Abstract

Wide-field fluorescence lifetime imaging allows for fast imaging of large sample areas at the cost of low sensitivity to weak fluorescence signals. To overcome this challenge, we developed an active wide-field illumination (AWFI) strategy to optimize the impinging spatial intensity for acquiring optimal fluorescence signals over the whole sample. We demonstrated the ability of AWFI to accurately estimate lifetimes from a multiwell plate sample with concentrations ranging over two orders of magnitude. We further reported its successful application to a quantitative Förster resonance energy transfer lifetime cell-based assay. Overall, this method allows for enhanced accuracy in lifetime-based imaging at high acquisition speed over samples with large fluorescence intensity distributions.

© 2013 Optical Society of America

Full Article  |  PDF Article

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  1. W. Becker, J. Microsc. 247, 119 (2012).
    [CrossRef]
  2. P. B. Jones, L. Herl, O. Berezovska, A. T. Kumar, B. J. Bacskai, and B. T. Hyman, J. Biomed. Opt. 11, 054024 (2006).
    [CrossRef]
  3. M. Kollner and J. Wolfrum, Chem. Phys. Lett. 200, 199 (1992).
    [CrossRef]
  4. R. A. Hoebe, C. H. Van Oven, T. W. Gadella, P. B. Dhonukshe, C. J. Van Noorden, and E. M. Manders, Nat. Biotechnol. 25, 249 (2007).
    [CrossRef]
  5. K. Chu, D. Lim, and J. Mertz, Biomed. Opt. Express 1, 236 (2010).
    [CrossRef]
  6. V. Venugopal, J. Chen, and X. Intes, Biomed. Opt. Express 1, 143 (2010).
    [CrossRef]
  7. H. Wallrabe, G. Bonamy, A. Periasamy, and M. Barroso, Mol. Biol. Cell 18, 2226 (2007).
    [CrossRef]
  8. A. Periasamy, H. Wallrabe, Y. Chen, and M. Barroso, Methods Cell Biol. 89, 569 (2008).
    [CrossRef]
  9. C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, C. Dunsby, M. A. Neil, and P. M. French, J. Biophotonics 1, 514 (2008).
    [CrossRef]
  10. D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineau, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A. A. Neil, C. Dunsby, and P. M. W. French, J. Biophotonics 6, 398 (2013).
    [CrossRef]
  11. A. Esposito, C. P. Dohm, M. Bahr, and F. S. Wouters, Mol. Cell Proteomics 6, 1446 (2007).
    [CrossRef]
  12. H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. Bastiaens, Nat. Methods 7, 467 (2010).
    [CrossRef]
  13. E. M. Manders, A. E. Visser, A. Koppen, W. C. de Leeuw, R. van Liere, G. J. Brakenhoff, and R. van Driel, Chromosom. Res. 11, 537 (2003).
    [CrossRef]
  14. V. Venugopal and X. Intes, J. Biomed. Opt. 18, 036006 (2013).
    [CrossRef]

2013 (2)

D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineau, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A. A. Neil, C. Dunsby, and P. M. W. French, J. Biophotonics 6, 398 (2013).
[CrossRef]

V. Venugopal and X. Intes, J. Biomed. Opt. 18, 036006 (2013).
[CrossRef]

2012 (1)

W. Becker, J. Microsc. 247, 119 (2012).
[CrossRef]

2010 (3)

K. Chu, D. Lim, and J. Mertz, Biomed. Opt. Express 1, 236 (2010).
[CrossRef]

V. Venugopal, J. Chen, and X. Intes, Biomed. Opt. Express 1, 143 (2010).
[CrossRef]

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. Bastiaens, Nat. Methods 7, 467 (2010).
[CrossRef]

2008 (2)

A. Periasamy, H. Wallrabe, Y. Chen, and M. Barroso, Methods Cell Biol. 89, 569 (2008).
[CrossRef]

C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, C. Dunsby, M. A. Neil, and P. M. French, J. Biophotonics 1, 514 (2008).
[CrossRef]

2007 (3)

A. Esposito, C. P. Dohm, M. Bahr, and F. S. Wouters, Mol. Cell Proteomics 6, 1446 (2007).
[CrossRef]

H. Wallrabe, G. Bonamy, A. Periasamy, and M. Barroso, Mol. Biol. Cell 18, 2226 (2007).
[CrossRef]

R. A. Hoebe, C. H. Van Oven, T. W. Gadella, P. B. Dhonukshe, C. J. Van Noorden, and E. M. Manders, Nat. Biotechnol. 25, 249 (2007).
[CrossRef]

2006 (1)

P. B. Jones, L. Herl, O. Berezovska, A. T. Kumar, B. J. Bacskai, and B. T. Hyman, J. Biomed. Opt. 11, 054024 (2006).
[CrossRef]

2003 (1)

E. M. Manders, A. E. Visser, A. Koppen, W. C. de Leeuw, R. van Liere, G. J. Brakenhoff, and R. van Driel, Chromosom. Res. 11, 537 (2003).
[CrossRef]

1992 (1)

M. Kollner and J. Wolfrum, Chem. Phys. Lett. 200, 199 (1992).
[CrossRef]

Alexandrov, Y.

D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineau, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A. A. Neil, C. Dunsby, and P. M. W. French, J. Biophotonics 6, 398 (2013).
[CrossRef]

Alibhai, D.

D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineau, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A. A. Neil, C. Dunsby, and P. M. W. French, J. Biophotonics 6, 398 (2013).
[CrossRef]

Bacskai, B. J.

P. B. Jones, L. Herl, O. Berezovska, A. T. Kumar, B. J. Bacskai, and B. T. Hyman, J. Biomed. Opt. 11, 054024 (2006).
[CrossRef]

Bahr, M.

A. Esposito, C. P. Dohm, M. Bahr, and F. S. Wouters, Mol. Cell Proteomics 6, 1446 (2007).
[CrossRef]

Barroso, M.

A. Periasamy, H. Wallrabe, Y. Chen, and M. Barroso, Methods Cell Biol. 89, 569 (2008).
[CrossRef]

H. Wallrabe, G. Bonamy, A. Periasamy, and M. Barroso, Mol. Biol. Cell 18, 2226 (2007).
[CrossRef]

Bastiaens, P. I.

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. Bastiaens, Nat. Methods 7, 467 (2010).
[CrossRef]

Becker, W.

W. Becker, J. Microsc. 247, 119 (2012).
[CrossRef]

Berezovska, O.

P. B. Jones, L. Herl, O. Berezovska, A. T. Kumar, B. J. Bacskai, and B. T. Hyman, J. Biomed. Opt. 11, 054024 (2006).
[CrossRef]

Bonamy, G.

H. Wallrabe, G. Bonamy, A. Periasamy, and M. Barroso, Mol. Biol. Cell 18, 2226 (2007).
[CrossRef]

Brakenhoff, G. J.

E. M. Manders, A. E. Visser, A. Koppen, W. C. de Leeuw, R. van Liere, G. J. Brakenhoff, and R. van Driel, Chromosom. Res. 11, 537 (2003).
[CrossRef]

Bunney, T. D.

C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, C. Dunsby, M. A. Neil, and P. M. French, J. Biophotonics 1, 514 (2008).
[CrossRef]

Chen, J.

Chen, Y.

A. Periasamy, H. Wallrabe, Y. Chen, and M. Barroso, Methods Cell Biol. 89, 569 (2008).
[CrossRef]

Chu, K.

de Leeuw, W. C.

E. M. Manders, A. E. Visser, A. Koppen, W. C. de Leeuw, R. van Liere, G. J. Brakenhoff, and R. van Driel, Chromosom. Res. 11, 537 (2003).
[CrossRef]

Dhonukshe, P. B.

R. A. Hoebe, C. H. Van Oven, T. W. Gadella, P. B. Dhonukshe, C. J. Van Noorden, and E. M. Manders, Nat. Biotechnol. 25, 249 (2007).
[CrossRef]

Dohm, C. P.

A. Esposito, C. P. Dohm, M. Bahr, and F. S. Wouters, Mol. Cell Proteomics 6, 1446 (2007).
[CrossRef]

Dunsby, C.

D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineau, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A. A. Neil, C. Dunsby, and P. M. W. French, J. Biophotonics 6, 398 (2013).
[CrossRef]

C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, C. Dunsby, M. A. Neil, and P. M. French, J. Biophotonics 1, 514 (2008).
[CrossRef]

Eagle, R.

C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, C. Dunsby, M. A. Neil, and P. M. French, J. Biophotonics 1, 514 (2008).
[CrossRef]

Esposito, A.

A. Esposito, C. P. Dohm, M. Bahr, and F. S. Wouters, Mol. Cell Proteomics 6, 1446 (2007).
[CrossRef]

Frahm, T.

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. Bastiaens, Nat. Methods 7, 467 (2010).
[CrossRef]

French, P. M.

C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, C. Dunsby, M. A. Neil, and P. M. French, J. Biophotonics 1, 514 (2008).
[CrossRef]

French, P. M. W.

D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineau, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A. A. Neil, C. Dunsby, and P. M. W. French, J. Biophotonics 6, 398 (2013).
[CrossRef]

Gadella, T. W.

R. A. Hoebe, C. H. Van Oven, T. W. Gadella, P. B. Dhonukshe, C. J. Van Noorden, and E. M. Manders, Nat. Biotechnol. 25, 249 (2007).
[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. Bastiaens, Nat. Methods 7, 467 (2010).
[CrossRef]

Grant, D. M.

C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, C. Dunsby, M. A. Neil, and P. M. French, J. Biophotonics 1, 514 (2008).
[CrossRef]

Grecco, H. E.

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. Bastiaens, Nat. Methods 7, 467 (2010).
[CrossRef]

Hargreaves, A.

C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, C. Dunsby, M. A. Neil, and P. M. French, J. Biophotonics 1, 514 (2008).
[CrossRef]

Herl, L.

P. B. Jones, L. Herl, O. Berezovska, A. T. Kumar, B. J. Bacskai, and B. T. Hyman, J. Biomed. Opt. 11, 054024 (2006).
[CrossRef]

Hoebe, R. A.

R. A. Hoebe, C. H. Van Oven, T. W. Gadella, P. B. Dhonukshe, C. J. Van Noorden, and E. M. Manders, Nat. Biotechnol. 25, 249 (2007).
[CrossRef]

Hou, J.

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. Bastiaens, Nat. Methods 7, 467 (2010).
[CrossRef]

Hyman, B. T.

P. B. Jones, L. Herl, O. Berezovska, A. T. Kumar, B. J. Bacskai, and B. T. Hyman, J. Biomed. Opt. 11, 054024 (2006).
[CrossRef]

Intes, X.

V. Venugopal and X. Intes, J. Biomed. Opt. 18, 036006 (2013).
[CrossRef]

V. Venugopal, J. Chen, and X. Intes, Biomed. Opt. Express 1, 143 (2010).
[CrossRef]

Isherwood, B.

C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, C. Dunsby, M. A. Neil, and P. M. French, J. Biophotonics 1, 514 (2008).
[CrossRef]

Jones, P. B.

P. B. Jones, L. Herl, O. Berezovska, A. T. Kumar, B. J. Bacskai, and B. T. Hyman, J. Biomed. Opt. 11, 054024 (2006).
[CrossRef]

Kelly, D. J.

D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineau, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A. A. Neil, C. Dunsby, and P. M. W. French, J. Biophotonics 6, 398 (2013).
[CrossRef]

Kollner, M.

M. Kollner and J. Wolfrum, Chem. Phys. Lett. 200, 199 (1992).
[CrossRef]

Koppen, A.

E. M. Manders, A. E. Visser, A. Koppen, W. C. de Leeuw, R. van Liere, G. J. Brakenhoff, and R. van Driel, Chromosom. Res. 11, 537 (2003).
[CrossRef]

Kumar, A. T.

P. B. Jones, L. Herl, O. Berezovska, A. T. Kumar, B. J. Bacskai, and B. T. Hyman, J. Biomed. Opt. 11, 054024 (2006).
[CrossRef]

Kumar, S.

D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineau, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A. A. Neil, C. Dunsby, and P. M. W. French, J. Biophotonics 6, 398 (2013).
[CrossRef]

Lim, D.

Manders, E. M.

R. A. Hoebe, C. H. Van Oven, T. W. Gadella, P. B. Dhonukshe, C. J. Van Noorden, and E. M. Manders, Nat. Biotechnol. 25, 249 (2007).
[CrossRef]

E. M. Manders, A. E. Visser, A. Koppen, W. C. de Leeuw, R. van Liere, G. J. Brakenhoff, and R. van Driel, Chromosom. Res. 11, 537 (2003).
[CrossRef]

Margineau, A.

D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineau, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A. A. Neil, C. Dunsby, and P. M. W. French, J. Biophotonics 6, 398 (2013).
[CrossRef]

McGhee, E. J.

C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, C. Dunsby, M. A. Neil, and P. M. French, J. Biophotonics 1, 514 (2008).
[CrossRef]

McGinty, J.

C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, C. Dunsby, M. A. Neil, and P. M. French, J. Biophotonics 1, 514 (2008).
[CrossRef]

Mertz, J.

Munro, I.

C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, C. Dunsby, M. A. Neil, and P. M. French, J. Biophotonics 1, 514 (2008).
[CrossRef]

Murray, E. J.

D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineau, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A. A. Neil, C. Dunsby, and P. M. W. French, J. Biophotonics 6, 398 (2013).
[CrossRef]

Neil, M. A.

C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, C. Dunsby, M. A. Neil, and P. M. French, J. Biophotonics 1, 514 (2008).
[CrossRef]

Neil, M. A. A.

D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineau, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A. A. Neil, C. Dunsby, and P. M. W. French, J. Biophotonics 6, 398 (2013).
[CrossRef]

Owen, D. M.

C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, C. Dunsby, M. A. Neil, and P. M. French, J. Biophotonics 1, 514 (2008).
[CrossRef]

Pepperkok, R.

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. Bastiaens, Nat. Methods 7, 467 (2010).
[CrossRef]

Periasamy, A.

A. Periasamy, H. Wallrabe, Y. Chen, and M. Barroso, Methods Cell Biol. 89, 569 (2008).
[CrossRef]

H. Wallrabe, G. Bonamy, A. Periasamy, and M. Barroso, Mol. Biol. Cell 18, 2226 (2007).
[CrossRef]

Roda-Navarro, P.

H. E. Grecco, P. Roda-Navarro, A. Girod, J. Hou, T. Frahm, D. C. Truxius, R. Pepperkok, A. Squire, and P. I. Bastiaens, Nat. Methods 7, 467 (2010).
[CrossRef]

Serwa, R. A.

D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineau, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A. A. Neil, C. Dunsby, and P. M. W. French, J. Biophotonics 6, 398 (2013).
[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. Bastiaens, Nat. Methods 7, 467 (2010).
[CrossRef]

Stuhmeier, F.

D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineau, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A. A. Neil, C. Dunsby, and P. M. W. French, J. Biophotonics 6, 398 (2013).
[CrossRef]

Talbot, C. B.

C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, C. Dunsby, M. A. Neil, and P. M. French, J. Biophotonics 1, 514 (2008).
[CrossRef]

Tate, E. W.

D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineau, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A. A. Neil, C. Dunsby, and P. M. W. French, J. Biophotonics 6, 398 (2013).
[CrossRef]

Thinon, E.

D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineau, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A. A. Neil, C. Dunsby, and P. M. W. French, J. Biophotonics 6, 398 (2013).
[CrossRef]

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. Bastiaens, Nat. Methods 7, 467 (2010).
[CrossRef]

van Driel, R.

E. M. Manders, A. E. Visser, A. Koppen, W. C. de Leeuw, R. van Liere, G. J. Brakenhoff, and R. van Driel, Chromosom. Res. 11, 537 (2003).
[CrossRef]

van Liere, R.

E. M. Manders, A. E. Visser, A. Koppen, W. C. de Leeuw, R. van Liere, G. J. Brakenhoff, and R. van Driel, Chromosom. Res. 11, 537 (2003).
[CrossRef]

Van Noorden, C. J.

R. A. Hoebe, C. H. Van Oven, T. W. Gadella, P. B. Dhonukshe, C. J. Van Noorden, and E. M. Manders, Nat. Biotechnol. 25, 249 (2007).
[CrossRef]

Van Oven, C. H.

R. A. Hoebe, C. H. Van Oven, T. W. Gadella, P. B. Dhonukshe, C. J. Van Noorden, and E. M. Manders, Nat. Biotechnol. 25, 249 (2007).
[CrossRef]

Venugopal, V.

V. Venugopal and X. Intes, J. Biomed. Opt. 18, 036006 (2013).
[CrossRef]

V. Venugopal, J. Chen, and X. Intes, Biomed. Opt. Express 1, 143 (2010).
[CrossRef]

Visser, A. E.

E. M. Manders, A. E. Visser, A. Koppen, W. C. de Leeuw, R. van Liere, G. J. Brakenhoff, and R. van Driel, Chromosom. Res. 11, 537 (2003).
[CrossRef]

Wallrabe, H.

A. Periasamy, H. Wallrabe, Y. Chen, and M. Barroso, Methods Cell Biol. 89, 569 (2008).
[CrossRef]

H. Wallrabe, G. Bonamy, A. Periasamy, and M. Barroso, Mol. Biol. Cell 18, 2226 (2007).
[CrossRef]

Warren, S.

D. Alibhai, D. J. Kelly, S. Warren, S. Kumar, A. Margineau, R. A. Serwa, E. Thinon, Y. Alexandrov, E. J. Murray, F. Stuhmeier, E. W. Tate, M. A. A. Neil, C. Dunsby, and P. M. W. French, J. Biophotonics 6, 398 (2013).
[CrossRef]

Wolfrum, J.

M. Kollner and J. Wolfrum, Chem. Phys. Lett. 200, 199 (1992).
[CrossRef]

Wouters, F. S.

A. Esposito, C. P. Dohm, M. Bahr, and F. S. Wouters, Mol. Cell Proteomics 6, 1446 (2007).
[CrossRef]

Zhang, W.

C. B. Talbot, J. McGinty, D. M. Grant, E. J. McGhee, D. M. Owen, W. Zhang, T. D. Bunney, I. Munro, B. Isherwood, R. Eagle, A. Hargreaves, C. Dunsby, M. A. Neil, and P. M. French, J. Biophotonics 1, 514 (2008).
[CrossRef]

Biomed. Opt. Express (2)

Chem. Phys. Lett. (1)

M. Kollner and J. Wolfrum, Chem. Phys. Lett. 200, 199 (1992).
[CrossRef]

Chromosom. Res. (1)

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Figures (4)

Fig. 1.
Fig. 1.

(a) AWFI algorithm flowchart at the nth iteration. (b) Comparison of photon counts (PC) versus laser power (LP), and comparison of detected grayscale (DG) versus expected grayscale. (c) One example of AWFI procedure used for fluorescence measurement. The initial illumination pattern and final optimized pattern are shown on the left with a 0–255 grayscale bar. The corresponding fluorescence detected images are shown on the right with a 0–4000 colorbar.

Fig. 2.
Fig. 2.

(a) Uniform pattern. (b) Gradient pattern. (c) Random pattern. (d) and (e) Comparison of Nm, γ, and uniformity versus different iteration numbers in. Colorbar, 0–255.

Fig. 3.
Fig. 3.

Fluorescence intensity image at the maximum of decay curve (a) without AWFI procedure and (b) with AWFI procedure. (c) Comparison of average estimated lifetime and standard deviation (STD) of lifetimes for each well. (d) Comparison of the fluorescence intensities with and without (w/o) correction (CORR.) versus different concentrations before and after AWFI. Linear regression applied to fluorescence intensity before AWFI w/o CORR. and after AWFI with CORR. to different concentrations yields R2=0.96 and R2=0.99, respectively.

Fig. 4.
Fig. 4.

Fluorescence intensity image at the maximum of decay curve (a) without AWFI procedure and (b) with AWFI procedure. Comparison of FDF versus different AD ratios (c) without AWFI procedure and (d) with AWFI procedure.

Tables (1)

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Table 1. FDF Estimates for a Subset of 6 Wellsa

Equations (3)

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A(n)(i,j)=N(n)S(n)(i,j)·P(n)(i,j),(A(n)(i,j)γ(n)),
P(n+1)(i,j)=A(n)(i,j)α(n).
Uniformity=100·(1σNm).

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