Abstract

Fluorescence microscopy is widely used to observe and quantify the inner workings of the cell. Traditionally, multiple types of cellular structures or biomolecules are visualized simultaneously in a sample by using spectrally distinct fluorescent labels. The wide emission spectra of most fluorophores limits spectral multiplexing to four or five labels in a standard fluorescence microscope. Further multiplexing requires another dimension of contrast. Here, we show that photostability differences can be used to distinguish between fluorescent labels. By combining photobleaching characteristics with a novel unmixing algorithm, we resolve up to three fluorescent labels in a single spectral channel and unmix fluorescent labels with nearly identical emission spectra. We apply our technique to organic dyes, autofluorescent biomolecules and fluorescent proteins. Our approach has the potential to triple the multiplexing capabilities of any digital widefield or confocal fluorescence microscope with no additional hardware, making it readily accessible to a wide range of researchers.

© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

Full Article  |  PDF Article
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References

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2017 (1)

L. Wei, Z. Chen, L. Shi, R. Long, A. V. Anzalone, L. Zhang, F. Hu, R. Yuste, V. W. Cornish, and W. Min, “Super-multiplex vibrational imaging,” Nature 544(7651), 465–470 (2017).
[Crossref] [PubMed]

2016 (3)

P. W. Tillberg, F. Chen, K. D. Piatkevich, Y. Zhao, C. C. Yu, B. P. English, L. Gao, A. Martorell, H. J. Suk, F. Yoshida, E. M. DeGennaro, D. H. Roossien, G. Gong, U. Seneviratne, S. R. Tannenbaum, R. Desimone, D. Cai, and E. S. Boyden, “Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies,” Nat. Biotechnol. 34(9), 987–992 (2016).
[Crossref] [PubMed]

P. Reineck, A. Francis, A. Orth, D. W. M. Lau, R. D. V. Nixon-Luke, I. D. Rastogi, W. A. W. Razali, N. M. Cordina, L. M. Parker, V. K. A. Sreenivasan, L. J. Brown, and B. C. Gibson, “Brightness and Photostability of Emerging Red and Near-IR Fluorescent Nanomaterials for Bioimaging,” Adv. Opt. Mater. 4(10), 1549–1557 (2016).
[Crossref]

T. Niehörster, A. Löschberger, I. Gregor, B. Krämer, H.-J. Rahn, M. Patting, F. Koberling, J. Enderlein, and M. Sauer, “Multi-target spectrally resolved fluorescence lifetime imaging microscopy,” Nat. Methods 13(3), 257–262 (2016).
[Crossref] [PubMed]

2015 (2)

F. Chen, P. W. Tillberg, and E. S. Boyden, “Optical imaging. Expansion microscopy,” Science 347(6221), 543–548 (2015).
[Crossref] [PubMed]

A. Orth, M. J. Tomaszewski, R. N. Ghosh, and E. Schonbrun, “Gigapixel multispectral microscopy,” Optica 2(7), 654–662 (2015).
[Crossref]

2014 (5)

A. Orth and K. B. Crozier, “High throughput multichannel fluorescence microscopy with microlens arrays,” Opt. Express 22(15), 18101–18112 (2014).
[Crossref] [PubMed]

D. Rönnlund, L. Xu, A. Perols, A. K. B. Gad, A. Eriksson Karlström, G. Auer, and J. Widengren, “Multicolor Fluorescence Nanoscopy by Photobleaching: Concept, Verification, and Its Application To Resolve Selective Storage of Proteins in Platelets,” ACS Nano 8(5), 4358–4365 (2014).
[Crossref] [PubMed]

Y. Lu, J. Lu, J. Zhao, J. Cusido, F. M. Raymo, J. Yuan, S. Yang, R. C. Leif, Y. Huo, J. A. Piper, J. Paul Robinson, E. M. Goldys, and D. Jin, “On-the-fly decoding luminescence lifetimes in the microsecond region for lanthanide-encoded suspension arrays,” Nat. Commun. 5(1), 3741 (2014).
[Crossref] [PubMed]

L. Gao, A. Garcia-Uribe, Y. Liu, C. Li, and L. V. Wang, “Photobleaching imprinting microscopy: seeing clearer and deeper,” J. Cell Sci. 127(2), 288–294 (2014).
[Crossref] [PubMed]

J. Yao, L. Wang, C. Li, C. Zhang, and L. V. Wang, “Photoimprint photoacoustic microscopy for three-dimensional label-free subdiffraction imaging,” Phys. Rev. Lett. 112(1), 014302 (2014).
[Crossref] [PubMed]

2013 (1)

P. Favreau, C. Hernandez, A. S. Lindsey, D. F. Alvarez, T. Rich, P. Prabhat, and S. J. Leavesley, “Thin-film tunable filters for hyperspectral fluorescence microscopy,” J. Biomed. Opt. 19(1), 011017 (2013).
[Crossref] [PubMed]

2011 (1)

D. T. Burnette, P. Sengupta, Y. Dai, J. Lippincott-Schwartz, and B. Kachar, “Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules,” Proc. Natl. Acad. Sci. U.S.A. 108(52), 21081–21086 (2011).
[Crossref] [PubMed]

2010 (1)

2008 (1)

2007 (2)

B. Kraus, M. Ziegler, and H. Wolff, “Linear fluorescence unmixing in cell biological research,” Mod. Res. Educ. Top. Microsc. 2, 863–873 (2007).

M. W. Berry, M. Browne, A. N. Langville, V. P. Pauca, and R. J. Plemmons, “Algorithms and applications for approximate nonnegative matrix factorization,” Comput. Stat. Data Anal. 52(1), 155–173 (2007).
[Crossref]

2006 (2)

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[Crossref] [PubMed]

2004 (1)

B. L. Sprague, R. L. Pego, D. A. Stavreva, and J. G. McNally, “Analysis of Binding Reactions by Fluorescence Recovery after Photobleaching,” Biophys. J. 86(6), 3473–3495 (2004).
[Crossref] [PubMed]

2003 (1)

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]

2000 (1)

G. H. Patterson and D. W. Piston, “Photobleaching in Two-Photon Excitation Microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[Crossref] [PubMed]

1997 (1)

R. I. Ghauharali, R. Van Driel, and G. J. Brakenhoff, “Structure-orientated fluorescence photobleaching analysis: a method for double fluorescent labelling studies,” J. Microsc. 185(3), 375–384 (1997).
[Crossref]

1995 (1)

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
[Crossref] [PubMed]

1994 (1)

G. J. Brakenhoff, K. Visscher, and E. J. Gijsbers, “Fluorescence bleach rate imaging,” J. Microsc. 175(2), 154–161 (1994).
[Crossref]

1984 (1)

C. J. G. Bakker and J. Vriend, “Multi-exponential water proton spin-lattice relaxation in biological tissues and its implications for quantitative NMR imaging,” Phys. Med. Biol. 29(5), 509–518 (1984).
[Crossref] [PubMed]

1974 (1)

A. Grinvald and I. Z. Steinberg, “On the analysis of fluorescence decay kinetics by the method of least-squares,” Anal. Biochem. 59(2), 583–598 (1974).
[Crossref] [PubMed]

1969 (1)

I. Isenberg and R. D. Dyson, “The Analysis of Fluorescence Decay by a Method of Moments,” Biophys. J. 9(11), 1337–1350 (1969).
[Crossref] [PubMed]

1968 (1)

P. H. R. Orth, W. R. Falk, and G. Jones, “Use of the maximum likelihood technique, for fitting counting distributions,” Nucl. Instrum. Methods 65(3), 301–306 (1968).
[Crossref]

1959 (1)

D. G. Gardner, J. C. Gardner, G. Laush, and W. W. Meinke, “Method for the analysis of multicomponent exponential decay curves,” J. Chem. Phys. 31(4), 978–986 (1959).
[Crossref]

Alvarez, D. F.

P. Favreau, C. Hernandez, A. S. Lindsey, D. F. Alvarez, T. Rich, P. Prabhat, and S. J. Leavesley, “Thin-film tunable filters for hyperspectral fluorescence microscopy,” J. Biomed. Opt. 19(1), 011017 (2013).
[Crossref] [PubMed]

Anzalone, A. V.

L. Wei, Z. Chen, L. Shi, R. Long, A. V. Anzalone, L. Zhang, F. Hu, R. Yuste, V. W. Cornish, and W. Min, “Super-multiplex vibrational imaging,” Nature 544(7651), 465–470 (2017).
[Crossref] [PubMed]

Auer, G.

D. Rönnlund, L. Xu, A. Perols, A. K. B. Gad, A. Eriksson Karlström, G. Auer, and J. Widengren, “Multicolor Fluorescence Nanoscopy by Photobleaching: Concept, Verification, and Its Application To Resolve Selective Storage of Proteins in Platelets,” ACS Nano 8(5), 4358–4365 (2014).
[Crossref] [PubMed]

Bakker, C. J. G.

C. J. G. Bakker and J. Vriend, “Multi-exponential water proton spin-lattice relaxation in biological tissues and its implications for quantitative NMR imaging,” Phys. Med. Biol. 29(5), 509–518 (1984).
[Crossref] [PubMed]

Bates, M.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

Berry, M. W.

M. W. Berry, M. Browne, A. N. Langville, V. P. Pauca, and R. J. Plemmons, “Algorithms and applications for approximate nonnegative matrix factorization,” Comput. Stat. Data Anal. 52(1), 155–173 (2007).
[Crossref]

Boyden, E. S.

P. W. Tillberg, F. Chen, K. D. Piatkevich, Y. Zhao, C. C. Yu, B. P. English, L. Gao, A. Martorell, H. J. Suk, F. Yoshida, E. M. DeGennaro, D. H. Roossien, G. Gong, U. Seneviratne, S. R. Tannenbaum, R. Desimone, D. Cai, and E. S. Boyden, “Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies,” Nat. Biotechnol. 34(9), 987–992 (2016).
[Crossref] [PubMed]

F. Chen, P. W. Tillberg, and E. S. Boyden, “Optical imaging. Expansion microscopy,” Science 347(6221), 543–548 (2015).
[Crossref] [PubMed]

Braeckmans, K.

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]

Brakenhoff, G. J.

R. I. Ghauharali, R. Van Driel, and G. J. Brakenhoff, “Structure-orientated fluorescence photobleaching analysis: a method for double fluorescent labelling studies,” J. Microsc. 185(3), 375–384 (1997).
[Crossref]

G. J. Brakenhoff, K. Visscher, and E. J. Gijsbers, “Fluorescence bleach rate imaging,” J. Microsc. 175(2), 154–161 (1994).
[Crossref]

Brown, L. J.

P. Reineck, A. Francis, A. Orth, D. W. M. Lau, R. D. V. Nixon-Luke, I. D. Rastogi, W. A. W. Razali, N. M. Cordina, L. M. Parker, V. K. A. Sreenivasan, L. J. Brown, and B. C. Gibson, “Brightness and Photostability of Emerging Red and Near-IR Fluorescent Nanomaterials for Bioimaging,” Adv. Opt. Mater. 4(10), 1549–1557 (2016).
[Crossref]

Browne, M.

M. W. Berry, M. Browne, A. N. Langville, V. P. Pauca, and R. J. Plemmons, “Algorithms and applications for approximate nonnegative matrix factorization,” Comput. Stat. Data Anal. 52(1), 155–173 (2007).
[Crossref]

Burnette, D. T.

D. T. Burnette, P. Sengupta, Y. Dai, J. Lippincott-Schwartz, and B. Kachar, “Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules,” Proc. Natl. Acad. Sci. U.S.A. 108(52), 21081–21086 (2011).
[Crossref] [PubMed]

Cai, D.

P. W. Tillberg, F. Chen, K. D. Piatkevich, Y. Zhao, C. C. Yu, B. P. English, L. Gao, A. Martorell, H. J. Suk, F. Yoshida, E. M. DeGennaro, D. H. Roossien, G. Gong, U. Seneviratne, S. R. Tannenbaum, R. Desimone, D. Cai, and E. S. Boyden, “Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies,” Nat. Biotechnol. 34(9), 987–992 (2016).
[Crossref] [PubMed]

Chen, F.

P. W. Tillberg, F. Chen, K. D. Piatkevich, Y. Zhao, C. C. Yu, B. P. English, L. Gao, A. Martorell, H. J. Suk, F. Yoshida, E. M. DeGennaro, D. H. Roossien, G. Gong, U. Seneviratne, S. R. Tannenbaum, R. Desimone, D. Cai, and E. S. Boyden, “Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies,” Nat. Biotechnol. 34(9), 987–992 (2016).
[Crossref] [PubMed]

F. Chen, P. W. Tillberg, and E. S. Boyden, “Optical imaging. Expansion microscopy,” Science 347(6221), 543–548 (2015).
[Crossref] [PubMed]

Chen, Z.

L. Wei, Z. Chen, L. Shi, R. Long, A. V. Anzalone, L. Zhang, F. Hu, R. Yuste, V. W. Cornish, and W. Min, “Super-multiplex vibrational imaging,” Nature 544(7651), 465–470 (2017).
[Crossref] [PubMed]

Cordina, N. M.

P. Reineck, A. Francis, A. Orth, D. W. M. Lau, R. D. V. Nixon-Luke, I. D. Rastogi, W. A. W. Razali, N. M. Cordina, L. M. Parker, V. K. A. Sreenivasan, L. J. Brown, and B. C. Gibson, “Brightness and Photostability of Emerging Red and Near-IR Fluorescent Nanomaterials for Bioimaging,” Adv. Opt. Mater. 4(10), 1549–1557 (2016).
[Crossref]

Cornish, V. W.

L. Wei, Z. Chen, L. Shi, R. Long, A. V. Anzalone, L. Zhang, F. Hu, R. Yuste, V. W. Cornish, and W. Min, “Super-multiplex vibrational imaging,” Nature 544(7651), 465–470 (2017).
[Crossref] [PubMed]

Crozier, K. B.

Cusido, J.

Y. Lu, J. Lu, J. Zhao, J. Cusido, F. M. Raymo, J. Yuan, S. Yang, R. C. Leif, Y. Huo, J. A. Piper, J. Paul Robinson, E. M. Goldys, and D. Jin, “On-the-fly decoding luminescence lifetimes in the microsecond region for lanthanide-encoded suspension arrays,” Nat. Commun. 5(1), 3741 (2014).
[Crossref] [PubMed]

Dai, Y.

D. T. Burnette, P. Sengupta, Y. Dai, J. Lippincott-Schwartz, and B. Kachar, “Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules,” Proc. Natl. Acad. Sci. U.S.A. 108(52), 21081–21086 (2011).
[Crossref] [PubMed]

De Smedt, S. C.

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]

DeGennaro, E. M.

P. W. Tillberg, F. Chen, K. D. Piatkevich, Y. Zhao, C. C. Yu, B. P. English, L. Gao, A. Martorell, H. J. Suk, F. Yoshida, E. M. DeGennaro, D. H. Roossien, G. Gong, U. Seneviratne, S. R. Tannenbaum, R. Desimone, D. Cai, and E. S. Boyden, “Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies,” Nat. Biotechnol. 34(9), 987–992 (2016).
[Crossref] [PubMed]

Demeester, J.

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]

Desimone, R.

P. W. Tillberg, F. Chen, K. D. Piatkevich, Y. Zhao, C. C. Yu, B. P. English, L. Gao, A. Martorell, H. J. Suk, F. Yoshida, E. M. DeGennaro, D. H. Roossien, G. Gong, U. Seneviratne, S. R. Tannenbaum, R. Desimone, D. Cai, and E. S. Boyden, “Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies,” Nat. Biotechnol. 34(9), 987–992 (2016).
[Crossref] [PubMed]

Dyson, R. D.

I. Isenberg and R. D. Dyson, “The Analysis of Fluorescence Decay by a Method of Moments,” Biophys. J. 9(11), 1337–1350 (1969).
[Crossref] [PubMed]

Enderlein, J.

T. Niehörster, A. Löschberger, I. Gregor, B. Krämer, H.-J. Rahn, M. Patting, F. Koberling, J. Enderlein, and M. Sauer, “Multi-target spectrally resolved fluorescence lifetime imaging microscopy,” Nat. Methods 13(3), 257–262 (2016).
[Crossref] [PubMed]

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Roossien, D. H.

P. W. Tillberg, F. Chen, K. D. Piatkevich, Y. Zhao, C. C. Yu, B. P. English, L. Gao, A. Martorell, H. J. Suk, F. Yoshida, E. M. DeGennaro, D. H. Roossien, G. Gong, U. Seneviratne, S. R. Tannenbaum, R. Desimone, D. Cai, and E. S. Boyden, “Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies,” Nat. Biotechnol. 34(9), 987–992 (2016).
[Crossref] [PubMed]

Rust, M. J.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[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]

Sauer, M.

T. Niehörster, A. Löschberger, I. Gregor, B. Krämer, H.-J. Rahn, M. Patting, F. Koberling, J. Enderlein, and M. Sauer, “Multi-target spectrally resolved fluorescence lifetime imaging microscopy,” Nat. Methods 13(3), 257–262 (2016).
[Crossref] [PubMed]

Schonbrun, E.

Seneviratne, U.

P. W. Tillberg, F. Chen, K. D. Piatkevich, Y. Zhao, C. C. Yu, B. P. English, L. Gao, A. Martorell, H. J. Suk, F. Yoshida, E. M. DeGennaro, D. H. Roossien, G. Gong, U. Seneviratne, S. R. Tannenbaum, R. Desimone, D. Cai, and E. S. Boyden, “Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies,” Nat. Biotechnol. 34(9), 987–992 (2016).
[Crossref] [PubMed]

Sengupta, P.

D. T. Burnette, P. Sengupta, Y. Dai, J. Lippincott-Schwartz, and B. Kachar, “Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules,” Proc. Natl. Acad. Sci. U.S.A. 108(52), 21081–21086 (2011).
[Crossref] [PubMed]

Shi, L.

L. Wei, Z. Chen, L. Shi, R. Long, A. V. Anzalone, L. Zhang, F. Hu, R. Yuste, V. W. Cornish, and W. Min, “Super-multiplex vibrational imaging,” Nature 544(7651), 465–470 (2017).
[Crossref] [PubMed]

Song, L.

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
[Crossref] [PubMed]

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B. L. Sprague, R. L. Pego, D. A. Stavreva, and J. G. McNally, “Analysis of Binding Reactions by Fluorescence Recovery after Photobleaching,” Biophys. J. 86(6), 3473–3495 (2004).
[Crossref] [PubMed]

Sreenivasan, V. K. A.

P. Reineck, A. Francis, A. Orth, D. W. M. Lau, R. D. V. Nixon-Luke, I. D. Rastogi, W. A. W. Razali, N. M. Cordina, L. M. Parker, V. K. A. Sreenivasan, L. J. Brown, and B. C. Gibson, “Brightness and Photostability of Emerging Red and Near-IR Fluorescent Nanomaterials for Bioimaging,” Adv. Opt. Mater. 4(10), 1549–1557 (2016).
[Crossref]

Stavreva, D. A.

B. L. Sprague, R. L. Pego, D. A. Stavreva, and J. G. McNally, “Analysis of Binding Reactions by Fluorescence Recovery after Photobleaching,” Biophys. J. 86(6), 3473–3495 (2004).
[Crossref] [PubMed]

Steinberg, I. Z.

A. Grinvald and I. Z. Steinberg, “On the analysis of fluorescence decay kinetics by the method of least-squares,” Anal. Biochem. 59(2), 583–598 (1974).
[Crossref] [PubMed]

Suk, H. J.

P. W. Tillberg, F. Chen, K. D. Piatkevich, Y. Zhao, C. C. Yu, B. P. English, L. Gao, A. Martorell, H. J. Suk, F. Yoshida, E. M. DeGennaro, D. H. Roossien, G. Gong, U. Seneviratne, S. R. Tannenbaum, R. Desimone, D. Cai, and E. S. Boyden, “Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies,” Nat. Biotechnol. 34(9), 987–992 (2016).
[Crossref] [PubMed]

Tanke, H. J.

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
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Tannenbaum, S. R.

P. W. Tillberg, F. Chen, K. D. Piatkevich, Y. Zhao, C. C. Yu, B. P. English, L. Gao, A. Martorell, H. J. Suk, F. Yoshida, E. M. DeGennaro, D. H. Roossien, G. Gong, U. Seneviratne, S. R. Tannenbaum, R. Desimone, D. Cai, and E. S. Boyden, “Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies,” Nat. Biotechnol. 34(9), 987–992 (2016).
[Crossref] [PubMed]

Thurman, S. T.

Tillberg, P. W.

P. W. Tillberg, F. Chen, K. D. Piatkevich, Y. Zhao, C. C. Yu, B. P. English, L. Gao, A. Martorell, H. J. Suk, F. Yoshida, E. M. DeGennaro, D. H. Roossien, G. Gong, U. Seneviratne, S. R. Tannenbaum, R. Desimone, D. Cai, and E. S. Boyden, “Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies,” Nat. Biotechnol. 34(9), 987–992 (2016).
[Crossref] [PubMed]

F. Chen, P. W. Tillberg, and E. S. Boyden, “Optical imaging. Expansion microscopy,” Science 347(6221), 543–548 (2015).
[Crossref] [PubMed]

Tkaczyk, T. S.

Tomaszewski, M. J.

Van Driel, R.

R. I. Ghauharali, R. Van Driel, and G. J. Brakenhoff, “Structure-orientated fluorescence photobleaching analysis: a method for double fluorescent labelling studies,” J. Microsc. 185(3), 375–384 (1997).
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G. J. Brakenhoff, K. Visscher, and E. J. Gijsbers, “Fluorescence bleach rate imaging,” J. Microsc. 175(2), 154–161 (1994).
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C. J. G. Bakker and J. Vriend, “Multi-exponential water proton spin-lattice relaxation in biological tissues and its implications for quantitative NMR imaging,” Phys. Med. Biol. 29(5), 509–518 (1984).
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J. Yao, L. Wang, C. Li, C. Zhang, and L. V. Wang, “Photoimprint photoacoustic microscopy for three-dimensional label-free subdiffraction imaging,” Phys. Rev. Lett. 112(1), 014302 (2014).
[Crossref] [PubMed]

Wang, L. V.

L. Gao, A. Garcia-Uribe, Y. Liu, C. Li, and L. V. Wang, “Photobleaching imprinting microscopy: seeing clearer and deeper,” J. Cell Sci. 127(2), 288–294 (2014).
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J. Yao, L. Wang, C. Li, C. Zhang, and L. V. Wang, “Photoimprint photoacoustic microscopy for three-dimensional label-free subdiffraction imaging,” Phys. Rev. Lett. 112(1), 014302 (2014).
[Crossref] [PubMed]

Wei, L.

L. Wei, Z. Chen, L. Shi, R. Long, A. V. Anzalone, L. Zhang, F. Hu, R. Yuste, V. W. Cornish, and W. Min, “Super-multiplex vibrational imaging,” Nature 544(7651), 465–470 (2017).
[Crossref] [PubMed]

Widengren, J.

D. Rönnlund, L. Xu, A. Perols, A. K. B. Gad, A. Eriksson Karlström, G. Auer, and J. Widengren, “Multicolor Fluorescence Nanoscopy by Photobleaching: Concept, Verification, and Its Application To Resolve Selective Storage of Proteins in Platelets,” ACS Nano 8(5), 4358–4365 (2014).
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B. Kraus, M. Ziegler, and H. Wolff, “Linear fluorescence unmixing in cell biological research,” Mod. Res. Educ. Top. Microsc. 2, 863–873 (2007).

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D. Rönnlund, L. Xu, A. Perols, A. K. B. Gad, A. Eriksson Karlström, G. Auer, and J. Widengren, “Multicolor Fluorescence Nanoscopy by Photobleaching: Concept, Verification, and Its Application To Resolve Selective Storage of Proteins in Platelets,” ACS Nano 8(5), 4358–4365 (2014).
[Crossref] [PubMed]

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Y. Lu, J. Lu, J. Zhao, J. Cusido, F. M. Raymo, J. Yuan, S. Yang, R. C. Leif, Y. Huo, J. A. Piper, J. Paul Robinson, E. M. Goldys, and D. Jin, “On-the-fly decoding luminescence lifetimes in the microsecond region for lanthanide-encoded suspension arrays,” Nat. Commun. 5(1), 3741 (2014).
[Crossref] [PubMed]

Yao, J.

J. Yao, L. Wang, C. Li, C. Zhang, and L. V. Wang, “Photoimprint photoacoustic microscopy for three-dimensional label-free subdiffraction imaging,” Phys. Rev. Lett. 112(1), 014302 (2014).
[Crossref] [PubMed]

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P. W. Tillberg, F. Chen, K. D. Piatkevich, Y. Zhao, C. C. Yu, B. P. English, L. Gao, A. Martorell, H. J. Suk, F. Yoshida, E. M. DeGennaro, D. H. Roossien, G. Gong, U. Seneviratne, S. R. Tannenbaum, R. Desimone, D. Cai, and E. S. Boyden, “Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies,” Nat. Biotechnol. 34(9), 987–992 (2016).
[Crossref] [PubMed]

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L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
[Crossref] [PubMed]

Yu, C. C.

P. W. Tillberg, F. Chen, K. D. Piatkevich, Y. Zhao, C. C. Yu, B. P. English, L. Gao, A. Martorell, H. J. Suk, F. Yoshida, E. M. DeGennaro, D. H. Roossien, G. Gong, U. Seneviratne, S. R. Tannenbaum, R. Desimone, D. Cai, and E. S. Boyden, “Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies,” Nat. Biotechnol. 34(9), 987–992 (2016).
[Crossref] [PubMed]

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Y. Lu, J. Lu, J. Zhao, J. Cusido, F. M. Raymo, J. Yuan, S. Yang, R. C. Leif, Y. Huo, J. A. Piper, J. Paul Robinson, E. M. Goldys, and D. Jin, “On-the-fly decoding luminescence lifetimes in the microsecond region for lanthanide-encoded suspension arrays,” Nat. Commun. 5(1), 3741 (2014).
[Crossref] [PubMed]

Yuste, R.

L. Wei, Z. Chen, L. Shi, R. Long, A. V. Anzalone, L. Zhang, F. Hu, R. Yuste, V. W. Cornish, and W. Min, “Super-multiplex vibrational imaging,” Nature 544(7651), 465–470 (2017).
[Crossref] [PubMed]

Zhang, C.

J. Yao, L. Wang, C. Li, C. Zhang, and L. V. Wang, “Photoimprint photoacoustic microscopy for three-dimensional label-free subdiffraction imaging,” Phys. Rev. Lett. 112(1), 014302 (2014).
[Crossref] [PubMed]

Zhang, L.

L. Wei, Z. Chen, L. Shi, R. Long, A. V. Anzalone, L. Zhang, F. Hu, R. Yuste, V. W. Cornish, and W. Min, “Super-multiplex vibrational imaging,” Nature 544(7651), 465–470 (2017).
[Crossref] [PubMed]

Zhao, J.

Y. Lu, J. Lu, J. Zhao, J. Cusido, F. M. Raymo, J. Yuan, S. Yang, R. C. Leif, Y. Huo, J. A. Piper, J. Paul Robinson, E. M. Goldys, and D. Jin, “On-the-fly decoding luminescence lifetimes in the microsecond region for lanthanide-encoded suspension arrays,” Nat. Commun. 5(1), 3741 (2014).
[Crossref] [PubMed]

Zhao, Y.

P. W. Tillberg, F. Chen, K. D. Piatkevich, Y. Zhao, C. C. Yu, B. P. English, L. Gao, A. Martorell, H. J. Suk, F. Yoshida, E. M. DeGennaro, D. H. Roossien, G. Gong, U. Seneviratne, S. R. Tannenbaum, R. Desimone, D. Cai, and E. S. Boyden, “Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies,” Nat. Biotechnol. 34(9), 987–992 (2016).
[Crossref] [PubMed]

Zhuang, X.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

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B. Kraus, M. Ziegler, and H. Wolff, “Linear fluorescence unmixing in cell biological research,” Mod. Res. Educ. Top. Microsc. 2, 863–873 (2007).

ACS Nano (1)

D. Rönnlund, L. Xu, A. Perols, A. K. B. Gad, A. Eriksson Karlström, G. Auer, and J. Widengren, “Multicolor Fluorescence Nanoscopy by Photobleaching: Concept, Verification, and Its Application To Resolve Selective Storage of Proteins in Platelets,” ACS Nano 8(5), 4358–4365 (2014).
[Crossref] [PubMed]

Adv. Opt. Mater. (1)

P. Reineck, A. Francis, A. Orth, D. W. M. Lau, R. D. V. Nixon-Luke, I. D. Rastogi, W. A. W. Razali, N. M. Cordina, L. M. Parker, V. K. A. Sreenivasan, L. J. Brown, and B. C. Gibson, “Brightness and Photostability of Emerging Red and Near-IR Fluorescent Nanomaterials for Bioimaging,” Adv. Opt. Mater. 4(10), 1549–1557 (2016).
[Crossref]

Anal. Biochem. (1)

A. Grinvald and I. Z. Steinberg, “On the analysis of fluorescence decay kinetics by the method of least-squares,” Anal. Biochem. 59(2), 583–598 (1974).
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L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
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S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (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]

B. L. Sprague, R. L. Pego, D. A. Stavreva, and J. G. McNally, “Analysis of Binding Reactions by Fluorescence Recovery after Photobleaching,” Biophys. J. 86(6), 3473–3495 (2004).
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Comput. Stat. Data Anal. (1)

M. W. Berry, M. Browne, A. N. Langville, V. P. Pauca, and R. J. Plemmons, “Algorithms and applications for approximate nonnegative matrix factorization,” Comput. Stat. Data Anal. 52(1), 155–173 (2007).
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P. Favreau, C. Hernandez, A. S. Lindsey, D. F. Alvarez, T. Rich, P. Prabhat, and S. J. Leavesley, “Thin-film tunable filters for hyperspectral fluorescence microscopy,” J. Biomed. Opt. 19(1), 011017 (2013).
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J. Cell Sci. (1)

L. Gao, A. Garcia-Uribe, Y. Liu, C. Li, and L. V. Wang, “Photobleaching imprinting microscopy: seeing clearer and deeper,” J. Cell Sci. 127(2), 288–294 (2014).
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G. J. Brakenhoff, K. Visscher, and E. J. Gijsbers, “Fluorescence bleach rate imaging,” J. Microsc. 175(2), 154–161 (1994).
[Crossref]

R. I. Ghauharali, R. Van Driel, and G. J. Brakenhoff, “Structure-orientated fluorescence photobleaching analysis: a method for double fluorescent labelling studies,” J. Microsc. 185(3), 375–384 (1997).
[Crossref]

Mod. Res. Educ. Top. Microsc. (1)

B. Kraus, M. Ziegler, and H. Wolff, “Linear fluorescence unmixing in cell biological research,” Mod. Res. Educ. Top. Microsc. 2, 863–873 (2007).

Nat. Biotechnol. (1)

P. W. Tillberg, F. Chen, K. D. Piatkevich, Y. Zhao, C. C. Yu, B. P. English, L. Gao, A. Martorell, H. J. Suk, F. Yoshida, E. M. DeGennaro, D. H. Roossien, G. Gong, U. Seneviratne, S. R. Tannenbaum, R. Desimone, D. Cai, and E. S. Boyden, “Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies,” Nat. Biotechnol. 34(9), 987–992 (2016).
[Crossref] [PubMed]

Nat. Commun. (1)

Y. Lu, J. Lu, J. Zhao, J. Cusido, F. M. Raymo, J. Yuan, S. Yang, R. C. Leif, Y. Huo, J. A. Piper, J. Paul Robinson, E. M. Goldys, and D. Jin, “On-the-fly decoding luminescence lifetimes in the microsecond region for lanthanide-encoded suspension arrays,” Nat. Commun. 5(1), 3741 (2014).
[Crossref] [PubMed]

Nat. Methods (2)

T. Niehörster, A. Löschberger, I. Gregor, B. Krämer, H.-J. Rahn, M. Patting, F. Koberling, J. Enderlein, and M. Sauer, “Multi-target spectrally resolved fluorescence lifetime imaging microscopy,” Nat. Methods 13(3), 257–262 (2016).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

Nature (1)

L. Wei, Z. Chen, L. Shi, R. Long, A. V. Anzalone, L. Zhang, F. Hu, R. Yuste, V. W. Cornish, and W. Min, “Super-multiplex vibrational imaging,” Nature 544(7651), 465–470 (2017).
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Opt. Express (2)

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Optica (1)

Phys. Med. Biol. (1)

C. J. G. Bakker and J. Vriend, “Multi-exponential water proton spin-lattice relaxation in biological tissues and its implications for quantitative NMR imaging,” Phys. Med. Biol. 29(5), 509–518 (1984).
[Crossref] [PubMed]

Phys. Rev. Lett. (1)

J. Yao, L. Wang, C. Li, C. Zhang, and L. V. Wang, “Photoimprint photoacoustic microscopy for three-dimensional label-free subdiffraction imaging,” Phys. Rev. Lett. 112(1), 014302 (2014).
[Crossref] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (1)

D. T. Burnette, P. Sengupta, Y. Dai, J. Lippincott-Schwartz, and B. Kachar, “Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules,” Proc. Natl. Acad. Sci. U.S.A. 108(52), 21081–21086 (2011).
[Crossref] [PubMed]

Science (1)

F. Chen, P. W. Tillberg, and E. S. Boyden, “Optical imaging. Expansion microscopy,” Science 347(6221), 543–548 (2015).
[Crossref] [PubMed]

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C. Lawson and R. Hanson, Solving Least Squares Problems, Classics in Applied Mathematics (Society for Industrial and Applied Mathematics, 1995).

J. Pawley, Handbook of Biological Confocal Microscopy (Springer, 2006).

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Figures (4)

Fig. 1
Fig. 1 BAMM with beads. a) The first frame of a 30-frame photobleaching experiment, with each frame consisting of a yellow/red dual spectral channel image of a mixture of 5 different fluorescent beads. Examples of different bead types are boxed and labeled as I-V. The red channel is gamma corrected to enhance dim pixels (for display only). Boxes II, IV and V have increased brightness for visibility. Scale bar is 50 μm. b) Time traces of beads I-V in (a), during the photobleaching experiment. Concatenated frames # 1-30 correspond to frames 1-30 in the yellow channel (570-620 nm) of image (a). Concatenated frames #31-60 correspond to frames 1-30 from the red channel (663-738 nm) of image (a). Each bead type has a unique spectral-bleaching “fingerprint”. c) False-coloured unmixed image of all five bead types. Abundance maps for bead types I-V are coloured red, yellow, purple, green and blue, respectively. d) The cross-talk matrix of the unmixing process. Cross-talk is generally low across all bead types except for bead type II into channel 1 (bead type I). This is due to the similarity in their bleaching rates, and low signal from type II beads (see (a) and (b)). For all other bead types, more than 97% of the bead signal is unmixed into the correct channel.
Fig. 2
Fig. 2 BAMM produces the correct abundance map with overlapping structures. a) First frame of a 45-frame bleaching timelapse of muntjac skin fibroblast cells under 473 nm illumination. A wide emission window is used (500-600 nm) to capture emission from both Alexa Fluor 488 and Alexa Fluor 555 dyes. Scale bar is 100 μm. b) Bleaching traces of Alexa Fluor 555 (red) and Alexa Fluor 488 (green) as estimated by NMF. c) and d) Alexa Fluor 555 and Alexa Fluor 488 abundance maps extracted from the single-channel timelapse (a) using the estimated bleaching curves in (b) along with the entire 45-frame bleaching timelapse. Even though structures in both channels overlap spatially, they are unmixed successfully. e) Image of Alexa Fluor 555 distribution acquired using spectral filtering (570-670 emission window). f) Image of Alexa Fluor 488 distribution acquired using conventional spectral filtering (480-542 nm emission window). Both (e) and (f) were recorded before bleaching.
Fig. 3
Fig. 3 Dual-channel BAMM in cells. a) First frame of a single spectral channel bleaching timelapse of a U2OS cell processed for Expansion Microscopy, labeled with Alexa Fluor 514 (tubulin) and GFP (mitochondria). b) Dual-channel BAMM image of (a) using NNLS with reference bleaching curves (40 frames) for GFP and Alexa Fluor 514. Alexa Fluor 514 is colored red and GFP is colored green. Color histograms are modified for visibility of dim features. Dotted white border indicates region shown in (c2). Scale bar indicates 60 μm after expansion (13 μm before expansion). c1) Intensity trace along the white line in (c2), intersecting two isolated microtubules. The black dots indicate data points and the blue curve is a Gaussian fit to the intensity profile. The full width at half maximum of each peak is indicated on the plot. c2) Magnified version of the region within the dotted region in (b). The white line indicates the path of the intensity trace in (c1). GFP channel not shown for clarity. Scale bar is 5 μm after expansion (1.09 μm before expansion). d) First frame of a single spectral channel bleaching timelapse of a HeLa cell labeled with Alexa Fluor 555 (Ki67) and RFP (Golgi). e) Dual-channel BAMM image of (c) using NMF (45 frames). Alexa Fluor 555 is coloured red and RFP is colored green. Scale bar is 10 μm. f) First frame of a bleaching timelapse mouse cumulus-oocyte-complex autofluorescence. The boxed region is enlarged 2.5x in the inset. g) False-coloured BAMM image using NMF (50 frames). Scale bar is 50 μm, inset scale bar is 5 μm.
Fig. 4
Fig. 4 BAMM unmixing of 3 fluorescent labels from a single spectral channel. a) The first frame (of 40) of the BAMM bleaching timelapse, using 485nm LED excitation. This LED excites Alexa Fluor 555 (Ki67), GFP (mitochondria) and Alexa Fluor 430 (microtubules). Scale bar is 20 μm. b) False-color BAMM image obtained via NI-NMF, showing the fluorescent emitter distributions. Alexa Fluor 555 in red, GFP in green and Alexa Fluor 430 in blue. c) Bleaching traces for each fluorophore type estimated by NI-NMF (solid) and NMF (dotted). Solid curve colors match the color scheme in (b).

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I k ( x,y )= i=1 N a i (x,y) v ik ,   k=1,2,,T

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