Abstract

Fluorescence Lifetime Imaging Microscopy (FLIM) is a quantitative technique to probe the nanoenvironment of fluorescent molecules. It is the most robust way to quantify Förster Resonance Energy Transfer (FRET) as it allows reliable differentiation between concentration changes and quenching. In this way, molecular interactions can be imaged in single living cells. The most common wide-field implementation is homodyne Frequency Domain (FD) FLIM, which determines the fluorescence lifetime by measuring the phase and modulation changes of the fluorescence in each pixel upon excitation with a light source modulated at a high frequency. The fluorescence lifetimes are derived from a stack of images acquired at different phase shifts between excitation and detection. In this work we describe a simple method to enhance the dynamic range of FD-FLIM based on precompensating the expected fluorescence modulation by varying the laser power through the phase stack. We show theoretically and experimentally that most of the dynamic range of the camera can be recovered to quantify cells with different intensities. This improvement can be added to any FD-FLIM setup with minimal modifications, enhancing the throughput of information content.

© 2012 OSA

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  1. H. C. Ishikawa-Ankerhold, R. Ankerhold, and G. P. C. Drummen, “Advanced fluorescence microscopy techniques-FRAP, FLIP, FLAP, FRET and FLIM.” Molecules (Basel, Switzerland)17, 4047–132 (2012).
    [CrossRef]
  2. R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox.” Trends Cell Biol.21, 682–91 (2011).
    [CrossRef] [PubMed]
  3. F. S. Wouters, P. J. Verveer, and P. I. H. Bastiaens, “Imaging biochemistry inside cells,” Trends Cell Biol.11, 203–211 (2001).
    [CrossRef] [PubMed]
  4. S. Ogikubo, T. Nakabayashi, T. Adachi, M. S. Islam, T. Yoshizawa, M. Kinjo, and N. Ohta, “Intracellular pH sensing using autofluorescence lifetime microscopy.” J. Phys. Chem. B115, 10385–90 (2011).
    [CrossRef] [PubMed]
  5. M. K. Kuimova, G. Yahioglu, J. A. Levitt, and K. Suhling, “Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging” J. Am. Chem. Soc.130, 6672–3 (2008).
    [CrossRef] [PubMed]
  6. T. Förster “Energy migration and fluorescence - 1946” J. Biomed. Opt.17, 011002–10 (2012).
    [CrossRef] [PubMed]
  7. E. A. Jares-Erijman and T. M. Jovin, “FRET imaging.” Nat. Biotechnol.21, 1387–95 (2003).
    [CrossRef] [PubMed]
  8. T. W. J. Gadella, T. M. Jovin, and R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM) Spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem.48, 221–239 (1993).
    [CrossRef]
  9. R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P.B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging” Nat. Biotechnol.25, 249–53 (2007).
    [CrossRef] [PubMed]
  10. W. Caarls, B. Rieger, A. H. B. De Vries, D. J. Arndt-Jovin, and T. M. Jovin, “Minimizing light exposure with the programmable array microscope” J. Microsc. (Oxford, U. K.)241, 101–10 (2011).
    [CrossRef]
  11. H. E. Grecco, P. Roda-Navarro, S. Fengler, and P. I. H. Bastiaens, “High-Throughput quantification of posttranslational modifications In Situ by CA-FLIM.” Methods Enzymol.500, 37–58 (2011).
    [CrossRef] [PubMed]
  12. E. B. van Munster and T. W. J. Gadella, “Suppression of photobleaching-induced artifacts in frequency-domain FLIM by permutation of the recording order.” Cytometry, Part A58, 185–94 (2004).
    [CrossRef]
  13. P. J. Verveer and P. I. H. Bastiaens, “Evaluation of global analysis algorithms for single frequency,” J. Microsc. (Oxford, U. K.)209, 1–7 (2003).
    [CrossRef]
  14. B. Q. Spring and R. M. Clegg, “Image analysis for denoising full-field frequency-domain fluorescence lifetime images” J. Microsc. (Oxford, U. K.)235, 221–237 (2009).
    [CrossRef]
  15. A. Esposito, C. P. Dohm, M. Bähr, and F. S. Wouters, “Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening.” Mol. Cell. Proteomics6, 1446–54 (2007).
    [CrossRef] [PubMed]
  16. H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. H. Bastiaens, “In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.” Nat. Methods7, 467–72 (2010).
    [CrossRef] [PubMed]

2012 (2)

H. C. Ishikawa-Ankerhold, R. Ankerhold, and G. P. C. Drummen, “Advanced fluorescence microscopy techniques-FRAP, FLIP, FLAP, FRET and FLIM.” Molecules (Basel, Switzerland)17, 4047–132 (2012).
[CrossRef]

T. Förster “Energy migration and fluorescence - 1946” J. Biomed. Opt.17, 011002–10 (2012).
[CrossRef] [PubMed]

2011 (4)

R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox.” Trends Cell Biol.21, 682–91 (2011).
[CrossRef] [PubMed]

S. Ogikubo, T. Nakabayashi, T. Adachi, M. S. Islam, T. Yoshizawa, M. Kinjo, and N. Ohta, “Intracellular pH sensing using autofluorescence lifetime microscopy.” J. Phys. Chem. B115, 10385–90 (2011).
[CrossRef] [PubMed]

W. Caarls, B. Rieger, A. H. B. De Vries, D. J. Arndt-Jovin, and T. M. Jovin, “Minimizing light exposure with the programmable array microscope” J. Microsc. (Oxford, U. K.)241, 101–10 (2011).
[CrossRef]

H. E. Grecco, P. Roda-Navarro, S. Fengler, and P. I. H. Bastiaens, “High-Throughput quantification of posttranslational modifications In Situ by CA-FLIM.” Methods Enzymol.500, 37–58 (2011).
[CrossRef] [PubMed]

2010 (1)

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. H. Bastiaens, “In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.” Nat. Methods7, 467–72 (2010).
[CrossRef] [PubMed]

2009 (1)

B. Q. Spring and R. M. Clegg, “Image analysis for denoising full-field frequency-domain fluorescence lifetime images” J. Microsc. (Oxford, U. K.)235, 221–237 (2009).
[CrossRef]

2008 (1)

M. K. Kuimova, G. Yahioglu, J. A. Levitt, and K. Suhling, “Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging” J. Am. Chem. Soc.130, 6672–3 (2008).
[CrossRef] [PubMed]

2007 (2)

A. Esposito, C. P. Dohm, M. Bähr, and F. S. Wouters, “Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening.” Mol. Cell. Proteomics6, 1446–54 (2007).
[CrossRef] [PubMed]

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P.B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging” Nat. Biotechnol.25, 249–53 (2007).
[CrossRef] [PubMed]

2004 (1)

E. B. van Munster and T. W. J. Gadella, “Suppression of photobleaching-induced artifacts in frequency-domain FLIM by permutation of the recording order.” Cytometry, Part A58, 185–94 (2004).
[CrossRef]

2003 (2)

P. J. Verveer and P. I. H. Bastiaens, “Evaluation of global analysis algorithms for single frequency,” J. Microsc. (Oxford, U. K.)209, 1–7 (2003).
[CrossRef]

E. A. Jares-Erijman and T. M. Jovin, “FRET imaging.” Nat. Biotechnol.21, 1387–95 (2003).
[CrossRef] [PubMed]

2001 (1)

F. S. Wouters, P. J. Verveer, and P. I. H. Bastiaens, “Imaging biochemistry inside cells,” Trends Cell Biol.11, 203–211 (2001).
[CrossRef] [PubMed]

1993 (1)

T. W. J. Gadella, T. M. Jovin, and R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM) Spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem.48, 221–239 (1993).
[CrossRef]

Adachi, T.

S. Ogikubo, T. Nakabayashi, T. Adachi, M. S. Islam, T. Yoshizawa, M. Kinjo, and N. Ohta, “Intracellular pH sensing using autofluorescence lifetime microscopy.” J. Phys. Chem. B115, 10385–90 (2011).
[CrossRef] [PubMed]

Ankerhold, R.

H. C. Ishikawa-Ankerhold, R. Ankerhold, and G. P. C. Drummen, “Advanced fluorescence microscopy techniques-FRAP, FLIP, FLAP, FRET and FLIM.” Molecules (Basel, Switzerland)17, 4047–132 (2012).
[CrossRef]

Arndt-Jovin, D. J.

W. Caarls, B. Rieger, A. H. B. De Vries, D. J. Arndt-Jovin, and T. M. Jovin, “Minimizing light exposure with the programmable array microscope” J. Microsc. (Oxford, U. K.)241, 101–10 (2011).
[CrossRef]

Bähr, M.

A. Esposito, C. P. Dohm, M. Bähr, and F. S. Wouters, “Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening.” Mol. Cell. Proteomics6, 1446–54 (2007).
[CrossRef] [PubMed]

Bastiaens, P. I. H.

H. E. Grecco, P. Roda-Navarro, S. Fengler, and P. I. H. Bastiaens, “High-Throughput quantification of posttranslational modifications In Situ by CA-FLIM.” Methods Enzymol.500, 37–58 (2011).
[CrossRef] [PubMed]

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. H. Bastiaens, “In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.” Nat. Methods7, 467–72 (2010).
[CrossRef] [PubMed]

P. J. Verveer and P. I. H. Bastiaens, “Evaluation of global analysis algorithms for single frequency,” J. Microsc. (Oxford, U. K.)209, 1–7 (2003).
[CrossRef]

F. S. Wouters, P. J. Verveer, and P. I. H. Bastiaens, “Imaging biochemistry inside cells,” Trends Cell Biol.11, 203–211 (2001).
[CrossRef] [PubMed]

Caarls, W.

W. Caarls, B. Rieger, A. H. B. De Vries, D. J. Arndt-Jovin, and T. M. Jovin, “Minimizing light exposure with the programmable array microscope” J. Microsc. (Oxford, U. K.)241, 101–10 (2011).
[CrossRef]

Clegg, R. M.

B. Q. Spring and R. M. Clegg, “Image analysis for denoising full-field frequency-domain fluorescence lifetime images” J. Microsc. (Oxford, U. K.)235, 221–237 (2009).
[CrossRef]

T. W. J. Gadella, T. M. Jovin, and R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM) Spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem.48, 221–239 (1993).
[CrossRef]

De Vries, A. H. B.

W. Caarls, B. Rieger, A. H. B. De Vries, D. J. Arndt-Jovin, and T. M. Jovin, “Minimizing light exposure with the programmable array microscope” J. Microsc. (Oxford, U. K.)241, 101–10 (2011).
[CrossRef]

Dhonukshe, P.B.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P.B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging” Nat. Biotechnol.25, 249–53 (2007).
[CrossRef] [PubMed]

Dohm, C. P.

A. Esposito, C. P. Dohm, M. Bähr, and F. S. Wouters, “Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening.” Mol. Cell. Proteomics6, 1446–54 (2007).
[CrossRef] [PubMed]

Drummen, G. P. C.

H. C. Ishikawa-Ankerhold, R. Ankerhold, and G. P. C. Drummen, “Advanced fluorescence microscopy techniques-FRAP, FLIP, FLAP, FRET and FLIM.” Molecules (Basel, Switzerland)17, 4047–132 (2012).
[CrossRef]

Esposito, A.

A. Esposito, C. P. Dohm, M. Bähr, and F. S. Wouters, “Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening.” Mol. Cell. Proteomics6, 1446–54 (2007).
[CrossRef] [PubMed]

Fengler, S.

H. E. Grecco, P. Roda-Navarro, S. Fengler, and P. I. H. Bastiaens, “High-Throughput quantification of posttranslational modifications In Situ by CA-FLIM.” Methods Enzymol.500, 37–58 (2011).
[CrossRef] [PubMed]

Fischer, R. S.

R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox.” Trends Cell Biol.21, 682–91 (2011).
[CrossRef] [PubMed]

Förster, T.

T. Förster “Energy migration and fluorescence - 1946” J. Biomed. Opt.17, 011002–10 (2012).
[CrossRef] [PubMed]

Frahm, T.

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. H. Bastiaens, “In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.” Nat. Methods7, 467–72 (2010).
[CrossRef] [PubMed]

Gadella, T. W. J.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P.B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging” Nat. Biotechnol.25, 249–53 (2007).
[CrossRef] [PubMed]

E. B. van Munster and T. W. J. Gadella, “Suppression of photobleaching-induced artifacts in frequency-domain FLIM by permutation of the recording order.” Cytometry, Part A58, 185–94 (2004).
[CrossRef]

T. W. J. Gadella, T. M. Jovin, and R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM) Spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem.48, 221–239 (1993).
[CrossRef]

Girod, A.

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. H. Bastiaens, “In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.” Nat. Methods7, 467–72 (2010).
[CrossRef] [PubMed]

Grecco, H. E.

H. E. Grecco, P. Roda-Navarro, S. Fengler, and P. I. H. Bastiaens, “High-Throughput quantification of posttranslational modifications In Situ by CA-FLIM.” Methods Enzymol.500, 37–58 (2011).
[CrossRef] [PubMed]

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. H. Bastiaens, “In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.” Nat. Methods7, 467–72 (2010).
[CrossRef] [PubMed]

Hoebe, R. A.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P.B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging” Nat. Biotechnol.25, 249–53 (2007).
[CrossRef] [PubMed]

Hou, J.

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. H. Bastiaens, “In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.” Nat. Methods7, 467–72 (2010).
[CrossRef] [PubMed]

Ishikawa-Ankerhold, H. C.

H. C. Ishikawa-Ankerhold, R. Ankerhold, and G. P. C. Drummen, “Advanced fluorescence microscopy techniques-FRAP, FLIP, FLAP, FRET and FLIM.” Molecules (Basel, Switzerland)17, 4047–132 (2012).
[CrossRef]

Islam, M. S.

S. Ogikubo, T. Nakabayashi, T. Adachi, M. S. Islam, T. Yoshizawa, M. Kinjo, and N. Ohta, “Intracellular pH sensing using autofluorescence lifetime microscopy.” J. Phys. Chem. B115, 10385–90 (2011).
[CrossRef] [PubMed]

Jares-Erijman, E. A.

E. A. Jares-Erijman and T. M. Jovin, “FRET imaging.” Nat. Biotechnol.21, 1387–95 (2003).
[CrossRef] [PubMed]

Jovin, T. M.

W. Caarls, B. Rieger, A. H. B. De Vries, D. J. Arndt-Jovin, and T. M. Jovin, “Minimizing light exposure with the programmable array microscope” J. Microsc. (Oxford, U. K.)241, 101–10 (2011).
[CrossRef]

E. A. Jares-Erijman and T. M. Jovin, “FRET imaging.” Nat. Biotechnol.21, 1387–95 (2003).
[CrossRef] [PubMed]

T. W. J. Gadella, T. M. Jovin, and R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM) Spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem.48, 221–239 (1993).
[CrossRef]

Kanchanawong, P.

R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox.” Trends Cell Biol.21, 682–91 (2011).
[CrossRef] [PubMed]

Kinjo, M.

S. Ogikubo, T. Nakabayashi, T. Adachi, M. S. Islam, T. Yoshizawa, M. Kinjo, and N. Ohta, “Intracellular pH sensing using autofluorescence lifetime microscopy.” J. Phys. Chem. B115, 10385–90 (2011).
[CrossRef] [PubMed]

Kuimova, M. K.

M. K. Kuimova, G. Yahioglu, J. A. Levitt, and K. Suhling, “Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging” J. Am. Chem. Soc.130, 6672–3 (2008).
[CrossRef] [PubMed]

Levitt, J. A.

M. K. Kuimova, G. Yahioglu, J. A. Levitt, and K. Suhling, “Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging” J. Am. Chem. Soc.130, 6672–3 (2008).
[CrossRef] [PubMed]

Manders, E. M. M.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P.B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging” Nat. Biotechnol.25, 249–53 (2007).
[CrossRef] [PubMed]

Nakabayashi, T.

S. Ogikubo, T. Nakabayashi, T. Adachi, M. S. Islam, T. Yoshizawa, M. Kinjo, and N. Ohta, “Intracellular pH sensing using autofluorescence lifetime microscopy.” J. Phys. Chem. B115, 10385–90 (2011).
[CrossRef] [PubMed]

Ogikubo, S.

S. Ogikubo, T. Nakabayashi, T. Adachi, M. S. Islam, T. Yoshizawa, M. Kinjo, and N. Ohta, “Intracellular pH sensing using autofluorescence lifetime microscopy.” J. Phys. Chem. B115, 10385–90 (2011).
[CrossRef] [PubMed]

Ohta, N.

S. Ogikubo, T. Nakabayashi, T. Adachi, M. S. Islam, T. Yoshizawa, M. Kinjo, and N. Ohta, “Intracellular pH sensing using autofluorescence lifetime microscopy.” J. Phys. Chem. B115, 10385–90 (2011).
[CrossRef] [PubMed]

Pepperkok, R.

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. H. Bastiaens, “In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.” Nat. Methods7, 467–72 (2010).
[CrossRef] [PubMed]

Rieger, B.

W. Caarls, B. Rieger, A. H. B. De Vries, D. J. Arndt-Jovin, and T. M. Jovin, “Minimizing light exposure with the programmable array microscope” J. Microsc. (Oxford, U. K.)241, 101–10 (2011).
[CrossRef]

Roda-Navarro, P.

H. E. Grecco, P. Roda-Navarro, S. Fengler, and P. I. H. Bastiaens, “High-Throughput quantification of posttranslational modifications In Situ by CA-FLIM.” Methods Enzymol.500, 37–58 (2011).
[CrossRef] [PubMed]

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. H. Bastiaens, “In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.” Nat. Methods7, 467–72 (2010).
[CrossRef] [PubMed]

Shroff, H.

R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox.” Trends Cell Biol.21, 682–91 (2011).
[CrossRef] [PubMed]

Spring, B. Q.

B. Q. Spring and R. M. Clegg, “Image analysis for denoising full-field frequency-domain fluorescence lifetime images” J. Microsc. (Oxford, U. K.)235, 221–237 (2009).
[CrossRef]

Squire, A.

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. H. Bastiaens, “In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.” Nat. Methods7, 467–72 (2010).
[CrossRef] [PubMed]

Suhling, K.

M. K. Kuimova, G. Yahioglu, J. A. Levitt, and K. Suhling, “Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging” J. Am. Chem. Soc.130, 6672–3 (2008).
[CrossRef] [PubMed]

Truxius, D. C.

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. H. Bastiaens, “In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.” Nat. Methods7, 467–72 (2010).
[CrossRef] [PubMed]

van Munster, E. B.

E. B. van Munster and T. W. J. Gadella, “Suppression of photobleaching-induced artifacts in frequency-domain FLIM by permutation of the recording order.” Cytometry, Part A58, 185–94 (2004).
[CrossRef]

Van Noorden, C. J. F.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P.B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging” Nat. Biotechnol.25, 249–53 (2007).
[CrossRef] [PubMed]

Van Oven, C. H.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P.B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging” Nat. Biotechnol.25, 249–53 (2007).
[CrossRef] [PubMed]

Verveer, P. J.

P. J. Verveer and P. I. H. Bastiaens, “Evaluation of global analysis algorithms for single frequency,” J. Microsc. (Oxford, U. K.)209, 1–7 (2003).
[CrossRef]

F. S. Wouters, P. J. Verveer, and P. I. H. Bastiaens, “Imaging biochemistry inside cells,” Trends Cell Biol.11, 203–211 (2001).
[CrossRef] [PubMed]

Waterman, C. M.

R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox.” Trends Cell Biol.21, 682–91 (2011).
[CrossRef] [PubMed]

Wouters, F. S.

A. Esposito, C. P. Dohm, M. Bähr, and F. S. Wouters, “Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening.” Mol. Cell. Proteomics6, 1446–54 (2007).
[CrossRef] [PubMed]

F. S. Wouters, P. J. Verveer, and P. I. H. Bastiaens, “Imaging biochemistry inside cells,” Trends Cell Biol.11, 203–211 (2001).
[CrossRef] [PubMed]

Wu, Y.

R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox.” Trends Cell Biol.21, 682–91 (2011).
[CrossRef] [PubMed]

Yahioglu, G.

M. K. Kuimova, G. Yahioglu, J. A. Levitt, and K. Suhling, “Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging” J. Am. Chem. Soc.130, 6672–3 (2008).
[CrossRef] [PubMed]

Yoshizawa, T.

S. Ogikubo, T. Nakabayashi, T. Adachi, M. S. Islam, T. Yoshizawa, M. Kinjo, and N. Ohta, “Intracellular pH sensing using autofluorescence lifetime microscopy.” J. Phys. Chem. B115, 10385–90 (2011).
[CrossRef] [PubMed]

Biophys. Chem. (1)

T. W. J. Gadella, T. M. Jovin, and R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM) Spatial resolution of microstructures on the nanosecond time scale,” Biophys. Chem.48, 221–239 (1993).
[CrossRef]

Cytometry, Part A (1)

E. B. van Munster and T. W. J. Gadella, “Suppression of photobleaching-induced artifacts in frequency-domain FLIM by permutation of the recording order.” Cytometry, Part A58, 185–94 (2004).
[CrossRef]

J. Am. Chem. Soc. (1)

M. K. Kuimova, G. Yahioglu, J. A. Levitt, and K. Suhling, “Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging” J. Am. Chem. Soc.130, 6672–3 (2008).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

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H. E. Grecco, P. Roda-Navarro, S. Fengler, and P. I. H. Bastiaens, “High-Throughput quantification of posttranslational modifications In Situ by CA-FLIM.” Methods Enzymol.500, 37–58 (2011).
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Nat. Biotechnol. (2)

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Nat. Methods (1)

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. H. Bastiaens, “In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.” Nat. Methods7, 467–72 (2010).
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R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox.” Trends Cell Biol.21, 682–91 (2011).
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Figures (6)

Fig. 1
Fig. 1

(a) FLIM setup (b) Principle of intensity compensated FLIM

Fig. 2
Fig. 2

Dynamic range enhancement of a single fluorophore with only one lifetime component, (a) Simulation of maximum dynamic range enhancement for a variety of lifetimes compensated with a range of assumed single lifetimes (sinusoidal excitation, M = 1), (b) Dynamic range enhancement for Alexa 488 (+) in water for various compensation lifetimes as compared to the corresponding simulated curve (red), (c) Simulation of maximum dynamic range enhancement with two fluorophores (2 ns and 4 ns) mixed, axes describe the fraction of 4 ns fluorophore, (d) Simulation of a FRET system with 3 ns lifetime as a function of FRET efficiency

Fig. 3
Fig. 3

(a) FLIM and flatFLIM images, over/underexposed regions are marked red/blue, for a variety of concentrations for a compensation lifetime of 4.1 ns (b) Percentage of usable pixels over the concentration range, flatFLIM 4.1 ns (gray area), 3 ns (blue, solid), 6 ns (blue, dashed), FLIM (green area)

Fig. 4
Fig. 4

Effect of photobleaching in the fluorescence lifetime determination: (a) dynamic range enhancement over FLIM (black) and increase in light dose for same SNR plotted versus the fluorescence lifetime (green) and comparison to the requirements for FLIM for the same dynamic range at 3.4ns lifetime and thus 3x dynamic range enhancement (b) effect of the reference phase on the lifetime calculation upon a bleaching of 10 % and a simulated lifetime of 3 ns; effect of different amounts of photobleaching for a simulated lifetime of 3 ns on the calculated phase and modulation lifetimes for FLIM and flatFLIM

Fig. 5
Fig. 5

(a) FLIM and flatFLIM images of HeLa cells transfected with EYFP showing over (red) and under (blue) exposed regions as well as modulation lifetime and phase lifetime images (b) Intensity projection across the yellow line in both FLIM and flatFLIM images

Fig. 6
Fig. 6

Phase lifetime images (FLIM and flatFLIM) of MCF-7 cells with EGFR-YFP receptor before and after addition of Cy3.5 labelled PY72 antibody (smaller images: under/overexposed regions within the cell)

Equations (11)

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F ( ϕ i ) = I C ( 1 + m 0 m τ cos ( ϕ i ϕ 0 ϕ τ ) ) ,
BG < F ( ϕ i ) < SAT
BG I ( 1 m 0 m τ ) < C < SAT I ( 1 + m 0 m τ )
D = 1 m 0 m τ 1 + m 0 m τ SAT BG ,
F ( ϕ i ) = I ( ϕ i ) C ( 1 + m 0 m τ cos ( ϕ i ϕ 0 ϕ τ ) ) ,
D flat = Min [ F ] Max [ F ] SAT BG
I ( ϕ i ) = I 0 ( 1 + m 0 m λ cos ( ϕ i ϕ 0 ϕ λ ) ) 1
L FLIM = N I
< F FLIM > = 1 N n = 1 N F ( ϕ i ) ~ I C N
L flatFLIM = N I 0 [ n I ( ϕ i ) ] 1
< F flatFLIM > ~ I 0 C N

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