M.

B. Chance, M. Cope, E. Gratton, N. Ramanujam, and B. Tromberg, “Phase measurement of light absorption and scatter in human tissue,” Rev. Sci. Instrum. 69, 3457-3481(1998).
[CrossRef]

DeGraff, B. A.

K. A. Kneas, J. N. Demas, B. A. DeGraff, and A. Periasamy, “Fluorescence microscopy study of heterogeneity in polymer-supported luminescence-based oxygen sensors,” Microsc. Microanal. 6, 551-561 (2000).

J. N. Demas, B. A. DeGraff, and W. Xu, “Modeling of luminescence quenching-based sensors: comparison of multi-site and non-linear gas solubility models,” Anal. Chem. 67, 1377-1380(1995).
[CrossRef]

E. R. Carraway, J. N. Demas, B. A. DeGraff, and R. Bacon, “Photophysics and oxygen quenching of transition-metal complexes on fumed silica,” Langmuir 7, 2991-2998 (1991).
[CrossRef]

Demas, J. N.

K. A. Kneas, J. N. Demas, B. A. DeGraff, and A. Periasamy, “Fluorescence microscopy study of heterogeneity in polymer-supported luminescence-based oxygen sensors,” Microsc. Microanal. 6, 551-561 (2000).

J. N. Demas, B. A. DeGraff, and W. Xu, “Modeling of luminescence quenching-based sensors: comparison of multi-site and non-linear gas solubility models,” Anal. Chem. 67, 1377-1380(1995).
[CrossRef]

E. R. Carraway, J. N. Demas, B. A. DeGraff, and R. Bacon, “Photophysics and oxygen quenching of transition-metal complexes on fumed silica,” Langmuir 7, 2991-2998 (1991).
[CrossRef]

Dieckmann, G. S.

T. Mock, G. S. Dieckmann, C. Haas, A. Krell, J. L. Tison, A. L. Belem, S. Papadimitriou, and D. N. Thomas, “Micro-optrodes in sea-ice: a new approach to investigate oxygen dynamics during sea ice formation,” Aquat. Microb. Ecol. 29, 297-306 (2002).
[CrossRef]

Gratton, E.

B. Chance, M. Cope, E. Gratton, N. Ramanujam, and B. Tromberg, “Phase measurement of light absorption and scatter in human tissue,” Rev. Sci. Instrum. 69, 3457-3481(1998).
[CrossRef]

Haas, C.

T. Mock, G. S. Dieckmann, C. Haas, A. Krell, J. L. Tison, A. L. Belem, S. Papadimitriou, and D. N. Thomas, “Micro-optrodes in sea-ice: a new approach to investigate oxygen dynamics during sea ice formation,” Aquat. Microb. Ecol. 29, 297-306 (2002).
[CrossRef]

Kneas, K. A.

K. A. Kneas, J. N. Demas, B. A. DeGraff, and A. Periasamy, “Fluorescence microscopy study of heterogeneity in polymer-supported luminescence-based oxygen sensors,” Microsc. Microanal. 6, 551-561 (2000).

Knudsen, O. S.

O. S. Knudsen, Biological Membranes: Theory of Transport Potentials and Electric Impulses (Cambridge U. Press, 2003).

Kolasinski, K.

K. Kolasinski, Surface Science: Foundations of Catalysis and Nanoscience (Wiley, 2002).

Kopelmann, R.

D. M. Porterfield, J. L. Rickus, and R. Kopelmann, “Non-invasive approaches to measuring respiratory patterns using a PtTFPP based phase-lifetime self-referencing oxygen optrode,” Proc. SPIE 6380, 63800S (2006).
[CrossRef]

Krell, A.

T. Mock, G. S. Dieckmann, C. Haas, A. Krell, J. L. Tison, A. L. Belem, S. Papadimitriou, and D. N. Thomas, “Micro-optrodes in sea-ice: a new approach to investigate oxygen dynamics during sea ice formation,” Aquat. Microb. Ecol. 29, 297-306 (2002).
[CrossRef]

Lackowicz, J. R.

J. R. Lackowicz, Principles of Fluorescence Spectroscopy (Springer, 2006).

Li, G.

A. ul Haque, M. R. Chatni, G. Li, and D. M. Porterfield, “Biochips and other microtechnologies for physiomics,” Expert Rev. Proteomics 4, 553-563 (2007).
[CrossRef]

Liljeborg, A.

K. Carlsson and A. Liljeborg, “Simultaneous confocal lifetime imaging of multiple imaging of fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique,” J. Microsc. 191, 119-127 (1998).
[CrossRef]

Lopez, G. P.

Maier, D. E.

M. R. Chatni, D. E. Maier, and D. M. Porterfield, “Evaluation of microparticle materials for enhancing the performance of fluorescence lifetime based optrodes,” Sens. Actuators B 141, 471-477 (2009).
[CrossRef]

Mock, T.

T. Mock, G. S. Dieckmann, C. Haas, A. Krell, J. L. Tison, A. L. Belem, S. Papadimitriou, and D. N. Thomas, “Micro-optrodes in sea-ice: a new approach to investigate oxygen dynamics during sea ice formation,” Aquat. Microb. Ecol. 29, 297-306 (2002).
[CrossRef]

O'Brien, M. J.

Ochoa-Acuna, H.

B. C. Sanchez, H. Ochoa-Acuna, D. M. Porterfield, and M. S. Sepulvada, “Oxygen flux as an indicator of physiological stress in fathead minnow embryos: a real-time biomonitoring system of water quality,” Environ. Sci. Technol. 42, 7010-7017(2008).
[CrossRef]

Ogurtsov, V. I.

V. I. Ogurtsov and D. B. Papkovsky, “Approximation of calibration for phase-fluorometric oxygen sensors on the basis of physical models,” Sens. Actuators B 88, 89-100(2003).
[CrossRef]

Papadimitriou, S.

T. Mock, G. S. Dieckmann, C. Haas, A. Krell, J. L. Tison, A. L. Belem, S. Papadimitriou, and D. N. Thomas, “Micro-optrodes in sea-ice: a new approach to investigate oxygen dynamics during sea ice formation,” Aquat. Microb. Ecol. 29, 297-306 (2002).
[CrossRef]

Papkovsky, D. B.

V. I. Ogurtsov and D. B. Papkovsky, “Approximation of calibration for phase-fluorometric oxygen sensors on the basis of physical models,” Sens. Actuators B 88, 89-100(2003).
[CrossRef]

Periasamy, A.

K. A. Kneas, J. N. Demas, B. A. DeGraff, and A. Periasamy, “Fluorescence microscopy study of heterogeneity in polymer-supported luminescence-based oxygen sensors,” Microsc. Microanal. 6, 551-561 (2000).

Porterfield, D. M.

M. R. Chatni, D. E. Maier, and D. M. Porterfield, “Evaluation of microparticle materials for enhancing the performance of fluorescence lifetime based optrodes,” Sens. Actuators B 141, 471-477 (2009).
[CrossRef]

B. C. Sanchez, H. Ochoa-Acuna, D. M. Porterfield, and M. S. Sepulvada, “Oxygen flux as an indicator of physiological stress in fathead minnow embryos: a real-time biomonitoring system of water quality,” Environ. Sci. Technol. 42, 7010-7017(2008).
[CrossRef]

A. ul Haque, M. R. Chatni, G. Li, and D. M. Porterfield, “Biochips and other microtechnologies for physiomics,” Expert Rev. Proteomics 4, 553-563 (2007).
[CrossRef]

D. M. Porterfield, J. L. Rickus, and R. Kopelmann, “Non-invasive approaches to measuring respiratory patterns using a PtTFPP based phase-lifetime self-referencing oxygen optrode,” Proc. SPIE 6380, 63800S (2006).
[CrossRef]

Rabinovich, E.

Ramanujam, N.

B. Chance, M. Cope, E. Gratton, N. Ramanujam, and B. Tromberg, “Phase measurement of light absorption and scatter in human tissue,” Rev. Sci. Instrum. 69, 3457-3481(1998).
[CrossRef]

Rickus, J. L.

D. M. Porterfield, J. L. Rickus, and R. Kopelmann, “Non-invasive approaches to measuring respiratory patterns using a PtTFPP based phase-lifetime self-referencing oxygen optrode,” Proc. SPIE 6380, 63800S (2006).
[CrossRef]

Sanchez, B. C.

B. C. Sanchez, H. Ochoa-Acuna, D. M. Porterfield, and M. S. Sepulvada, “Oxygen flux as an indicator of physiological stress in fathead minnow embryos: a real-time biomonitoring system of water quality,” Environ. Sci. Technol. 42, 7010-7017(2008).
[CrossRef]

Sepulvada, M. S.

B. C. Sanchez, H. Ochoa-Acuna, D. M. Porterfield, and M. S. Sepulvada, “Oxygen flux as an indicator of physiological stress in fathead minnow embryos: a real-time biomonitoring system of water quality,” Environ. Sci. Technol. 42, 7010-7017(2008).
[CrossRef]

Thomas, D. N.

T. Mock, G. S. Dieckmann, C. Haas, A. Krell, J. L. Tison, A. L. Belem, S. Papadimitriou, and D. N. Thomas, “Micro-optrodes in sea-ice: a new approach to investigate oxygen dynamics during sea ice formation,” Aquat. Microb. Ecol. 29, 297-306 (2002).
[CrossRef]

Tison, J. L.

T. Mock, G. S. Dieckmann, C. Haas, A. Krell, J. L. Tison, A. L. Belem, S. Papadimitriou, and D. N. Thomas, “Micro-optrodes in sea-ice: a new approach to investigate oxygen dynamics during sea ice formation,” Aquat. Microb. Ecol. 29, 297-306 (2002).
[CrossRef]

Tromberg, B.

B. Chance, M. Cope, E. Gratton, N. Ramanujam, and B. Tromberg, “Phase measurement of light absorption and scatter in human tissue,” Rev. Sci. Instrum. 69, 3457-3481(1998).
[CrossRef]

ul Haque, A.

A. ul Haque, M. R. Chatni, G. Li, and D. M. Porterfield, “Biochips and other microtechnologies for physiomics,” Expert Rev. Proteomics 4, 553-563 (2007).
[CrossRef]

Wolfbeis, O. S.

S. M. Borisov and O. S. Wolfbeis, “Optical biosensors,” Chem. Rev. 108, 423-461 (2008).
[CrossRef]

O. S. Wolfbeis, “Fiber optic chemical sensors and biosensors,” Anal. Chem. 76, 3269-3284 (2004).
[CrossRef]

Xu, W.

J. N. Demas, B. A. DeGraff, and W. Xu, “Modeling of luminescence quenching-based sensors: comparison of multi-site and non-linear gas solubility models,” Anal. Chem. 67, 1377-1380(1995).
[CrossRef]

Anal. Chem. (2)

O. S. Wolfbeis, “Fiber optic chemical sensors and biosensors,” Anal. Chem. 76, 3269-3284 (2004).
[CrossRef]

J. N. Demas, B. A. DeGraff, and W. Xu, “Modeling of luminescence quenching-based sensors: comparison of multi-site and non-linear gas solubility models,” Anal. Chem. 67, 1377-1380(1995).
[CrossRef]

Aquat. Microb. Ecol. (1)

T. Mock, G. S. Dieckmann, C. Haas, A. Krell, J. L. Tison, A. L. Belem, S. Papadimitriou, and D. N. Thomas, “Micro-optrodes in sea-ice: a new approach to investigate oxygen dynamics during sea ice formation,” Aquat. Microb. Ecol. 29, 297-306 (2002).
[CrossRef]

Chem. Rev. (1)

S. M. Borisov and O. S. Wolfbeis, “Optical biosensors,” Chem. Rev. 108, 423-461 (2008).
[CrossRef]

Environ. Sci. Technol. (1)

B. C. Sanchez, H. Ochoa-Acuna, D. M. Porterfield, and M. S. Sepulvada, “Oxygen flux as an indicator of physiological stress in fathead minnow embryos: a real-time biomonitoring system of water quality,” Environ. Sci. Technol. 42, 7010-7017(2008).
[CrossRef]

Expert Rev. Proteomics (1)

A. ul Haque, M. R. Chatni, G. Li, and D. M. Porterfield, “Biochips and other microtechnologies for physiomics,” Expert Rev. Proteomics 4, 553-563 (2007).
[CrossRef]

J. Microsc. (1)

K. Carlsson and A. Liljeborg, “Simultaneous confocal lifetime imaging of multiple imaging of fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique,” J. Microsc. 191, 119-127 (1998).
[CrossRef]

Langmuir (1)

E. R. Carraway, J. N. Demas, B. A. DeGraff, and R. Bacon, “Photophysics and oxygen quenching of transition-metal complexes on fumed silica,” Langmuir 7, 2991-2998 (1991).
[CrossRef]

Microsc. Microanal. (1)

K. A. Kneas, J. N. Demas, B. A. DeGraff, and A. Periasamy, “Fluorescence microscopy study of heterogeneity in polymer-supported luminescence-based oxygen sensors,” Microsc. Microanal. 6, 551-561 (2000).

Opt. Lett. (1)

Proc. SPIE (1)

D. M. Porterfield, J. L. Rickus, and R. Kopelmann, “Non-invasive approaches to measuring respiratory patterns using a PtTFPP based phase-lifetime self-referencing oxygen optrode,” Proc. SPIE 6380, 63800S (2006).
[CrossRef]

Rev. Sci. Instrum. (1)

B. Chance, M. Cope, E. Gratton, N. Ramanujam, and B. Tromberg, “Phase measurement of light absorption and scatter in human tissue,” Rev. Sci. Instrum. 69, 3457-3481(1998).
[CrossRef]

Sens. Actuators B (2)

M. R. Chatni, D. E. Maier, and D. M. Porterfield, “Evaluation of microparticle materials for enhancing the performance of fluorescence lifetime based optrodes,” Sens. Actuators B 141, 471-477 (2009).
[CrossRef]

V. I. Ogurtsov and D. B. Papkovsky, “Approximation of calibration for phase-fluorometric oxygen sensors on the basis of physical models,” Sens. Actuators B 88, 89-100(2003).
[CrossRef]

Other (4)

O. S. Knudsen, Biological Membranes: Theory of Transport Potentials and Electric Impulses (Cambridge U. Press, 2003).

K. Kolasinski, Surface Science: Foundations of Catalysis and Nanoscience (Wiley, 2002).

J. E. Cantle, Atomic Absorption Spectrometry (Elsevier, 1982).

J. R. Lackowicz, Principles of Fluorescence Spectroscopy (Springer, 2006).

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Figures (9)

Fig. 1
Fig. 1

Schematic of the oxygen optrode system. SRS 830 provides the sinusoidal input to the LED driver, which drives the blue LED, which is fitted into a LED fiber coupler. The microscope objective in the LED fiber coupler focuses the excitation light onto the 2 × 2 or 2 × 1 coupler. The excitation light is coupled to the optrode, where it excites the PtTFPP molecules immobilized on the optrode tip. The emitted radiation is guided from the optrode to the PMT via a fiber coupler. The PMT voltage output is connected to the input of the SRS 830. Using the input sinusoidal signal as an internal reference, SRS 830 measures the fluorescence intensity and phase shift. SRS 830 parameters are controlled via a GPIB interface which is controlled by LabVIEW 8.0. The fluorescence intensity and phase-shift data are acquired every 1 s and stored in EXCEL.

Fig. 2
Fig. 2

Circuit diagram of LED driver. The circuit is divided into three stages—buffer stage, summing amplifier, and VCCS. In the buffer stage, the inputs from both the SRS 830 and the DC source are buffered using unity gain followers (AD 713). The output from the two buffers is fed into the input of a summing amplifier (AD 8599) with gain of unity. The output from the summing amplifier is input to two independent VCCS. Each VCCS (AD 8599) unit is capable of driving one LED. The amount of current flowing through the LED is the ratio of the input voltage and the current-limiting resistor at the source of BS 170. The LED state (ON or OFF) can be controlled by applying voltage input to the BS 250.

Fig. 3
Fig. 3

Inherent system phase-shift characterization of the oxygen optrode system. The system phase noise has to be calibrated with approximately the same wavelength of light at which the emission of the fluorophore is being detected. In this system, PtTFPP emission at 640 nm is being used for measurement of fluorescence intensity and phase shift. A red ( 637 640 nm ) LED is aligned with the blank optrode fiber, and red intensity and phase shifts are measured by the SRS 830. Varying PMT control voltages did not have any significant effects on phase shifts. Also, varying input sinusoidal ( 1 5 V ) signals did not have any significant effect on phase shifts. Red intensity was proportional to the input voltage; the higher the input voltage, the brighter the LED and the more photoelectric current generated by the PMT. The optical phase shift is linear with modulation frequency: Φ OPT = 0.5302 × frequency ( kHz ) + 0.459 .

Fig. 4
Fig. 4

Determination of optimum modulation frequency for PtTFPP fluorescence. The PtTFPP oxygen optrode was dipped in 0% oxygen solution and 21% oxygen deionized water. The modulation frequency was swept from 1 70 kHz . Phase shift 4a and fluorescence intensity 4b at each frequency were measured for 45 s and then averaged. Figure 4c shows the tangent of measured phase shift in both the 21% and the 0% oxygen environments. “U” denotes the unadjusted measurements without compensation of the inherent system phase shift, and “A” denotes the adjusted measurements with compensation of the inherent system phase shift. The inset in (c) shows the linear range of the tangent of phase shift. From (a) it was observed that the maximum difference occurs at less than 10 kHz . From the data, the modulation frequency was fixed at 5 kHz . The fluorescence intensity distribution reduces as modulation frequency is increased. The following parameters were used for the measurements: PMT control voltage = 0.8 V , input sinusoidal signal = 3 V , and DC offset = 4.25 V .

Fig. 5
Fig. 5

Determination of optimum LED driver input sinusoidal voltage. The oxygen optrode was dipped in 21% oxygen deionized water. The fluorescence intensity and phase shift were measured under varying input voltages from 0.5 to 5 V with 0.5 V steps. Fluorescence intensity and phase angle at each input voltage was measured for 45 s and averaged. The magnitude of input voltage has a noticeable effect on fluorescence intensity but does not affect the phase angle measurements. Fluorescence intensity increases rapidly between 0.5 and 2 V , as compared to between 2 and 5 V . The goal is to achieve the highest possible fluorescence signal that will impact the SNR for the phase measurements. An input voltage of 3 V was chosen as the operating point.

Fig. 6
Fig. 6

Optimal fiber coupler configuration and splitting ratio for the oxygen optrode system. The oxygen optrode was dipped in 21% deionized water. The fluorescence intensity was measured under varying control voltage from 0 1 V with 0.05 V steps for 45 s and the data were averaged. It can be seen that the fluorescence intensity is not detectable beyond a certain threshold control voltage. The fluorescence increases as the control voltage is increased beyond the threshold voltage. The splitting ratios are given in brackets, and the letter with the splitting ratio specifies the location of the splitting arm ( L = Blue   LED , P = PMT ). 1 × 2 ( 30 L / 70 P ) is the best fiber configuration for the oxygen optrode system. It provides minimum trade-off radiation loss at splitting junctions and maximum fluorescence signal at the PMT.

Fig. 7
Fig. 7

Oxygen optrode calibration. Different ratios of oxygen and nitrogen gas were bubbled into deionized water for 20 min . The oxygen optrode was dipped in the deionized water. Fluorescence lifetime ( mean ± standard diviation ) and fluorescence intensity (inset) were measured for 45 s and averaged. In the homodyne detection technique, the standard deviations of measured fluorescence lifetimes were lower than that of the heterodyne technique. Fluorescence intensity measured at 0% and 21% oxygen were higher in the homodyne detection technique and the SNR was higher compared to the heterodyne detection technique. Parameters: PMT control v oltage = 0.8 V , input sinusoidal signal = 3 V , and modulation frequency = 5 kHz .

Fig. 8
Fig. 8

Stern–Volmer relationship of the oxygen optrode. Because of the nonlinear nature of the calibration curve, the Stern–Volmer relationship for the oxygen optrode is also nonlinear. The three-parameter nonlinear oxygen solubility model fits the data very well. The three data points must be chosen in a way that they include the entire range. The best approach is to choose the two end points and one point in the middle. The interpolating data points will be unequally spaced, which greatly reduces interpolation errors. The values for the fitting parameter at STP for the oxygen optrode: A = 0.43 E 2 l i t e r s ( μ mol ) 1 , B = 0.014 l i t e r ( μ mol ) 1 , and C = 0.018 l i t e r ( μ mol ) 1 .

Fig. 9
Fig. 9

Oxygen optrode response time measurement. The optrode was dipped in a 25 mm petri dish containing deionized water. Nitrogen and compressed air were bubbled into the petri dish at different times. Nitrogen deoxygenated the deionized water while compressed air oxygenated the deionized water. The optode measurements were also alternated and phase angle measurements were recorded. Parameters: input sinusoidal voltage = 3 V , PMT control voltage = 0.8 V , and modulation frequency = 5 kHz .

Equations (4)

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τ = tan ( θ ϕ ) 2 π f mod ,
τ = tan ( θ ) 2 π f mod .
[ O 2 ] μ mol / L = [ p atm p W ( T ) p N Q α ( T ) 1000 M ( O 2 ) V M ] × ( 31.25 ) ,
τ 0 τ = 1 + A [ O 2 ] + B [ O 2 ] 1 + C [ O 2 ] ,

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