Abstract

Confocal or multi-photon laser scanning microscopes are convenient tools to perform FRAP diffusion measurements. Despite its popularity, accurate FRAP remains often challenging since current methods are either limited to relatively large bleach regions or can be complicated for non-specialists. In order to bring reliable quantitative FRAP measurements to the broad community of laser scanning microscopy users, here we have revised FRAP theory and present a new pixel based FRAP method relying on the photo bleaching of rectangular regions of any size and aspect ratio. The method allows for fast and straightforward quantitative diffusion measurements due to a closed–form expression for the recovery process utilizing all available spatial and temporal data. After a detailed validation, its versatility is demonstrated by diffusion studies in heterogeneous biopolymer mixtures.

© 2010 OSA

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2010 (1)

J. K. Jonasson, J. Hagman, N. Lorén, D. Bernin, M. Nydén, and M. Rudemo, “Pixel-based analysis of FRAP data with a general initial bleaching profile,” J. Microsc. 239(2), 142–153 (2010).
[PubMed]

2009 (5)

A. Tannert, S. Tannert, S. Burgold, and M. Schaefer, “Convolution-based one and two component FRAP analysis: theory and application,” Eur. Biophys. J. 38(5), 649–661 (2009).
[CrossRef] [PubMed]

M. Kang, C. A. Day, K. Drake, A. K. Kenworthy, and E. DiBenedetto, “A generalization of theory for two-dimensional fluorescence recovery after photobleaching applicable to confocal laser scanning microscopes,” Biophys. J. 97(5), 1501–1511 (2009).
[CrossRef] [PubMed]

S. Fransson, N. Lorén, A. Altskär, and A. M. Hermansson, “Effect of confinement and kinetics on the morphology of phase separating gelatin-maltodextrin droplets,” Biomacromolecules 10(6), 1446–1453 (2009).
[CrossRef] [PubMed]

F. van de Manakker, K. Braeckmans, N. Morabit, S. C. De Smedt, C. F. van Nostrum, and W. E. Hennink, “Protein-Release Behavior of Self-Assembled PEG-beta-Cyclodextrin/PEG-Cholesterol Hydrogels,” Adv. Funct. Mater. 19(18), 2992–3001 (2009).
[CrossRef]

R. Censi, T. Vermonden, M. J. van Steenbergen, H. Deschout, K. Braeckmans, S. C. De Smedt, C. F. van Nostrum, P. di Martino, and W. E. Hennink, “Photopolymerized thermosensitive hydrogels for tailorable diffusion-controlled protein delivery,” J. Control. Release 140(3), 230–236 (2009).
[CrossRef] [PubMed]

2008 (3)

D. Mazza, K. Braeckmans, F. Cella, I. Testa, D. Vercauteren, J. Demeester, S. S. De Smedt, and A. Diaspro, “A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy,” Biophys. J. 95(7), 3457–3469 (2008).
[CrossRef] [PubMed]

P. Jönsson, M. P. Jonsson, J. O. Tegenfeldt, and F. Höök, “A method improving the accuracy of fluorescence recovery after photobleaching analysis,” Biophys. J. 95(11), 5334–5348 (2008).
[CrossRef] [PubMed]

J. K. Jonasson, N. Lorén, P. Olofsson, M. Nydén, and M. Rudemo, “A pixel-based likelihood framework for analysis of fluorescence recovery after photobleaching data,” J. Microsc. 232(2), 260–269 (2008).
[CrossRef] [PubMed]

2007 (1)

K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. De Smedt, and J. Demeester, “Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples,” Biophys. J. 92(6), 2172–2183 (2007).
[CrossRef] [PubMed]

2006 (2)

K. Braeckmans, B. G. Stubbe, K. Remaut, J. Demeester, and S. C. De Smedt, “Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation--a case study for fluorescein,” J. Biomed. Opt. 11(4), 044013 (2006).
[CrossRef] [PubMed]

F. Alvarez-Manceñido, K. Braeckmans, S. C. De Smedt, J. Demeester, M. Landin, and R. Martínez-Pacheco, “Characterization of diffusion of macromolecules in konjac glucomannan solutions and gels by fluorescence recovery after photobleaching technique,” Int. J. Pharm. 316(1-2), 37–46 (2006).
[CrossRef] [PubMed]

2005 (1)

S. R. Van Tomme, B. G. De Geest, K. Braeckmans, S. C. De Smedt, F. Siepmann, J. Siepmann, C. F. van Nostrum, and W. E. Hennink, “Mobility of model proteins in hydrogels composed of oppositely charged dextran microspheres studied by protein release and fluorescence recovery after photobleaching,” J. Control. Release 110(1), 67–78 (2005).
[CrossRef] [PubMed]

2004 (1)

J. Braga, J. M. P. Desterro, and M. Carmo-Fonseca, “Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes,” Mol. Biol. Cell 15(10), 4749–4760 (2004).
[CrossRef] [PubMed]

2003 (2)

K. Braeckmans, L. Peeters, N. N. Sanders, S. C. De Smedt, and J. Demeester, “Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope,” Biophys. J. 85(4), 2240–2252 (2003).
[CrossRef] [PubMed]

A. S. Verkman, “Diffusion in cells measured by fluorescence recovery after photobleaching,” Methods Enzymol. 360, 635–648 (2003).
[CrossRef] [PubMed]

2001 (1)

N. Lorén, A. Altskar, and A. M. Hermansson, “Structure evolution during gelation at later stages of spinodal decomposition in gelatin/maltodextrin mixtures,” Macromolecules 34(23), 8117–8128 (2001).
[CrossRef]

2000 (3)

N. Lorén and A. M. Hermansson, “Phase separation and gel formation in kinetically trapped gelatin/maltodextrin gels,” Int. J. Biol. Macromol. 27(4), 249–262 (2000).
[CrossRef] [PubMed]

M. D. Burke, J. O. Park, M. Srinivasarao, and S. A. Khan, “Diffusion of macromolecules in polymer solutions and gels: A laser scanning confocal microscopy study,” Macromolecules 33(20), 7500–7507 (2000).
[CrossRef]

F. Umenishi, J. M. Verbavatz, and A. S. Verkman, “cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera,” Biophys. J. 78(2), 1024–1035 (2000).
[CrossRef] [PubMed]

1999 (1)

E. B. Brown, E. S. Wu, W. Zipfel, and W. W. Webb, “Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery,” Biophys. J. 77(5), 2837–2849 (1999).
[CrossRef] [PubMed]

1997 (2)

O. Seksek, J. Biwersi, and A. S. Verkman, “Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus,” J. Cell Biol. 138(1), 131–142 (1997).
[CrossRef] [PubMed]

S. C. De Smedt, T. K. L. Meyvis, J. Demeester, P. VanOostveldt, J. C. G. Blonk, and W. E. Hennink, “Diffusion of macromolecules in dextran methacrylate solutions and gels as studied by confocal scanning laser microscopy,” Macromolecules 30(17), 4863–4870 (1997).
[CrossRef]

1996 (1)

P. Wedekind, U. Kubitscheck, O. Heinrich, and R. Peters, “Line-scanning microphotolysis for diffraction-limited measurements of lateral diffusion,” Biophys. J. 71(3), 1621–1632 (1996).
[CrossRef] [PubMed]

1994 (1)

P. Wedekind, U. Kubitscheck, and R. Peters, “Scanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging,” J. Microsc. 176(Pt 1), 23–33 (1994).
[CrossRef] [PubMed]

1993 (3)

J. C. G. Blonk, A. Don, H. Vanaalst, and J. J. Birmingham, “Fluorescence Photobleaching Recovery in the Confocal Scanning Light-Microscope,” J. Microsc. 169, 363–374 (1993).
[CrossRef]

D. A. Berk, F. Yuan, M. Leunig, and R. K. Jain, “Fluorescence photobleaching with spatial Fourier analysis: measurement of diffusion in light-scattering media,” Biophys. J. 65(6), 2428–2436 (1993).
[CrossRef] [PubMed]

A. Ishihara and K. Jacobson, “A closer look at how membrane proteins move,” Biophys. J. 65(5), 1754–1755 (1993).
[CrossRef] [PubMed]

1991 (1)

T. T. Tsay and K. A. Jacobson, “Spatial Fourier analysis of video photobleaching measurements. Principles and optimization,” Biophys. J. 60(2), 360–368 (1991).
[CrossRef] [PubMed]

1976 (1)

D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb, “Mobility measurement by analysis of fluorescence photobleaching recovery kinetics,” Biophys. J. 16(9), 1055–1069 (1976).
[CrossRef] [PubMed]

Altskar, A.

N. Lorén, A. Altskar, and A. M. Hermansson, “Structure evolution during gelation at later stages of spinodal decomposition in gelatin/maltodextrin mixtures,” Macromolecules 34(23), 8117–8128 (2001).
[CrossRef]

Altskär, A.

S. Fransson, N. Lorén, A. Altskär, and A. M. Hermansson, “Effect of confinement and kinetics on the morphology of phase separating gelatin-maltodextrin droplets,” Biomacromolecules 10(6), 1446–1453 (2009).
[CrossRef] [PubMed]

Alvarez-Manceñido, F.

F. Alvarez-Manceñido, K. Braeckmans, S. C. De Smedt, J. Demeester, M. Landin, and R. Martínez-Pacheco, “Characterization of diffusion of macromolecules in konjac glucomannan solutions and gels by fluorescence recovery after photobleaching technique,” Int. J. Pharm. 316(1-2), 37–46 (2006).
[CrossRef] [PubMed]

Axelrod, D.

D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb, “Mobility measurement by analysis of fluorescence photobleaching recovery kinetics,” Biophys. J. 16(9), 1055–1069 (1976).
[CrossRef] [PubMed]

Berk, D. A.

D. A. Berk, F. Yuan, M. Leunig, and R. K. Jain, “Fluorescence photobleaching with spatial Fourier analysis: measurement of diffusion in light-scattering media,” Biophys. J. 65(6), 2428–2436 (1993).
[CrossRef] [PubMed]

Bernin, D.

J. K. Jonasson, J. Hagman, N. Lorén, D. Bernin, M. Nydén, and M. Rudemo, “Pixel-based analysis of FRAP data with a general initial bleaching profile,” J. Microsc. 239(2), 142–153 (2010).
[PubMed]

Birmingham, J. J.

J. C. G. Blonk, A. Don, H. Vanaalst, and J. J. Birmingham, “Fluorescence Photobleaching Recovery in the Confocal Scanning Light-Microscope,” J. Microsc. 169, 363–374 (1993).
[CrossRef]

Biwersi, J.

O. Seksek, J. Biwersi, and A. S. Verkman, “Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus,” J. Cell Biol. 138(1), 131–142 (1997).
[CrossRef] [PubMed]

Blonk, J. C. G.

S. C. De Smedt, T. K. L. Meyvis, J. Demeester, P. VanOostveldt, J. C. G. Blonk, and W. E. Hennink, “Diffusion of macromolecules in dextran methacrylate solutions and gels as studied by confocal scanning laser microscopy,” Macromolecules 30(17), 4863–4870 (1997).
[CrossRef]

J. C. G. Blonk, A. Don, H. Vanaalst, and J. J. Birmingham, “Fluorescence Photobleaching Recovery in the Confocal Scanning Light-Microscope,” J. Microsc. 169, 363–374 (1993).
[CrossRef]

Braeckmans, K.

R. Censi, T. Vermonden, M. J. van Steenbergen, H. Deschout, K. Braeckmans, S. C. De Smedt, C. F. van Nostrum, P. di Martino, and W. E. Hennink, “Photopolymerized thermosensitive hydrogels for tailorable diffusion-controlled protein delivery,” J. Control. Release 140(3), 230–236 (2009).
[CrossRef] [PubMed]

F. van de Manakker, K. Braeckmans, N. Morabit, S. C. De Smedt, C. F. van Nostrum, and W. E. Hennink, “Protein-Release Behavior of Self-Assembled PEG-beta-Cyclodextrin/PEG-Cholesterol Hydrogels,” Adv. Funct. Mater. 19(18), 2992–3001 (2009).
[CrossRef]

D. Mazza, K. Braeckmans, F. Cella, I. Testa, D. Vercauteren, J. Demeester, S. S. De Smedt, and A. Diaspro, “A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy,” Biophys. J. 95(7), 3457–3469 (2008).
[CrossRef] [PubMed]

K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. De Smedt, and J. Demeester, “Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples,” Biophys. J. 92(6), 2172–2183 (2007).
[CrossRef] [PubMed]

F. Alvarez-Manceñido, K. Braeckmans, S. C. De Smedt, J. Demeester, M. Landin, and R. Martínez-Pacheco, “Characterization of diffusion of macromolecules in konjac glucomannan solutions and gels by fluorescence recovery after photobleaching technique,” Int. J. Pharm. 316(1-2), 37–46 (2006).
[CrossRef] [PubMed]

K. Braeckmans, B. G. Stubbe, K. Remaut, J. Demeester, and S. C. De Smedt, “Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation--a case study for fluorescein,” J. Biomed. Opt. 11(4), 044013 (2006).
[CrossRef] [PubMed]

S. R. Van Tomme, B. G. De Geest, K. Braeckmans, S. C. De Smedt, F. Siepmann, J. Siepmann, C. F. van Nostrum, and W. E. Hennink, “Mobility of model proteins in hydrogels composed of oppositely charged dextran microspheres studied by protein release and fluorescence recovery after photobleaching,” J. Control. Release 110(1), 67–78 (2005).
[CrossRef] [PubMed]

K. Braeckmans, L. Peeters, N. N. Sanders, S. C. De Smedt, and J. Demeester, “Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope,” Biophys. J. 85(4), 2240–2252 (2003).
[CrossRef] [PubMed]

Braga, J.

J. Braga, J. M. P. Desterro, and M. Carmo-Fonseca, “Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes,” Mol. Biol. Cell 15(10), 4749–4760 (2004).
[CrossRef] [PubMed]

Brown, E. B.

E. B. Brown, E. S. Wu, W. Zipfel, and W. W. Webb, “Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery,” Biophys. J. 77(5), 2837–2849 (1999).
[CrossRef] [PubMed]

Burgold, S.

A. Tannert, S. Tannert, S. Burgold, and M. Schaefer, “Convolution-based one and two component FRAP analysis: theory and application,” Eur. Biophys. J. 38(5), 649–661 (2009).
[CrossRef] [PubMed]

Burke, M. D.

M. D. Burke, J. O. Park, M. Srinivasarao, and S. A. Khan, “Diffusion of macromolecules in polymer solutions and gels: A laser scanning confocal microscopy study,” Macromolecules 33(20), 7500–7507 (2000).
[CrossRef]

Carmo-Fonseca, M.

J. Braga, J. M. P. Desterro, and M. Carmo-Fonseca, “Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes,” Mol. Biol. Cell 15(10), 4749–4760 (2004).
[CrossRef] [PubMed]

Cella, F.

D. Mazza, K. Braeckmans, F. Cella, I. Testa, D. Vercauteren, J. Demeester, S. S. De Smedt, and A. Diaspro, “A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy,” Biophys. J. 95(7), 3457–3469 (2008).
[CrossRef] [PubMed]

Censi, R.

R. Censi, T. Vermonden, M. J. van Steenbergen, H. Deschout, K. Braeckmans, S. C. De Smedt, C. F. van Nostrum, P. di Martino, and W. E. Hennink, “Photopolymerized thermosensitive hydrogels for tailorable diffusion-controlled protein delivery,” J. Control. Release 140(3), 230–236 (2009).
[CrossRef] [PubMed]

Day, C. A.

M. Kang, C. A. Day, K. Drake, A. K. Kenworthy, and E. DiBenedetto, “A generalization of theory for two-dimensional fluorescence recovery after photobleaching applicable to confocal laser scanning microscopes,” Biophys. J. 97(5), 1501–1511 (2009).
[CrossRef] [PubMed]

De Geest, B. G.

S. R. Van Tomme, B. G. De Geest, K. Braeckmans, S. C. De Smedt, F. Siepmann, J. Siepmann, C. F. van Nostrum, and W. E. Hennink, “Mobility of model proteins in hydrogels composed of oppositely charged dextran microspheres studied by protein release and fluorescence recovery after photobleaching,” J. Control. Release 110(1), 67–78 (2005).
[CrossRef] [PubMed]

De Smedt, S. C.

F. van de Manakker, K. Braeckmans, N. Morabit, S. C. De Smedt, C. F. van Nostrum, and W. E. Hennink, “Protein-Release Behavior of Self-Assembled PEG-beta-Cyclodextrin/PEG-Cholesterol Hydrogels,” Adv. Funct. Mater. 19(18), 2992–3001 (2009).
[CrossRef]

R. Censi, T. Vermonden, M. J. van Steenbergen, H. Deschout, K. Braeckmans, S. C. De Smedt, C. F. van Nostrum, P. di Martino, and W. E. Hennink, “Photopolymerized thermosensitive hydrogels for tailorable diffusion-controlled protein delivery,” J. Control. Release 140(3), 230–236 (2009).
[CrossRef] [PubMed]

K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. De Smedt, and J. Demeester, “Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples,” Biophys. J. 92(6), 2172–2183 (2007).
[CrossRef] [PubMed]

F. Alvarez-Manceñido, K. Braeckmans, S. C. De Smedt, J. Demeester, M. Landin, and R. Martínez-Pacheco, “Characterization of diffusion of macromolecules in konjac glucomannan solutions and gels by fluorescence recovery after photobleaching technique,” Int. J. Pharm. 316(1-2), 37–46 (2006).
[CrossRef] [PubMed]

K. Braeckmans, B. G. Stubbe, K. Remaut, J. Demeester, and S. C. De Smedt, “Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation--a case study for fluorescein,” J. Biomed. Opt. 11(4), 044013 (2006).
[CrossRef] [PubMed]

S. R. Van Tomme, B. G. De Geest, K. Braeckmans, S. C. De Smedt, F. Siepmann, J. Siepmann, C. F. van Nostrum, and W. E. Hennink, “Mobility of model proteins in hydrogels composed of oppositely charged dextran microspheres studied by protein release and fluorescence recovery after photobleaching,” J. Control. Release 110(1), 67–78 (2005).
[CrossRef] [PubMed]

K. Braeckmans, L. Peeters, N. N. Sanders, S. C. De Smedt, and J. Demeester, “Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope,” Biophys. J. 85(4), 2240–2252 (2003).
[CrossRef] [PubMed]

S. C. De Smedt, T. K. L. Meyvis, J. Demeester, P. VanOostveldt, J. C. G. Blonk, and W. E. Hennink, “Diffusion of macromolecules in dextran methacrylate solutions and gels as studied by confocal scanning laser microscopy,” Macromolecules 30(17), 4863–4870 (1997).
[CrossRef]

De Smedt, S. S.

D. Mazza, K. Braeckmans, F. Cella, I. Testa, D. Vercauteren, J. Demeester, S. S. De Smedt, and A. Diaspro, “A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy,” Biophys. J. 95(7), 3457–3469 (2008).
[CrossRef] [PubMed]

Demeester, J.

D. Mazza, K. Braeckmans, F. Cella, I. Testa, D. Vercauteren, J. Demeester, S. S. De Smedt, and A. Diaspro, “A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy,” Biophys. J. 95(7), 3457–3469 (2008).
[CrossRef] [PubMed]

K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. De Smedt, and J. Demeester, “Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples,” Biophys. J. 92(6), 2172–2183 (2007).
[CrossRef] [PubMed]

F. Alvarez-Manceñido, K. Braeckmans, S. C. De Smedt, J. Demeester, M. Landin, and R. Martínez-Pacheco, “Characterization of diffusion of macromolecules in konjac glucomannan solutions and gels by fluorescence recovery after photobleaching technique,” Int. J. Pharm. 316(1-2), 37–46 (2006).
[CrossRef] [PubMed]

K. Braeckmans, B. G. Stubbe, K. Remaut, J. Demeester, and S. C. De Smedt, “Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation--a case study for fluorescein,” J. Biomed. Opt. 11(4), 044013 (2006).
[CrossRef] [PubMed]

K. Braeckmans, L. Peeters, N. N. Sanders, S. C. De Smedt, and J. Demeester, “Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope,” Biophys. J. 85(4), 2240–2252 (2003).
[CrossRef] [PubMed]

S. C. De Smedt, T. K. L. Meyvis, J. Demeester, P. VanOostveldt, J. C. G. Blonk, and W. E. Hennink, “Diffusion of macromolecules in dextran methacrylate solutions and gels as studied by confocal scanning laser microscopy,” Macromolecules 30(17), 4863–4870 (1997).
[CrossRef]

Deschout, H.

R. Censi, T. Vermonden, M. J. van Steenbergen, H. Deschout, K. Braeckmans, S. C. De Smedt, C. F. van Nostrum, P. di Martino, and W. E. Hennink, “Photopolymerized thermosensitive hydrogels for tailorable diffusion-controlled protein delivery,” J. Control. Release 140(3), 230–236 (2009).
[CrossRef] [PubMed]

Desterro, J. M. P.

J. Braga, J. M. P. Desterro, and M. Carmo-Fonseca, “Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes,” Mol. Biol. Cell 15(10), 4749–4760 (2004).
[CrossRef] [PubMed]

di Martino, P.

R. Censi, T. Vermonden, M. J. van Steenbergen, H. Deschout, K. Braeckmans, S. C. De Smedt, C. F. van Nostrum, P. di Martino, and W. E. Hennink, “Photopolymerized thermosensitive hydrogels for tailorable diffusion-controlled protein delivery,” J. Control. Release 140(3), 230–236 (2009).
[CrossRef] [PubMed]

Diaspro, A.

D. Mazza, K. Braeckmans, F. Cella, I. Testa, D. Vercauteren, J. Demeester, S. S. De Smedt, and A. Diaspro, “A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy,” Biophys. J. 95(7), 3457–3469 (2008).
[CrossRef] [PubMed]

DiBenedetto, E.

M. Kang, C. A. Day, K. Drake, A. K. Kenworthy, and E. DiBenedetto, “A generalization of theory for two-dimensional fluorescence recovery after photobleaching applicable to confocal laser scanning microscopes,” Biophys. J. 97(5), 1501–1511 (2009).
[CrossRef] [PubMed]

Don, A.

J. C. G. Blonk, A. Don, H. Vanaalst, and J. J. Birmingham, “Fluorescence Photobleaching Recovery in the Confocal Scanning Light-Microscope,” J. Microsc. 169, 363–374 (1993).
[CrossRef]

Drake, K.

M. Kang, C. A. Day, K. Drake, A. K. Kenworthy, and E. DiBenedetto, “A generalization of theory for two-dimensional fluorescence recovery after photobleaching applicable to confocal laser scanning microscopes,” Biophys. J. 97(5), 1501–1511 (2009).
[CrossRef] [PubMed]

Elson, E.

D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb, “Mobility measurement by analysis of fluorescence photobleaching recovery kinetics,” Biophys. J. 16(9), 1055–1069 (1976).
[CrossRef] [PubMed]

Fransson, S.

S. Fransson, N. Lorén, A. Altskär, and A. M. Hermansson, “Effect of confinement and kinetics on the morphology of phase separating gelatin-maltodextrin droplets,” Biomacromolecules 10(6), 1446–1453 (2009).
[CrossRef] [PubMed]

Hagman, J.

J. K. Jonasson, J. Hagman, N. Lorén, D. Bernin, M. Nydén, and M. Rudemo, “Pixel-based analysis of FRAP data with a general initial bleaching profile,” J. Microsc. 239(2), 142–153 (2010).
[PubMed]

Heinrich, O.

P. Wedekind, U. Kubitscheck, O. Heinrich, and R. Peters, “Line-scanning microphotolysis for diffraction-limited measurements of lateral diffusion,” Biophys. J. 71(3), 1621–1632 (1996).
[CrossRef] [PubMed]

Hennink, W. E.

R. Censi, T. Vermonden, M. J. van Steenbergen, H. Deschout, K. Braeckmans, S. C. De Smedt, C. F. van Nostrum, P. di Martino, and W. E. Hennink, “Photopolymerized thermosensitive hydrogels for tailorable diffusion-controlled protein delivery,” J. Control. Release 140(3), 230–236 (2009).
[CrossRef] [PubMed]

F. van de Manakker, K. Braeckmans, N. Morabit, S. C. De Smedt, C. F. van Nostrum, and W. E. Hennink, “Protein-Release Behavior of Self-Assembled PEG-beta-Cyclodextrin/PEG-Cholesterol Hydrogels,” Adv. Funct. Mater. 19(18), 2992–3001 (2009).
[CrossRef]

S. R. Van Tomme, B. G. De Geest, K. Braeckmans, S. C. De Smedt, F. Siepmann, J. Siepmann, C. F. van Nostrum, and W. E. Hennink, “Mobility of model proteins in hydrogels composed of oppositely charged dextran microspheres studied by protein release and fluorescence recovery after photobleaching,” J. Control. Release 110(1), 67–78 (2005).
[CrossRef] [PubMed]

S. C. De Smedt, T. K. L. Meyvis, J. Demeester, P. VanOostveldt, J. C. G. Blonk, and W. E. Hennink, “Diffusion of macromolecules in dextran methacrylate solutions and gels as studied by confocal scanning laser microscopy,” Macromolecules 30(17), 4863–4870 (1997).
[CrossRef]

Hermansson, A. M.

S. Fransson, N. Lorén, A. Altskär, and A. M. Hermansson, “Effect of confinement and kinetics on the morphology of phase separating gelatin-maltodextrin droplets,” Biomacromolecules 10(6), 1446–1453 (2009).
[CrossRef] [PubMed]

N. Lorén, A. Altskar, and A. M. Hermansson, “Structure evolution during gelation at later stages of spinodal decomposition in gelatin/maltodextrin mixtures,” Macromolecules 34(23), 8117–8128 (2001).
[CrossRef]

N. Lorén and A. M. Hermansson, “Phase separation and gel formation in kinetically trapped gelatin/maltodextrin gels,” Int. J. Biol. Macromol. 27(4), 249–262 (2000).
[CrossRef] [PubMed]

Höök, F.

P. Jönsson, M. P. Jonsson, J. O. Tegenfeldt, and F. Höök, “A method improving the accuracy of fluorescence recovery after photobleaching analysis,” Biophys. J. 95(11), 5334–5348 (2008).
[CrossRef] [PubMed]

Ishihara, A.

A. Ishihara and K. Jacobson, “A closer look at how membrane proteins move,” Biophys. J. 65(5), 1754–1755 (1993).
[CrossRef] [PubMed]

Jacobson, K.

A. Ishihara and K. Jacobson, “A closer look at how membrane proteins move,” Biophys. J. 65(5), 1754–1755 (1993).
[CrossRef] [PubMed]

Jacobson, K. A.

T. T. Tsay and K. A. Jacobson, “Spatial Fourier analysis of video photobleaching measurements. Principles and optimization,” Biophys. J. 60(2), 360–368 (1991).
[CrossRef] [PubMed]

Jain, R. K.

D. A. Berk, F. Yuan, M. Leunig, and R. K. Jain, “Fluorescence photobleaching with spatial Fourier analysis: measurement of diffusion in light-scattering media,” Biophys. J. 65(6), 2428–2436 (1993).
[CrossRef] [PubMed]

Jonasson, J. K.

J. K. Jonasson, J. Hagman, N. Lorén, D. Bernin, M. Nydén, and M. Rudemo, “Pixel-based analysis of FRAP data with a general initial bleaching profile,” J. Microsc. 239(2), 142–153 (2010).
[PubMed]

J. K. Jonasson, N. Lorén, P. Olofsson, M. Nydén, and M. Rudemo, “A pixel-based likelihood framework for analysis of fluorescence recovery after photobleaching data,” J. Microsc. 232(2), 260–269 (2008).
[CrossRef] [PubMed]

Jonsson, M. P.

P. Jönsson, M. P. Jonsson, J. O. Tegenfeldt, and F. Höök, “A method improving the accuracy of fluorescence recovery after photobleaching analysis,” Biophys. J. 95(11), 5334–5348 (2008).
[CrossRef] [PubMed]

Jönsson, P.

P. Jönsson, M. P. Jonsson, J. O. Tegenfeldt, and F. Höök, “A method improving the accuracy of fluorescence recovery after photobleaching analysis,” Biophys. J. 95(11), 5334–5348 (2008).
[CrossRef] [PubMed]

Kang, M.

M. Kang, C. A. Day, K. Drake, A. K. Kenworthy, and E. DiBenedetto, “A generalization of theory for two-dimensional fluorescence recovery after photobleaching applicable to confocal laser scanning microscopes,” Biophys. J. 97(5), 1501–1511 (2009).
[CrossRef] [PubMed]

Kenworthy, A. K.

M. Kang, C. A. Day, K. Drake, A. K. Kenworthy, and E. DiBenedetto, “A generalization of theory for two-dimensional fluorescence recovery after photobleaching applicable to confocal laser scanning microscopes,” Biophys. J. 97(5), 1501–1511 (2009).
[CrossRef] [PubMed]

Khan, S. A.

M. D. Burke, J. O. Park, M. Srinivasarao, and S. A. Khan, “Diffusion of macromolecules in polymer solutions and gels: A laser scanning confocal microscopy study,” Macromolecules 33(20), 7500–7507 (2000).
[CrossRef]

Koppel, D. E.

D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb, “Mobility measurement by analysis of fluorescence photobleaching recovery kinetics,” Biophys. J. 16(9), 1055–1069 (1976).
[CrossRef] [PubMed]

Kubitscheck, U.

P. Wedekind, U. Kubitscheck, O. Heinrich, and R. Peters, “Line-scanning microphotolysis for diffraction-limited measurements of lateral diffusion,” Biophys. J. 71(3), 1621–1632 (1996).
[CrossRef] [PubMed]

P. Wedekind, U. Kubitscheck, and R. Peters, “Scanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging,” J. Microsc. 176(Pt 1), 23–33 (1994).
[CrossRef] [PubMed]

Landin, M.

F. Alvarez-Manceñido, K. Braeckmans, S. C. De Smedt, J. Demeester, M. Landin, and R. Martínez-Pacheco, “Characterization of diffusion of macromolecules in konjac glucomannan solutions and gels by fluorescence recovery after photobleaching technique,” Int. J. Pharm. 316(1-2), 37–46 (2006).
[CrossRef] [PubMed]

Leunig, M.

D. A. Berk, F. Yuan, M. Leunig, and R. K. Jain, “Fluorescence photobleaching with spatial Fourier analysis: measurement of diffusion in light-scattering media,” Biophys. J. 65(6), 2428–2436 (1993).
[CrossRef] [PubMed]

Lorén, N.

J. K. Jonasson, J. Hagman, N. Lorén, D. Bernin, M. Nydén, and M. Rudemo, “Pixel-based analysis of FRAP data with a general initial bleaching profile,” J. Microsc. 239(2), 142–153 (2010).
[PubMed]

S. Fransson, N. Lorén, A. Altskär, and A. M. Hermansson, “Effect of confinement and kinetics on the morphology of phase separating gelatin-maltodextrin droplets,” Biomacromolecules 10(6), 1446–1453 (2009).
[CrossRef] [PubMed]

J. K. Jonasson, N. Lorén, P. Olofsson, M. Nydén, and M. Rudemo, “A pixel-based likelihood framework for analysis of fluorescence recovery after photobleaching data,” J. Microsc. 232(2), 260–269 (2008).
[CrossRef] [PubMed]

N. Lorén, A. Altskar, and A. M. Hermansson, “Structure evolution during gelation at later stages of spinodal decomposition in gelatin/maltodextrin mixtures,” Macromolecules 34(23), 8117–8128 (2001).
[CrossRef]

N. Lorén and A. M. Hermansson, “Phase separation and gel formation in kinetically trapped gelatin/maltodextrin gels,” Int. J. Biol. Macromol. 27(4), 249–262 (2000).
[CrossRef] [PubMed]

Lucas, B.

K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. De Smedt, and J. Demeester, “Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples,” Biophys. J. 92(6), 2172–2183 (2007).
[CrossRef] [PubMed]

Martínez-Pacheco, R.

F. Alvarez-Manceñido, K. Braeckmans, S. C. De Smedt, J. Demeester, M. Landin, and R. Martínez-Pacheco, “Characterization of diffusion of macromolecules in konjac glucomannan solutions and gels by fluorescence recovery after photobleaching technique,” Int. J. Pharm. 316(1-2), 37–46 (2006).
[CrossRef] [PubMed]

Mazza, D.

D. Mazza, K. Braeckmans, F. Cella, I. Testa, D. Vercauteren, J. Demeester, S. S. De Smedt, and A. Diaspro, “A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy,” Biophys. J. 95(7), 3457–3469 (2008).
[CrossRef] [PubMed]

Meyvis, T. K. L.

S. C. De Smedt, T. K. L. Meyvis, J. Demeester, P. VanOostveldt, J. C. G. Blonk, and W. E. Hennink, “Diffusion of macromolecules in dextran methacrylate solutions and gels as studied by confocal scanning laser microscopy,” Macromolecules 30(17), 4863–4870 (1997).
[CrossRef]

Morabit, N.

F. van de Manakker, K. Braeckmans, N. Morabit, S. C. De Smedt, C. F. van Nostrum, and W. E. Hennink, “Protein-Release Behavior of Self-Assembled PEG-beta-Cyclodextrin/PEG-Cholesterol Hydrogels,” Adv. Funct. Mater. 19(18), 2992–3001 (2009).
[CrossRef]

Nydén, M.

J. K. Jonasson, J. Hagman, N. Lorén, D. Bernin, M. Nydén, and M. Rudemo, “Pixel-based analysis of FRAP data with a general initial bleaching profile,” J. Microsc. 239(2), 142–153 (2010).
[PubMed]

J. K. Jonasson, N. Lorén, P. Olofsson, M. Nydén, and M. Rudemo, “A pixel-based likelihood framework for analysis of fluorescence recovery after photobleaching data,” J. Microsc. 232(2), 260–269 (2008).
[CrossRef] [PubMed]

Olofsson, P.

J. K. Jonasson, N. Lorén, P. Olofsson, M. Nydén, and M. Rudemo, “A pixel-based likelihood framework for analysis of fluorescence recovery after photobleaching data,” J. Microsc. 232(2), 260–269 (2008).
[CrossRef] [PubMed]

Park, J. O.

M. D. Burke, J. O. Park, M. Srinivasarao, and S. A. Khan, “Diffusion of macromolecules in polymer solutions and gels: A laser scanning confocal microscopy study,” Macromolecules 33(20), 7500–7507 (2000).
[CrossRef]

Peeters, L.

K. Braeckmans, L. Peeters, N. N. Sanders, S. C. De Smedt, and J. Demeester, “Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope,” Biophys. J. 85(4), 2240–2252 (2003).
[CrossRef] [PubMed]

Peters, R.

P. Wedekind, U. Kubitscheck, O. Heinrich, and R. Peters, “Line-scanning microphotolysis for diffraction-limited measurements of lateral diffusion,” Biophys. J. 71(3), 1621–1632 (1996).
[CrossRef] [PubMed]

P. Wedekind, U. Kubitscheck, and R. Peters, “Scanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging,” J. Microsc. 176(Pt 1), 23–33 (1994).
[CrossRef] [PubMed]

Remaut, K.

K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. De Smedt, and J. Demeester, “Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples,” Biophys. J. 92(6), 2172–2183 (2007).
[CrossRef] [PubMed]

K. Braeckmans, B. G. Stubbe, K. Remaut, J. Demeester, and S. C. De Smedt, “Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation--a case study for fluorescein,” J. Biomed. Opt. 11(4), 044013 (2006).
[CrossRef] [PubMed]

Rudemo, M.

J. K. Jonasson, J. Hagman, N. Lorén, D. Bernin, M. Nydén, and M. Rudemo, “Pixel-based analysis of FRAP data with a general initial bleaching profile,” J. Microsc. 239(2), 142–153 (2010).
[PubMed]

J. K. Jonasson, N. Lorén, P. Olofsson, M. Nydén, and M. Rudemo, “A pixel-based likelihood framework for analysis of fluorescence recovery after photobleaching data,” J. Microsc. 232(2), 260–269 (2008).
[CrossRef] [PubMed]

Sanders, N. N.

K. Braeckmans, L. Peeters, N. N. Sanders, S. C. De Smedt, and J. Demeester, “Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope,” Biophys. J. 85(4), 2240–2252 (2003).
[CrossRef] [PubMed]

Schaefer, M.

A. Tannert, S. Tannert, S. Burgold, and M. Schaefer, “Convolution-based one and two component FRAP analysis: theory and application,” Eur. Biophys. J. 38(5), 649–661 (2009).
[CrossRef] [PubMed]

Schlessinger, J.

D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb, “Mobility measurement by analysis of fluorescence photobleaching recovery kinetics,” Biophys. J. 16(9), 1055–1069 (1976).
[CrossRef] [PubMed]

Seksek, O.

O. Seksek, J. Biwersi, and A. S. Verkman, “Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus,” J. Cell Biol. 138(1), 131–142 (1997).
[CrossRef] [PubMed]

Siepmann, F.

S. R. Van Tomme, B. G. De Geest, K. Braeckmans, S. C. De Smedt, F. Siepmann, J. Siepmann, C. F. van Nostrum, and W. E. Hennink, “Mobility of model proteins in hydrogels composed of oppositely charged dextran microspheres studied by protein release and fluorescence recovery after photobleaching,” J. Control. Release 110(1), 67–78 (2005).
[CrossRef] [PubMed]

Siepmann, J.

S. R. Van Tomme, B. G. De Geest, K. Braeckmans, S. C. De Smedt, F. Siepmann, J. Siepmann, C. F. van Nostrum, and W. E. Hennink, “Mobility of model proteins in hydrogels composed of oppositely charged dextran microspheres studied by protein release and fluorescence recovery after photobleaching,” J. Control. Release 110(1), 67–78 (2005).
[CrossRef] [PubMed]

Srinivasarao, M.

M. D. Burke, J. O. Park, M. Srinivasarao, and S. A. Khan, “Diffusion of macromolecules in polymer solutions and gels: A laser scanning confocal microscopy study,” Macromolecules 33(20), 7500–7507 (2000).
[CrossRef]

Stubbe, B. G.

K. Braeckmans, B. G. Stubbe, K. Remaut, J. Demeester, and S. C. De Smedt, “Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation--a case study for fluorescein,” J. Biomed. Opt. 11(4), 044013 (2006).
[CrossRef] [PubMed]

Tannert, A.

A. Tannert, S. Tannert, S. Burgold, and M. Schaefer, “Convolution-based one and two component FRAP analysis: theory and application,” Eur. Biophys. J. 38(5), 649–661 (2009).
[CrossRef] [PubMed]

Tannert, S.

A. Tannert, S. Tannert, S. Burgold, and M. Schaefer, “Convolution-based one and two component FRAP analysis: theory and application,” Eur. Biophys. J. 38(5), 649–661 (2009).
[CrossRef] [PubMed]

Tegenfeldt, J. O.

P. Jönsson, M. P. Jonsson, J. O. Tegenfeldt, and F. Höök, “A method improving the accuracy of fluorescence recovery after photobleaching analysis,” Biophys. J. 95(11), 5334–5348 (2008).
[CrossRef] [PubMed]

Testa, I.

D. Mazza, K. Braeckmans, F. Cella, I. Testa, D. Vercauteren, J. Demeester, S. S. De Smedt, and A. Diaspro, “A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy,” Biophys. J. 95(7), 3457–3469 (2008).
[CrossRef] [PubMed]

Tsay, T. T.

T. T. Tsay and K. A. Jacobson, “Spatial Fourier analysis of video photobleaching measurements. Principles and optimization,” Biophys. J. 60(2), 360–368 (1991).
[CrossRef] [PubMed]

Umenishi, F.

F. Umenishi, J. M. Verbavatz, and A. S. Verkman, “cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera,” Biophys. J. 78(2), 1024–1035 (2000).
[CrossRef] [PubMed]

van de Manakker, F.

F. van de Manakker, K. Braeckmans, N. Morabit, S. C. De Smedt, C. F. van Nostrum, and W. E. Hennink, “Protein-Release Behavior of Self-Assembled PEG-beta-Cyclodextrin/PEG-Cholesterol Hydrogels,” Adv. Funct. Mater. 19(18), 2992–3001 (2009).
[CrossRef]

van Nostrum, C. F.

F. van de Manakker, K. Braeckmans, N. Morabit, S. C. De Smedt, C. F. van Nostrum, and W. E. Hennink, “Protein-Release Behavior of Self-Assembled PEG-beta-Cyclodextrin/PEG-Cholesterol Hydrogels,” Adv. Funct. Mater. 19(18), 2992–3001 (2009).
[CrossRef]

R. Censi, T. Vermonden, M. J. van Steenbergen, H. Deschout, K. Braeckmans, S. C. De Smedt, C. F. van Nostrum, P. di Martino, and W. E. Hennink, “Photopolymerized thermosensitive hydrogels for tailorable diffusion-controlled protein delivery,” J. Control. Release 140(3), 230–236 (2009).
[CrossRef] [PubMed]

S. R. Van Tomme, B. G. De Geest, K. Braeckmans, S. C. De Smedt, F. Siepmann, J. Siepmann, C. F. van Nostrum, and W. E. Hennink, “Mobility of model proteins in hydrogels composed of oppositely charged dextran microspheres studied by protein release and fluorescence recovery after photobleaching,” J. Control. Release 110(1), 67–78 (2005).
[CrossRef] [PubMed]

van Steenbergen, M. J.

R. Censi, T. Vermonden, M. J. van Steenbergen, H. Deschout, K. Braeckmans, S. C. De Smedt, C. F. van Nostrum, P. di Martino, and W. E. Hennink, “Photopolymerized thermosensitive hydrogels for tailorable diffusion-controlled protein delivery,” J. Control. Release 140(3), 230–236 (2009).
[CrossRef] [PubMed]

Van Tomme, S. R.

S. R. Van Tomme, B. G. De Geest, K. Braeckmans, S. C. De Smedt, F. Siepmann, J. Siepmann, C. F. van Nostrum, and W. E. Hennink, “Mobility of model proteins in hydrogels composed of oppositely charged dextran microspheres studied by protein release and fluorescence recovery after photobleaching,” J. Control. Release 110(1), 67–78 (2005).
[CrossRef] [PubMed]

Vanaalst, H.

J. C. G. Blonk, A. Don, H. Vanaalst, and J. J. Birmingham, “Fluorescence Photobleaching Recovery in the Confocal Scanning Light-Microscope,” J. Microsc. 169, 363–374 (1993).
[CrossRef]

Vandenbroucke, R. E.

K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. De Smedt, and J. Demeester, “Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples,” Biophys. J. 92(6), 2172–2183 (2007).
[CrossRef] [PubMed]

VanOostveldt, P.

S. C. De Smedt, T. K. L. Meyvis, J. Demeester, P. VanOostveldt, J. C. G. Blonk, and W. E. Hennink, “Diffusion of macromolecules in dextran methacrylate solutions and gels as studied by confocal scanning laser microscopy,” Macromolecules 30(17), 4863–4870 (1997).
[CrossRef]

Verbavatz, J. M.

F. Umenishi, J. M. Verbavatz, and A. S. Verkman, “cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera,” Biophys. J. 78(2), 1024–1035 (2000).
[CrossRef] [PubMed]

Vercauteren, D.

D. Mazza, K. Braeckmans, F. Cella, I. Testa, D. Vercauteren, J. Demeester, S. S. De Smedt, and A. Diaspro, “A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy,” Biophys. J. 95(7), 3457–3469 (2008).
[CrossRef] [PubMed]

Verkman, A. S.

A. S. Verkman, “Diffusion in cells measured by fluorescence recovery after photobleaching,” Methods Enzymol. 360, 635–648 (2003).
[CrossRef] [PubMed]

F. Umenishi, J. M. Verbavatz, and A. S. Verkman, “cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera,” Biophys. J. 78(2), 1024–1035 (2000).
[CrossRef] [PubMed]

O. Seksek, J. Biwersi, and A. S. Verkman, “Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus,” J. Cell Biol. 138(1), 131–142 (1997).
[CrossRef] [PubMed]

Vermonden, T.

R. Censi, T. Vermonden, M. J. van Steenbergen, H. Deschout, K. Braeckmans, S. C. De Smedt, C. F. van Nostrum, P. di Martino, and W. E. Hennink, “Photopolymerized thermosensitive hydrogels for tailorable diffusion-controlled protein delivery,” J. Control. Release 140(3), 230–236 (2009).
[CrossRef] [PubMed]

Webb, W. W.

E. B. Brown, E. S. Wu, W. Zipfel, and W. W. Webb, “Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery,” Biophys. J. 77(5), 2837–2849 (1999).
[CrossRef] [PubMed]

D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb, “Mobility measurement by analysis of fluorescence photobleaching recovery kinetics,” Biophys. J. 16(9), 1055–1069 (1976).
[CrossRef] [PubMed]

Wedekind, P.

P. Wedekind, U. Kubitscheck, O. Heinrich, and R. Peters, “Line-scanning microphotolysis for diffraction-limited measurements of lateral diffusion,” Biophys. J. 71(3), 1621–1632 (1996).
[CrossRef] [PubMed]

P. Wedekind, U. Kubitscheck, and R. Peters, “Scanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging,” J. Microsc. 176(Pt 1), 23–33 (1994).
[CrossRef] [PubMed]

Wu, E. S.

E. B. Brown, E. S. Wu, W. Zipfel, and W. W. Webb, “Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery,” Biophys. J. 77(5), 2837–2849 (1999).
[CrossRef] [PubMed]

Yuan, F.

D. A. Berk, F. Yuan, M. Leunig, and R. K. Jain, “Fluorescence photobleaching with spatial Fourier analysis: measurement of diffusion in light-scattering media,” Biophys. J. 65(6), 2428–2436 (1993).
[CrossRef] [PubMed]

Zipfel, W.

E. B. Brown, E. S. Wu, W. Zipfel, and W. W. Webb, “Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery,” Biophys. J. 77(5), 2837–2849 (1999).
[CrossRef] [PubMed]

Adv. Funct. Mater. (1)

F. van de Manakker, K. Braeckmans, N. Morabit, S. C. De Smedt, C. F. van Nostrum, and W. E. Hennink, “Protein-Release Behavior of Self-Assembled PEG-beta-Cyclodextrin/PEG-Cholesterol Hydrogels,” Adv. Funct. Mater. 19(18), 2992–3001 (2009).
[CrossRef]

Biomacromolecules (1)

S. Fransson, N. Lorén, A. Altskär, and A. M. Hermansson, “Effect of confinement and kinetics on the morphology of phase separating gelatin-maltodextrin droplets,” Biomacromolecules 10(6), 1446–1453 (2009).
[CrossRef] [PubMed]

Biophys. J. (12)

M. Kang, C. A. Day, K. Drake, A. K. Kenworthy, and E. DiBenedetto, “A generalization of theory for two-dimensional fluorescence recovery after photobleaching applicable to confocal laser scanning microscopes,” Biophys. J. 97(5), 1501–1511 (2009).
[CrossRef] [PubMed]

D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb, “Mobility measurement by analysis of fluorescence photobleaching recovery kinetics,” Biophys. J. 16(9), 1055–1069 (1976).
[CrossRef] [PubMed]

P. Wedekind, U. Kubitscheck, O. Heinrich, and R. Peters, “Line-scanning microphotolysis for diffraction-limited measurements of lateral diffusion,” Biophys. J. 71(3), 1621–1632 (1996).
[CrossRef] [PubMed]

E. B. Brown, E. S. Wu, W. Zipfel, and W. W. Webb, “Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery,” Biophys. J. 77(5), 2837–2849 (1999).
[CrossRef] [PubMed]

A. Ishihara and K. Jacobson, “A closer look at how membrane proteins move,” Biophys. J. 65(5), 1754–1755 (1993).
[CrossRef] [PubMed]

F. Umenishi, J. M. Verbavatz, and A. S. Verkman, “cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera,” Biophys. J. 78(2), 1024–1035 (2000).
[CrossRef] [PubMed]

K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. De Smedt, and J. Demeester, “Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples,” Biophys. J. 92(6), 2172–2183 (2007).
[CrossRef] [PubMed]

D. Mazza, K. Braeckmans, F. Cella, I. Testa, D. Vercauteren, J. Demeester, S. S. De Smedt, and A. Diaspro, “A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy,” Biophys. J. 95(7), 3457–3469 (2008).
[CrossRef] [PubMed]

K. Braeckmans, L. Peeters, N. N. Sanders, S. C. De Smedt, and J. Demeester, “Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope,” Biophys. J. 85(4), 2240–2252 (2003).
[CrossRef] [PubMed]

D. A. Berk, F. Yuan, M. Leunig, and R. K. Jain, “Fluorescence photobleaching with spatial Fourier analysis: measurement of diffusion in light-scattering media,” Biophys. J. 65(6), 2428–2436 (1993).
[CrossRef] [PubMed]

P. Jönsson, M. P. Jonsson, J. O. Tegenfeldt, and F. Höök, “A method improving the accuracy of fluorescence recovery after photobleaching analysis,” Biophys. J. 95(11), 5334–5348 (2008).
[CrossRef] [PubMed]

T. T. Tsay and K. A. Jacobson, “Spatial Fourier analysis of video photobleaching measurements. Principles and optimization,” Biophys. J. 60(2), 360–368 (1991).
[CrossRef] [PubMed]

Eur. Biophys. J. (1)

A. Tannert, S. Tannert, S. Burgold, and M. Schaefer, “Convolution-based one and two component FRAP analysis: theory and application,” Eur. Biophys. J. 38(5), 649–661 (2009).
[CrossRef] [PubMed]

Int. J. Biol. Macromol. (1)

N. Lorén and A. M. Hermansson, “Phase separation and gel formation in kinetically trapped gelatin/maltodextrin gels,” Int. J. Biol. Macromol. 27(4), 249–262 (2000).
[CrossRef] [PubMed]

Int. J. Pharm. (1)

F. Alvarez-Manceñido, K. Braeckmans, S. C. De Smedt, J. Demeester, M. Landin, and R. Martínez-Pacheco, “Characterization of diffusion of macromolecules in konjac glucomannan solutions and gels by fluorescence recovery after photobleaching technique,” Int. J. Pharm. 316(1-2), 37–46 (2006).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

K. Braeckmans, B. G. Stubbe, K. Remaut, J. Demeester, and S. C. De Smedt, “Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation--a case study for fluorescein,” J. Biomed. Opt. 11(4), 044013 (2006).
[CrossRef] [PubMed]

J. Cell Biol. (1)

O. Seksek, J. Biwersi, and A. S. Verkman, “Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus,” J. Cell Biol. 138(1), 131–142 (1997).
[CrossRef] [PubMed]

J. Control. Release (2)

S. R. Van Tomme, B. G. De Geest, K. Braeckmans, S. C. De Smedt, F. Siepmann, J. Siepmann, C. F. van Nostrum, and W. E. Hennink, “Mobility of model proteins in hydrogels composed of oppositely charged dextran microspheres studied by protein release and fluorescence recovery after photobleaching,” J. Control. Release 110(1), 67–78 (2005).
[CrossRef] [PubMed]

R. Censi, T. Vermonden, M. J. van Steenbergen, H. Deschout, K. Braeckmans, S. C. De Smedt, C. F. van Nostrum, P. di Martino, and W. E. Hennink, “Photopolymerized thermosensitive hydrogels for tailorable diffusion-controlled protein delivery,” J. Control. Release 140(3), 230–236 (2009).
[CrossRef] [PubMed]

J. Microsc. (4)

J. K. Jonasson, N. Lorén, P. Olofsson, M. Nydén, and M. Rudemo, “A pixel-based likelihood framework for analysis of fluorescence recovery after photobleaching data,” J. Microsc. 232(2), 260–269 (2008).
[CrossRef] [PubMed]

J. K. Jonasson, J. Hagman, N. Lorén, D. Bernin, M. Nydén, and M. Rudemo, “Pixel-based analysis of FRAP data with a general initial bleaching profile,” J. Microsc. 239(2), 142–153 (2010).
[PubMed]

P. Wedekind, U. Kubitscheck, and R. Peters, “Scanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging,” J. Microsc. 176(Pt 1), 23–33 (1994).
[CrossRef] [PubMed]

J. C. G. Blonk, A. Don, H. Vanaalst, and J. J. Birmingham, “Fluorescence Photobleaching Recovery in the Confocal Scanning Light-Microscope,” J. Microsc. 169, 363–374 (1993).
[CrossRef]

Macromolecules (3)

N. Lorén, A. Altskar, and A. M. Hermansson, “Structure evolution during gelation at later stages of spinodal decomposition in gelatin/maltodextrin mixtures,” Macromolecules 34(23), 8117–8128 (2001).
[CrossRef]

S. C. De Smedt, T. K. L. Meyvis, J. Demeester, P. VanOostveldt, J. C. G. Blonk, and W. E. Hennink, “Diffusion of macromolecules in dextran methacrylate solutions and gels as studied by confocal scanning laser microscopy,” Macromolecules 30(17), 4863–4870 (1997).
[CrossRef]

M. D. Burke, J. O. Park, M. Srinivasarao, and S. A. Khan, “Diffusion of macromolecules in polymer solutions and gels: A laser scanning confocal microscopy study,” Macromolecules 33(20), 7500–7507 (2000).
[CrossRef]

Methods Enzymol. (1)

A. S. Verkman, “Diffusion in cells measured by fluorescence recovery after photobleaching,” Methods Enzymol. 360, 635–648 (2003).
[CrossRef] [PubMed]

Mol. Biol. Cell (1)

J. Braga, J. M. P. Desterro, and M. Carmo-Fonseca, “Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes,” Mol. Biol. Cell 15(10), 4749–4760 (2004).
[CrossRef] [PubMed]

Other (5)

J. Crank, The Mathematics of Diffusion, (Clarendon Press, Oxford, 1975).

N. Loren, and A. M. Hermansson, “Structure evolution during phase separation and gelation of biopolymer mixtures,” in Food Colloids - Biopolymers and Materials, Dickinson E. and Van Vliet T., eds., (The Royal Society of Chemistry, Cambridge, 2003), pp. 298–308.

Y. Pawitan, In All Likelihood, (Clarendon Press, Oxford, 2001).

W. H. Press, S. A. Teukolsky, W. T. Vetterling, and B. P. Flannery, “Confidence Limits on Estimated Model Parameters,” in Numerical Recipes in C, (Cambridge University Press, Cambridge, 1992).

M. Edidin, “Translational diffusion of membrane proteins,” in The Structure of Biological Membranes, P. Yeagle, ed., (CRC Press, Boca Raton, 1992), pp. 539–572.

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Figures (8)

Fig. 1
Fig. 1

An illustration of the use of a confocal laser scanning microscope in performing a FRAP measurement. The scanning speed is v and the distance between the consecutive scanning lines is Δy. (A) Images are acquired by scanning the imaging point spread function (open circle) across the focal plane. (B) By increasing the laser intensity within the indicated rectangle, a rectangular area can be photo bleached.

Fig. 2
Fig. 2

Illustration of an rFRAP experiment on a 150 kD FITC-dextran solution in HEPES buffer with 60% sucrose. (A) Several frames (512 by 512 pixels) of the time lapse movie are shown. The first frame shows the pre-bleach image. At time t = 0 s, a square region is bleached (30 by 30 µm) at the left side of the field of view, as is illustrated in the second frame. The following frames show the fluorescence recovery at four different times after bleaching. The dashed square around the bleached area indicates the region that was taken into account in the analysis. The dashed rectangle to the right shows the region that was used for the background correction. (B) The intensity values with the result from the fitting procedure (solid line) are shown for a cross section along the x-direction of the square.

Fig. 3
Fig. 3

(A) The average diffusion coefficient of 5 rFRAP measurements on a FD150 solution (24% sucrose) is plotted as a function of the time interval Δt (relative to the characteristic recovery time τ) between the images. In all cases a square region of 50 by 50 µm was bleached. The solid horizontal line indicates the diffusion coefficient of a uniform disk FRAP reference measurement (D = 9.8 ± 0.5 µm2/s). The dashed lines indicate the corresponding standard deviation of 0.5 µm2/s. (B) The average diffusion coefficient for rFRAP measurements on FD150 solutions with 16% sucrose in function of the bleaching time (expressed as the percentage of the characteristic recovery time τ) that was needed in order to bleach a square of 20 by 20 µm. The dashed line indicates the average value of the data points (D = 12.88 ± 1.0 µm2/s).

Fig. 4
Fig. 4

rFRAP measurements for different bleaching percentages on an FITC-dextran solution. (A) The average diffusion coefficient is plotted in function of percentage of bleaching. The straight dashed line represents the average value over the first 3 data points. (B) The resolution parameter is shown in function of the percentage of bleaching. The dashed line represents a linear fit to the measured average values (the data point at 10% was excluded).

Fig. 5
Fig. 5

(A) The average diffusion coefficient for rFRAP measurements on FITC-dextran solutions with 60% sucrose in function of the size of the bleached square with side length l. The dashed line indicates the average value of the data points. (B) The diffusion coefficient is calculated from rFRAP measurements on an FD150 solution (60% sucrose) using rectangles of length 10 µm but with a varying height ly . The dashed line indicates the average value over all measurements.

Fig. 6
Fig. 6

The average diffusion coefficient D as determined by the rFRAP method is plotted vs. the average diffusion coefficient D determined by the disk model for FD150 solutions with different amounts of sucrose. The dashed line represents the ideal case in which both methods predict exactly the same diffusion coefficient.

Fig. 7
Fig. 7

Comparison of least squares estimates (black) and maximum likelihood estimates (red) for rFRAP experiments performed on an FD150 solution (60% sucrose) using a constant bleach region but an increasing laser intensity between 2 mW and 10 mW. Averages of 10 measurements are shown with error bars corresponding to one standard deviation.

Fig. 8
Fig. 8

(A) CLSM image of a kinetically trapped and phase separated gelatin/maltodextrin mixture. The maltodextrin phase is bright and the gelatin phase is dark. The scale bar is 20 µm. (B) An rFRAP measurement in the maltodextrin phase of a phase separated gelatin/maltodextrin system. The bleached square is 7 by 7 µm and the field of view is 60 by 60 µm. (C) Diffusion coefficients determined using rFRAP in pure gelatine and maltodextrin, as well as in the pase-separated gelatin/maltodextrin mixture.

Equations (20)

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C b ( x , y , z ) = C 0 e σ n q n n v Δ y K ( x , y , z ) ,
K ( x , y , z ) = + + B ( x ' , y ' ) I b n ( x x ' , y y ' , z , t ) d x ' d y ' .
B ( x , y ) = { 1 ,      l x 2 x + l x 2  and  l y 2 y + l y 2 0 ,     elsewhere
I b n ( x , y , z , t ) = I b n ( 0 , 0 , 0 , t ) e 2 n ( x 2 + y 2 r b , 0 2 + z 2 z b , 0 2 ) ,
K ( x , y , z ) = I b n ( 0 , 0 , 0 , t ) e 2 n z b , 0 2 z 2 l x 2 + l x 2 e 2 n r b , 0 2 ( x x ' ) 2 d x ' l y 2 + l y 2 e 2 n r b , 0 2 ( y y ' ) 2 d y ' .
C b ( x , y , z ) = C 0 C 0 σ n q n n v Δ y K ( x , y , z ) .
C ( x , y , z , t ) =   1 ( 4 π D t ) 3 2 + + + C b ( x x ' , y y ' , z z ' ) e 1 4 D t ( x ' 2 + y ' 2 + z ' 2 ) d x ' d y ' d z ' .
C ( x , y , z , t ) C 0 = 1 σ n q n n v Δ y I b n ( 0 , 0 , 0 , t ) z b , 0 r b , 0 2 1 z b , 0 2 + 8 n D t 1 r b , 0 2 + 8 n D t                        × e 2 n z b , 0 2 + 8 n D t z 2 l x 2 + l x 2 e 2 n r b , 0 2 + 8 n D t ( x x ' ) 2 d x ' l y 2 + l y 2 e 2 n r b , 0 2 + 8 n D t ( y y ' ) 2 dy' ,
F ( x , y , z , t ) = q + + + I d m ( x , y , z ) C ( x x ' , y y ' , z z ' , t ) d x ' d y ' d z ' ,
I d m ( x , y , z , t ) = I d m ( 0 , 0 , 0 , t ) e 2 m ( x 2 + y 2 r d , 0 2 + z 2 z d , 0 2 ) ,
r m , n 2 = r d , 0 2 m + r b , 0 2 n 2 ,   z m , n 2 = z d , 0 2 m + z b , 0 2 n 2 ,   K 0 = π 2 σ n q n n v Δ y I b n ( 0 , 0 , 0 , t ) ( r b , 0 n ) 2 .
F ( x , y , z , t ) F 0 = 1 K 0 4 z b , 0 2 n 1 z m , n 2 + 4 D t e 1 z m , n 2 + 4 D t z 2                        × [ erf ( x + l x 2 r m , n 2 + 4 D t ) erf ( x l x 2 r m , n 2 + 4 D t ) ]                        × [ erf ( y + l y 2 r m , n 2 + 4 D t ) erf ( y l y 2 r m , n 2 + 4 D t ) ] ,
F ( x , y , t ) F 0 = 1 K 0 4 [ erf ( x + l x 2 r 2 + 4 D t ) erf ( x l x 2 r 2 + 4 D t ) ]                                    × [ erf ( y + l y 2 r 2 + 4 D t ) erf ( y l y 2 r 2 + 4 D t ) ] .
P = 100 × ( 1 F ( 0 , 0 , 0 ) F 0 ) = 100 × ( 1 K 0 erf ( l x 2 r ) erf ( l y 2 r ) ) .
F k ( x , y , t ) = F ( x , y , 0 ) + k [ F ( x , y , t ) F ( x , y , 0 ) ] .
L ( x , y , t ; θ ) = ( x , y ) S t T 1 2 π β F ( x , y , t ; θ ) exp { ( p ( x , y , t ) F ( x , y , t ; θ ) ) 2 2 β F ( x , y , t ; θ ) } .
l ( θ ; x , y , t ) = | S | | T | 2 log 2 π β 1 2 ( x , y ) S t T log F ( x , y , t ; θ )                            1 2 β ( x , y ) S t T ( p ( x , y , t ) F ( x , y , t ; θ ) ) 2 F ( x , y , t ; θ ) ,
2 θ i θ j l ( θ ) | θ = θ ^ .
Q ( θ ) = ( x , y ) S t T ( p ( x , y , t ) F ( x , y , t ; θ ) ) 2 .
τ = ( L / 2 ) 2 4 D ,

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