Abstract

Characterizing the photoactivation performance of optical highlighter fluorescent proteins is crucial to the realization of photoactivation localization microscopy. In contrast to those fluorescence-based approaches that require complex data processing and calibration procedures, here we report a simple and quantitative alternative, which relies on the measurement of small absorption spectra changes over time with a fiber-optic system. Using Dronpa as a representative highlighter protein, we have investigated the capacity of this system in monitoring the fast photoactivation process.

© 2011 OSA

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    [CrossRef] [PubMed]

2011 (1)

E. Pastrana, “Fast 3D super-resolution fluorescence microscopy,” Nat. Methods 8(1), 46 (2011).
[CrossRef]

2010 (5)

A. R. Faro, V. Adam, P. Carpentier, C. Darnault, D. Bourgeois, and E. de Rosny, “Low-temperature switching by photoinduced protonation in photochromic fluorescent proteins,” Photochem. Photobiol. Sci. 9(2), 254–262 (2010).
[CrossRef] [PubMed]

B. Huang, H. Babcock, and X. Zhuang, “Breaking the diffraction barrier: super-resolution imaging of cells,” Cell 143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

P. Kanchanawong, G. Shtengel, A. M. Pasapera, E. B. Ramko, M. W. Davidson, H. F. Hess, and C. M. Waterman, “Nanoscale architecture of integrin-based cell adhesions,” Nature 468(7323), 580–584 (2010).
[CrossRef] [PubMed]

J. Liu, Z. G. Pei, L. Wang, Z. H. Zhang, S. Q. Zeng, and Z. L. Huang, “A straightforward and quantitative approach for characterizing the photoactivation performance of optical highlighter fluorescent proteins,” Appl. Phys. Lett. 97(20), 203701 (2010).
[CrossRef]

G. Patterson, M. Davidson, S. Manley, and J. Lippincott-Schwartz, “Superresolution imaging using single-molecule localization,” Annu. Rev. Phys. Chem. 61(1), 345–367 (2010).
[CrossRef] [PubMed]

2009 (1)

F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V. Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods 6(2), 153–159 (2009).
[CrossRef] [PubMed]

2008 (1)

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods 5(5), 417–423 (2008).
[CrossRef] [PubMed]

2007 (3)

S. W. Hell, “Far-field optical nanoscopy,” Science 316(5828), 1153–1158 (2007).
[CrossRef] [PubMed]

C. Flors, J.-i. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc. 129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

2006 (2)

S. Habuchi, P. Dedecker, J.-i. Hotta, C. Flors, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching,” Photochem. Photobiol. Sci. 5(6), 567–576 (2006).
[CrossRef] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

2005 (1)

S. Habuchi, R. Ando, P. Dedecker, W. Verheijen, H. Mizuno, A. Miyawaki, and J. Hofkens, “Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 102(27), 9511–9516 (2005).
[CrossRef] [PubMed]

2004 (1)

R. Ando, H. Mizuno, and A. Miyawaki, “Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting,” Science 306(5700), 1370–1373 (2004).
[CrossRef] [PubMed]

Adam, V.

A. R. Faro, V. Adam, P. Carpentier, C. Darnault, D. Bourgeois, and E. de Rosny, “Low-temperature switching by photoinduced protonation in photochromic fluorescent proteins,” Photochem. Photobiol. Sci. 9(2), 254–262 (2010).
[CrossRef] [PubMed]

Ando, R.

C. Flors, J.-i. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc. 129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

S. Habuchi, P. Dedecker, J.-i. Hotta, C. Flors, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching,” Photochem. Photobiol. Sci. 5(6), 567–576 (2006).
[CrossRef] [PubMed]

S. Habuchi, R. Ando, P. Dedecker, W. Verheijen, H. Mizuno, A. Miyawaki, and J. Hofkens, “Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 102(27), 9511–9516 (2005).
[CrossRef] [PubMed]

R. Ando, H. Mizuno, and A. Miyawaki, “Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting,” Science 306(5700), 1370–1373 (2004).
[CrossRef] [PubMed]

Andresen, M.

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

Babcock, H.

B. Huang, H. Babcock, and X. Zhuang, “Breaking the diffraction barrier: super-resolution imaging of cells,” Cell 143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

Betzig, E.

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods 5(5), 417–423 (2008).
[CrossRef] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Bonifacino, J. S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Bourgeois, D.

A. R. Faro, V. Adam, P. Carpentier, C. Darnault, D. Bourgeois, and E. de Rosny, “Low-temperature switching by photoinduced protonation in photochromic fluorescent proteins,” Photochem. Photobiol. Sci. 9(2), 254–262 (2010).
[CrossRef] [PubMed]

Carpentier, P.

A. R. Faro, V. Adam, P. Carpentier, C. Darnault, D. Bourgeois, and E. de Rosny, “Low-temperature switching by photoinduced protonation in photochromic fluorescent proteins,” Photochem. Photobiol. Sci. 9(2), 254–262 (2010).
[CrossRef] [PubMed]

Darnault, C.

A. R. Faro, V. Adam, P. Carpentier, C. Darnault, D. Bourgeois, and E. de Rosny, “Low-temperature switching by photoinduced protonation in photochromic fluorescent proteins,” Photochem. Photobiol. Sci. 9(2), 254–262 (2010).
[CrossRef] [PubMed]

Davidson, M.

G. Patterson, M. Davidson, S. Manley, and J. Lippincott-Schwartz, “Superresolution imaging using single-molecule localization,” Annu. Rev. Phys. Chem. 61(1), 345–367 (2010).
[CrossRef] [PubMed]

Davidson, M. W.

P. Kanchanawong, G. Shtengel, A. M. Pasapera, E. B. Ramko, M. W. Davidson, H. F. Hess, and C. M. Waterman, “Nanoscale architecture of integrin-based cell adhesions,” Nature 468(7323), 580–584 (2010).
[CrossRef] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

de Rosny, E.

A. R. Faro, V. Adam, P. Carpentier, C. Darnault, D. Bourgeois, and E. de Rosny, “Low-temperature switching by photoinduced protonation in photochromic fluorescent proteins,” Photochem. Photobiol. Sci. 9(2), 254–262 (2010).
[CrossRef] [PubMed]

Dedecker, P.

C. Flors, J.-i. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc. 129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

S. Habuchi, P. Dedecker, J.-i. Hotta, C. Flors, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching,” Photochem. Photobiol. Sci. 5(6), 567–576 (2006).
[CrossRef] [PubMed]

S. Habuchi, R. Ando, P. Dedecker, W. Verheijen, H. Mizuno, A. Miyawaki, and J. Hofkens, “Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 102(27), 9511–9516 (2005).
[CrossRef] [PubMed]

Eggeling, C.

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

Faro, A. R.

A. R. Faro, V. Adam, P. Carpentier, C. Darnault, D. Bourgeois, and E. de Rosny, “Low-temperature switching by photoinduced protonation in photochromic fluorescent proteins,” Photochem. Photobiol. Sci. 9(2), 254–262 (2010).
[CrossRef] [PubMed]

Flors, C.

C. Flors, J.-i. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc. 129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

S. Habuchi, P. Dedecker, J.-i. Hotta, C. Flors, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching,” Photochem. Photobiol. Sci. 5(6), 567–576 (2006).
[CrossRef] [PubMed]

Galbraith, C. G.

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods 5(5), 417–423 (2008).
[CrossRef] [PubMed]

Galbraith, J. A.

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods 5(5), 417–423 (2008).
[CrossRef] [PubMed]

Gillette, J. M.

F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V. Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods 6(2), 153–159 (2009).
[CrossRef] [PubMed]

Habuchi, S.

S. Habuchi, P. Dedecker, J.-i. Hotta, C. Flors, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching,” Photochem. Photobiol. Sci. 5(6), 567–576 (2006).
[CrossRef] [PubMed]

S. Habuchi, R. Ando, P. Dedecker, W. Verheijen, H. Mizuno, A. Miyawaki, and J. Hofkens, “Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 102(27), 9511–9516 (2005).
[CrossRef] [PubMed]

Hell, S. W.

S. W. Hell, “Far-field optical nanoscopy,” Science 316(5828), 1153–1158 (2007).
[CrossRef] [PubMed]

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

Hess, H. F.

P. Kanchanawong, G. Shtengel, A. M. Pasapera, E. B. Ramko, M. W. Davidson, H. F. Hess, and C. M. Waterman, “Nanoscale architecture of integrin-based cell adhesions,” Nature 468(7323), 580–584 (2010).
[CrossRef] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Hofkens, J.

C. Flors, J.-i. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc. 129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

S. Habuchi, P. Dedecker, J.-i. Hotta, C. Flors, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching,” Photochem. Photobiol. Sci. 5(6), 567–576 (2006).
[CrossRef] [PubMed]

S. Habuchi, R. Ando, P. Dedecker, W. Verheijen, H. Mizuno, A. Miyawaki, and J. Hofkens, “Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 102(27), 9511–9516 (2005).
[CrossRef] [PubMed]

Hotta, J.-i.

C. Flors, J.-i. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc. 129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

S. Habuchi, P. Dedecker, J.-i. Hotta, C. Flors, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching,” Photochem. Photobiol. Sci. 5(6), 567–576 (2006).
[CrossRef] [PubMed]

Huang, B.

B. Huang, H. Babcock, and X. Zhuang, “Breaking the diffraction barrier: super-resolution imaging of cells,” Cell 143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

Huang, Z. L.

J. Liu, Z. G. Pei, L. Wang, Z. H. Zhang, S. Q. Zeng, and Z. L. Huang, “A straightforward and quantitative approach for characterizing the photoactivation performance of optical highlighter fluorescent proteins,” Appl. Phys. Lett. 97(20), 203701 (2010).
[CrossRef]

Jakobs, S.

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

Kanchanawong, P.

P. Kanchanawong, G. Shtengel, A. M. Pasapera, E. B. Ramko, M. W. Davidson, H. F. Hess, and C. M. Waterman, “Nanoscale architecture of integrin-based cell adhesions,” Nature 468(7323), 580–584 (2010).
[CrossRef] [PubMed]

Lindwasser, O. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Lippincott-Schwartz, J.

G. Patterson, M. Davidson, S. Manley, and J. Lippincott-Schwartz, “Superresolution imaging using single-molecule localization,” Annu. Rev. Phys. Chem. 61(1), 345–367 (2010).
[CrossRef] [PubMed]

F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V. Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods 6(2), 153–159 (2009).
[CrossRef] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Liu, J.

J. Liu, Z. G. Pei, L. Wang, Z. H. Zhang, S. Q. Zeng, and Z. L. Huang, “A straightforward and quantitative approach for characterizing the photoactivation performance of optical highlighter fluorescent proteins,” Appl. Phys. Lett. 97(20), 203701 (2010).
[CrossRef]

Manley, S.

G. Patterson, M. Davidson, S. Manley, and J. Lippincott-Schwartz, “Superresolution imaging using single-molecule localization,” Annu. Rev. Phys. Chem. 61(1), 345–367 (2010).
[CrossRef] [PubMed]

F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V. Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods 6(2), 153–159 (2009).
[CrossRef] [PubMed]

Miyawaki, A.

C. Flors, J.-i. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc. 129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

S. Habuchi, P. Dedecker, J.-i. Hotta, C. Flors, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching,” Photochem. Photobiol. Sci. 5(6), 567–576 (2006).
[CrossRef] [PubMed]

S. Habuchi, R. Ando, P. Dedecker, W. Verheijen, H. Mizuno, A. Miyawaki, and J. Hofkens, “Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 102(27), 9511–9516 (2005).
[CrossRef] [PubMed]

R. Ando, H. Mizuno, and A. Miyawaki, “Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting,” Science 306(5700), 1370–1373 (2004).
[CrossRef] [PubMed]

Mizuno, H.

C. Flors, J.-i. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc. 129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

S. Habuchi, P. Dedecker, J.-i. Hotta, C. Flors, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching,” Photochem. Photobiol. Sci. 5(6), 567–576 (2006).
[CrossRef] [PubMed]

S. Habuchi, R. Ando, P. Dedecker, W. Verheijen, H. Mizuno, A. Miyawaki, and J. Hofkens, “Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 102(27), 9511–9516 (2005).
[CrossRef] [PubMed]

R. Ando, H. Mizuno, and A. Miyawaki, “Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting,” Science 306(5700), 1370–1373 (2004).
[CrossRef] [PubMed]

Olenych, S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Pasapera, A. M.

P. Kanchanawong, G. Shtengel, A. M. Pasapera, E. B. Ramko, M. W. Davidson, H. F. Hess, and C. M. Waterman, “Nanoscale architecture of integrin-based cell adhesions,” Nature 468(7323), 580–584 (2010).
[CrossRef] [PubMed]

Pastrana, E.

E. Pastrana, “Fast 3D super-resolution fluorescence microscopy,” Nat. Methods 8(1), 46 (2011).
[CrossRef]

Patterson, G.

G. Patterson, M. Davidson, S. Manley, and J. Lippincott-Schwartz, “Superresolution imaging using single-molecule localization,” Annu. Rev. Phys. Chem. 61(1), 345–367 (2010).
[CrossRef] [PubMed]

Patterson, G. H.

F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V. Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods 6(2), 153–159 (2009).
[CrossRef] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Pei, Z. G.

J. Liu, Z. G. Pei, L. Wang, Z. H. Zhang, S. Q. Zeng, and Z. L. Huang, “A straightforward and quantitative approach for characterizing the photoactivation performance of optical highlighter fluorescent proteins,” Appl. Phys. Lett. 97(20), 203701 (2010).
[CrossRef]

Ramko, E. B.

P. Kanchanawong, G. Shtengel, A. M. Pasapera, E. B. Ramko, M. W. Davidson, H. F. Hess, and C. M. Waterman, “Nanoscale architecture of integrin-based cell adhesions,” Nature 468(7323), 580–584 (2010).
[CrossRef] [PubMed]

Shroff, H.

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods 5(5), 417–423 (2008).
[CrossRef] [PubMed]

Shtengel, G.

P. Kanchanawong, G. Shtengel, A. M. Pasapera, E. B. Ramko, M. W. Davidson, H. F. Hess, and C. M. Waterman, “Nanoscale architecture of integrin-based cell adhesions,” Nature 468(7323), 580–584 (2010).
[CrossRef] [PubMed]

Sougrat, R.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Stiel, A. C.

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

Subach, F. V.

F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V. Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods 6(2), 153–159 (2009).
[CrossRef] [PubMed]

Trowitzsch, S.

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

Uji-i, H.

C. Flors, J.-i. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc. 129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

Verheijen, W.

S. Habuchi, R. Ando, P. Dedecker, W. Verheijen, H. Mizuno, A. Miyawaki, and J. Hofkens, “Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 102(27), 9511–9516 (2005).
[CrossRef] [PubMed]

Verkhusha, V. V.

F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V. Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods 6(2), 153–159 (2009).
[CrossRef] [PubMed]

Wahl, M. C.

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

Wang, L.

J. Liu, Z. G. Pei, L. Wang, Z. H. Zhang, S. Q. Zeng, and Z. L. Huang, “A straightforward and quantitative approach for characterizing the photoactivation performance of optical highlighter fluorescent proteins,” Appl. Phys. Lett. 97(20), 203701 (2010).
[CrossRef]

Waterman, C. M.

P. Kanchanawong, G. Shtengel, A. M. Pasapera, E. B. Ramko, M. W. Davidson, H. F. Hess, and C. M. Waterman, “Nanoscale architecture of integrin-based cell adhesions,” Nature 468(7323), 580–584 (2010).
[CrossRef] [PubMed]

Weber, G.

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

Zeng, S. Q.

J. Liu, Z. G. Pei, L. Wang, Z. H. Zhang, S. Q. Zeng, and Z. L. Huang, “A straightforward and quantitative approach for characterizing the photoactivation performance of optical highlighter fluorescent proteins,” Appl. Phys. Lett. 97(20), 203701 (2010).
[CrossRef]

Zhang, Z. H.

J. Liu, Z. G. Pei, L. Wang, Z. H. Zhang, S. Q. Zeng, and Z. L. Huang, “A straightforward and quantitative approach for characterizing the photoactivation performance of optical highlighter fluorescent proteins,” Appl. Phys. Lett. 97(20), 203701 (2010).
[CrossRef]

Zhuang, X.

B. Huang, H. Babcock, and X. Zhuang, “Breaking the diffraction barrier: super-resolution imaging of cells,” Cell 143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

Annu. Rev. Phys. Chem. (1)

G. Patterson, M. Davidson, S. Manley, and J. Lippincott-Schwartz, “Superresolution imaging using single-molecule localization,” Annu. Rev. Phys. Chem. 61(1), 345–367 (2010).
[CrossRef] [PubMed]

Appl. Phys. Lett. (1)

J. Liu, Z. G. Pei, L. Wang, Z. H. Zhang, S. Q. Zeng, and Z. L. Huang, “A straightforward and quantitative approach for characterizing the photoactivation performance of optical highlighter fluorescent proteins,” Appl. Phys. Lett. 97(20), 203701 (2010).
[CrossRef]

Cell (1)

B. Huang, H. Babcock, and X. Zhuang, “Breaking the diffraction barrier: super-resolution imaging of cells,” Cell 143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

J. Am. Chem. Soc. (1)

C. Flors, J.-i. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants,” J. Am. Chem. Soc. 129(45), 13970–13977 (2007).
[CrossRef] [PubMed]

Nat. Methods (3)

E. Pastrana, “Fast 3D super-resolution fluorescence microscopy,” Nat. Methods 8(1), 46 (2011).
[CrossRef]

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods 5(5), 417–423 (2008).
[CrossRef] [PubMed]

F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V. Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods 6(2), 153–159 (2009).
[CrossRef] [PubMed]

Nature (1)

P. Kanchanawong, G. Shtengel, A. M. Pasapera, E. B. Ramko, M. W. Davidson, H. F. Hess, and C. M. Waterman, “Nanoscale architecture of integrin-based cell adhesions,” Nature 468(7323), 580–584 (2010).
[CrossRef] [PubMed]

Photochem. Photobiol. Sci. (2)

S. Habuchi, P. Dedecker, J.-i. Hotta, C. Flors, R. Ando, H. Mizuno, A. Miyawaki, and J. Hofkens, “Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching,” Photochem. Photobiol. Sci. 5(6), 567–576 (2006).
[CrossRef] [PubMed]

A. R. Faro, V. Adam, P. Carpentier, C. Darnault, D. Bourgeois, and E. de Rosny, “Low-temperature switching by photoinduced protonation in photochromic fluorescent proteins,” Photochem. Photobiol. Sci. 9(2), 254–262 (2010).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (2)

S. Habuchi, R. Ando, P. Dedecker, W. Verheijen, H. Mizuno, A. Miyawaki, and J. Hofkens, “Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 102(27), 9511–9516 (2005).
[CrossRef] [PubMed]

M. Andresen, A. C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M. C. Wahl, S. W. Hell, and S. Jakobs, “Structural basis for reversible photoswitching in Dronpa,” Proc. Natl. Acad. Sci. U.S.A. 104(32), 13005–13009 (2007).
[CrossRef] [PubMed]

Science (3)

S. W. Hell, “Far-field optical nanoscopy,” Science 316(5828), 1153–1158 (2007).
[CrossRef] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

R. Ando, H. Mizuno, and A. Miyawaki, “Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting,” Science 306(5700), 1370–1373 (2004).
[CrossRef] [PubMed]

Other (6)

Ocean Optics catalog (Ocean Optics, Inc., 2011).

N. Irwin and K. A. Janssen, Molecular Cloning: a Laboratory Manual, 3ed., J. Argentine and N. Irwin, eds. (Cold Spring Harbor Laboratory Press, New York 2001).

“CCD noise sources and signal-to-noise ratio,” http://learn.hamamatsu.com/articles/ccdsnr.html , accessed Mar. 2011.

“HL-2000 tungsten halogen light sources,” http://www.oceanoptics.com/Products/hl2000.asp , accessed Mar. 2011.

“USB2000+ Miniature Fiber Optic Spectrometer,” http://www.oceanoptics.com/Products/usb2000+.asp , accessed Mar. 2011.

S. W. Smith, The Scientist and Engineer's Guide to Digital Signal Processing, 2nd ed. (California Technical Publishing, 2003).

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Figures (6)

Fig. 1
Fig. 1

The optical set-up for monitoring fast photoactivation dynamics. SHG, BBO crystal; SF, short pass filter; ND, neutral density filters; M, mirror; BS, beam splitter; LS, light source; L, focusing lens; CF, color filters; Block, beam block.

Fig. 2
Fig. 2

The absorption spectra of Dronpa measured with a commercial Lambda 950 spectrophotometer and our system based on a USB2000+ miniature fiber-optic spectrometer.

Fig. 3
Fig. 3

The measured output of the tungsten-halogen lamp with (a) no filter, (b) color filter QB2, and (c) QB2 and SP650 (short pass filter). The inset shows the intensity changes after inserting the filters.

Fig. 4
Fig. 4

The absorption spectrum of Dronpa changes with time during the photoactivation process (a) and the evolution of peak absorption at 503 nm wavelength versus time can be fitted well with a first-order kinetic model (b).

Fig. 5
Fig. 5

Time evolvement of Dronpa’s peak absorption during the activation process under low (a) and high (b) intensity of activation light (mW cm−2). Note that under high activation intensities, the absorption changes were recorded every 10 ms (see the inset of (b), which is for an activation intensity of 333.3 mW cm−2).

Fig. 6
Fig. 6

The photoswiching rate constant depends linearly on the intensity of activation light. Note that the data points are from the mean of three independent experiments, and the reproducity of these experiments is shown with the error bars.

Equations (4)

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

N C C D = S + D t + N r 2
S N R s y s = n S ( n a S ) 2 + n S = 1 a 2 + 1 n S
A ( λ ) = log 10 ( I ( λ ) I 0 ( λ ) ) = log 10 ( 1 Δ I ( λ ) I 0 ( λ ) ) = ln ( 1 Δ I ( λ ) I 0 ( λ ) ) / ln 10 0.43 × ln ( 1 Δ I ( λ ) I 0 ( λ ) )
A min ( λ ) = Δ A ( λ ) 0.43 Δ I min ( λ ) I 0 ( λ ) = 0.43 S N R s y s max

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