Abstract

We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.

© 2016 Optical Society of America

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References

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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref]
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    [Crossref] [PubMed]

2016 (1)

F. Greiss, M. Deligiannaki, C. Jung, U. Gaul, and D. Braun, “Single-Molecule Imaging in Living Drosophila Embryos with Reflected Light-Sheet Microscopy,” Biophys. J. 110, 939–946 (2016).
[Crossref] [PubMed]

2015 (3)

R. Galland, G. Grenci, A. Aravind, V. Viasnoff, V. Studer, and J.-B. Sibarita, “3D high- and super-resolution imaging using single-objective SPIM,” Nat. Methods 12, 641–644 (2015).
[Crossref] [PubMed]

C. C. Valley, S. Liu, D. S. Lidke, and K. A. Lidke, “Sequential Superresolution Imaging of Multiple Targets Using a Single Fluorophore,” PLoS One 10, e0123941 (2015).
[Crossref] [PubMed]

Y. Lin, J. J. Long, F. Huang, W. C. Duim, S. Kirschbaum, Y. Zhang, L. K. Schroeder, A. A. Rebane, M. G. M. Velasco, A. Virrueta, D. W. Moonan, J. Jiao, S. Y. Hernandez, Y. Zhang, and J. Bewersdorf, “Quantifying and optimizing single-molecule switching nanoscopy at high speeds,” PLoS One 10, e0128135 (2015).
[Crossref] [PubMed]

2014 (1)

B.-C. Chen, W. R. Legant, K. Wang, L. Shao, D. E. Milkie, M. W. Davidson, C. Janetopoulos, X. S. Wu, J. A. Hammer, Z. Liu, B. P. English, Y. Mimori-Kiyosue, D. P. Romero, A. T. Ritter, J. Lippincott-Schwartz, L. Fritz-Laylin, R. D. Mullins, D. M. Mitchell, J. N. Bembenek, A.-C. Reymann, R. Bohme, S. W. Grill, J. T. Wang, G. Seydoux, U. S. Tulu, D. P. Kiehart, and E. Betzig, “Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution,” Science 3461257998 (2014).
[Crossref]

2013 (5)

Y. S. Hu, Q. Zhu, K. Elkins, K. Tse, Y. Li, J. A. J. Fitzpatrick, I. M. Verma, and H. Cang, “Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells,” Opt. Nanosc. 2, 1–12 (2013).
[Crossref]

J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, “Single-molecule imaging of transcription factor binding to DNA in live mammalian cells,” Nat. Methods 10, 421–426 (2013).
[Crossref] [PubMed]

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10, 653–658 (2013).
[Crossref] [PubMed]

K. P. Rola, K. Ptasiński, A. Zakrzewski, and I. Zubel, “Silicon 45 micromirrors fabricated by etching in alkaline solutions with organic additives,” Microsyst. Technol. 20, 221–226 (2013).
[Crossref]

S. Liu, E. B. Kromann, W. D. Krueger, J. Bewersdorf, and K. A. Lidke, “Three dimensional single molecule localization using a phase retrieved pupil function,” Opt. Express 21, 29462–29487 (2013).
[Crossref]

2012 (1)

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9, 676–682 (2012).
[Crossref] [PubMed]

2011 (5)

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8, 1027–1036 (2011).
[Crossref] [PubMed]

F. Huang, S. L. Schwartz, J. M. Byars, and K. A. Lidke, “Simultaneous multiple-emitter fitting for single molecule super-resolution imaging,” Biomed. Opt. Express 2, 1377–1393 (2011).
[Crossref] [PubMed]

Y. W. Xu, A. Michael, and C. Y. Kwok, “Formation of ultra-smooth 45 micromirror on (100) silicon with low concentration TMAH and surfactant: Techniques for enlarging the truly 45 portion,” Sens. Actuators, A 166, 164–171 (2011).
[Crossref]

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8, 417–423 (2011).
[Crossref] [PubMed]

F. Cella Zanacchi, Z. Lavagnino, M. Perrone Donnorso, A. Del Bue, L. Furia, M. Faretta, and A. Diaspro, “Live-cell 3D super-resolution imaging in thick biological samples,” Nat. Methods 8, 1047–1049 (2011).
[Crossref] [PubMed]

2010 (1)

C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, “Fast, single-molecule localization that achieves theoretically minimum uncertainty,” Nat. Methods 7, 373–375 (2010).
[Crossref] [PubMed]

2008 (4)

C. a. Konopka and S. Y. Bednarek, “Variable-angle epifluorescence microscopy: A new way to look at protein dynamics in the plant cell cortex,” Plant J. 53, 186–196 (2008).
[Crossref]

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, “Highly inclined thin illumination enables clear single-molecule imaging in cells,” Nat. Methods 5, 159–161 (2008).
[Crossref] [PubMed]

K. T. Seale, R. S. Reiserer, D. a. Markov, I. a. Ges, C. Wright, C. Janetopoulos, and J. P. Wikswo, “Mirrored pyramidal wells for simultaneous multiple vantage point microscopy,” J. Microsc. 232, 1–6 (2008).
[Crossref] [PubMed]

M. G. L. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94, 4957–4970 (2008).
[Crossref] [PubMed]

2004 (1)

R. J. Ober, S. Ram, and E. S. Ward, “Localization accuracy in single-molecule microscopy,” Biophys. J. 86, 1185–1200 (2004).
[Crossref] [PubMed]

1977 (1)

G. W. Zack, W. E. Rogers, and S. A. Latt, “Automatic measurement of sister chromatid exchange frequency,” J. Histochem. Cytochem. 25, 741–753 (1977).
[Crossref] [PubMed]

Agard, D. A.

M. G. L. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94, 4957–4970 (2008).
[Crossref] [PubMed]

Aravind, A.

R. Galland, G. Grenci, A. Aravind, V. Viasnoff, V. Studer, and J.-B. Sibarita, “3D high- and super-resolution imaging using single-objective SPIM,” Nat. Methods 12, 641–644 (2015).
[Crossref] [PubMed]

Arganda-Carreras, I.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9, 676–682 (2012).
[Crossref] [PubMed]

Baird, M. A.

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10, 653–658 (2013).
[Crossref] [PubMed]

Basu, S.

J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, “Single-molecule imaging of transcription factor binding to DNA in live mammalian cells,” Nat. Methods 10, 421–426 (2013).
[Crossref] [PubMed]

Bates, M.

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8, 1027–1036 (2011).
[Crossref] [PubMed]

Bednarek, S. Y.

C. a. Konopka and S. Y. Bednarek, “Variable-angle epifluorescence microscopy: A new way to look at protein dynamics in the plant cell cortex,” Plant J. 53, 186–196 (2008).
[Crossref]

Bembenek, J. N.

B.-C. Chen, W. R. Legant, K. Wang, L. Shao, D. E. Milkie, M. W. Davidson, C. Janetopoulos, X. S. Wu, J. A. Hammer, Z. Liu, B. P. English, Y. Mimori-Kiyosue, D. P. Romero, A. T. Ritter, J. Lippincott-Schwartz, L. Fritz-Laylin, R. D. Mullins, D. M. Mitchell, J. N. Bembenek, A.-C. Reymann, R. Bohme, S. W. Grill, J. T. Wang, G. Seydoux, U. S. Tulu, D. P. Kiehart, and E. Betzig, “Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution,” Science 3461257998 (2014).
[Crossref]

Betzig, E.

B.-C. Chen, W. R. Legant, K. Wang, L. Shao, D. E. Milkie, M. W. Davidson, C. Janetopoulos, X. S. Wu, J. A. Hammer, Z. Liu, B. P. English, Y. Mimori-Kiyosue, D. P. Romero, A. T. Ritter, J. Lippincott-Schwartz, L. Fritz-Laylin, R. D. Mullins, D. M. Mitchell, J. N. Bembenek, A.-C. Reymann, R. Bohme, S. W. Grill, J. T. Wang, G. Seydoux, U. S. Tulu, D. P. Kiehart, and E. Betzig, “Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution,” Science 3461257998 (2014).
[Crossref]

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8, 417–423 (2011).
[Crossref] [PubMed]

Bewersdorf, J.

Y. Lin, J. J. Long, F. Huang, W. C. Duim, S. Kirschbaum, Y. Zhang, L. K. Schroeder, A. A. Rebane, M. G. M. Velasco, A. Virrueta, D. W. Moonan, J. Jiao, S. Y. Hernandez, Y. Zhang, and J. Bewersdorf, “Quantifying and optimizing single-molecule switching nanoscopy at high speeds,” PLoS One 10, e0128135 (2015).
[Crossref] [PubMed]

S. Liu, E. B. Kromann, W. D. Krueger, J. Bewersdorf, and K. A. Lidke, “Three dimensional single molecule localization using a phase retrieved pupil function,” Opt. Express 21, 29462–29487 (2013).
[Crossref]

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10, 653–658 (2013).
[Crossref] [PubMed]

Bohme, R.

B.-C. Chen, W. R. Legant, K. Wang, L. Shao, D. E. Milkie, M. W. Davidson, C. Janetopoulos, X. S. Wu, J. A. Hammer, Z. Liu, B. P. English, Y. Mimori-Kiyosue, D. P. Romero, A. T. Ritter, J. Lippincott-Schwartz, L. Fritz-Laylin, R. D. Mullins, D. M. Mitchell, J. N. Bembenek, A.-C. Reymann, R. Bohme, S. W. Grill, J. T. Wang, G. Seydoux, U. S. Tulu, D. P. Kiehart, and E. Betzig, “Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution,” Science 3461257998 (2014).
[Crossref]

Braun, D.

F. Greiss, M. Deligiannaki, C. Jung, U. Gaul, and D. Braun, “Single-Molecule Imaging in Living Drosophila Embryos with Reflected Light-Sheet Microscopy,” Biophys. J. 110, 939–946 (2016).
[Crossref] [PubMed]

Byars, J. M.

Cande, W. Z.

M. G. L. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94, 4957–4970 (2008).
[Crossref] [PubMed]

Cang, H.

Y. S. Hu, Q. Zhu, K. Elkins, K. Tse, Y. Li, J. A. J. Fitzpatrick, I. M. Verma, and H. Cang, “Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells,” Opt. Nanosc. 2, 1–12 (2013).
[Crossref]

Cardona, A.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9, 676–682 (2012).
[Crossref] [PubMed]

Carlton, P. M.

M. G. L. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94, 4957–4970 (2008).
[Crossref] [PubMed]

Cella Zanacchi, F.

F. Cella Zanacchi, Z. Lavagnino, M. Perrone Donnorso, A. Del Bue, L. Furia, M. Faretta, and A. Diaspro, “Live-cell 3D super-resolution imaging in thick biological samples,” Nat. Methods 8, 1047–1049 (2011).
[Crossref] [PubMed]

Chapman, A. R.

J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, “Single-molecule imaging of transcription factor binding to DNA in live mammalian cells,” Nat. Methods 10, 421–426 (2013).
[Crossref] [PubMed]

Chen, B.-C.

B.-C. Chen, W. R. Legant, K. Wang, L. Shao, D. E. Milkie, M. W. Davidson, C. Janetopoulos, X. S. Wu, J. A. Hammer, Z. Liu, B. P. English, Y. Mimori-Kiyosue, D. P. Romero, A. T. Ritter, J. Lippincott-Schwartz, L. Fritz-Laylin, R. D. Mullins, D. M. Mitchell, J. N. Bembenek, A.-C. Reymann, R. Bohme, S. W. Grill, J. T. Wang, G. Seydoux, U. S. Tulu, D. P. Kiehart, and E. Betzig, “Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution,” Science 3461257998 (2014).
[Crossref]

Chen, K. H.

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8, 1027–1036 (2011).
[Crossref] [PubMed]

Davidson, M. W.

B.-C. Chen, W. R. Legant, K. Wang, L. Shao, D. E. Milkie, M. W. Davidson, C. Janetopoulos, X. S. Wu, J. A. Hammer, Z. Liu, B. P. English, Y. Mimori-Kiyosue, D. P. Romero, A. T. Ritter, J. Lippincott-Schwartz, L. Fritz-Laylin, R. D. Mullins, D. M. Mitchell, J. N. Bembenek, A.-C. Reymann, R. Bohme, S. W. Grill, J. T. Wang, G. Seydoux, U. S. Tulu, D. P. Kiehart, and E. Betzig, “Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution,” Science 3461257998 (2014).
[Crossref]

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10, 653–658 (2013).
[Crossref] [PubMed]

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8, 417–423 (2011).
[Crossref] [PubMed]

Del Bue, A.

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J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, “Single-molecule imaging of transcription factor binding to DNA in live mammalian cells,” Nat. Methods 10, 421–426 (2013).
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Xu, Y. W.

Y. W. Xu, A. Michael, and C. Y. Kwok, “Formation of ultra-smooth 45 micromirror on (100) silicon with low concentration TMAH and surfactant: Techniques for enlarging the truly 45 portion,” Sens. Actuators, A 166, 164–171 (2011).
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G. W. Zack, W. E. Rogers, and S. A. Latt, “Automatic measurement of sister chromatid exchange frequency,” J. Histochem. Cytochem. 25, 741–753 (1977).
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K. P. Rola, K. Ptasiński, A. Zakrzewski, and I. Zubel, “Silicon 45 micromirrors fabricated by etching in alkaline solutions with organic additives,” Microsyst. Technol. 20, 221–226 (2013).
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Y. Lin, J. J. Long, F. Huang, W. C. Duim, S. Kirschbaum, Y. Zhang, L. K. Schroeder, A. A. Rebane, M. G. M. Velasco, A. Virrueta, D. W. Moonan, J. Jiao, S. Y. Hernandez, Y. Zhang, and J. Bewersdorf, “Quantifying and optimizing single-molecule switching nanoscopy at high speeds,” PLoS One 10, e0128135 (2015).
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J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, “Single-molecule imaging of transcription factor binding to DNA in live mammalian cells,” Nat. Methods 10, 421–426 (2013).
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Y. S. Hu, Q. Zhu, K. Elkins, K. Tse, Y. Li, J. A. J. Fitzpatrick, I. M. Verma, and H. Cang, “Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells,” Opt. Nanosc. 2, 1–12 (2013).
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G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8, 1027–1036 (2011).
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K. P. Rola, K. Ptasiński, A. Zakrzewski, and I. Zubel, “Silicon 45 micromirrors fabricated by etching in alkaline solutions with organic additives,” Microsyst. Technol. 20, 221–226 (2013).
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Biomed. Opt. Express (1)

Biophys. J. (3)

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G. W. Zack, W. E. Rogers, and S. A. Latt, “Automatic measurement of sister chromatid exchange frequency,” J. Histochem. Cytochem. 25, 741–753 (1977).
[Crossref] [PubMed]

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K. T. Seale, R. S. Reiserer, D. a. Markov, I. a. Ges, C. Wright, C. Janetopoulos, and J. P. Wikswo, “Mirrored pyramidal wells for simultaneous multiple vantage point microscopy,” J. Microsc. 232, 1–6 (2008).
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K. P. Rola, K. Ptasiński, A. Zakrzewski, and I. Zubel, “Silicon 45 micromirrors fabricated by etching in alkaline solutions with organic additives,” Microsyst. Technol. 20, 221–226 (2013).
[Crossref]

Nat. Methods (9)

J. C. M. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X. S. Xie, “Single-molecule imaging of transcription factor binding to DNA in live mammalian cells,” Nat. Methods 10, 421–426 (2013).
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G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8, 1027–1036 (2011).
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Opt. Express (1)

Opt. Nanosc. (1)

Y. S. Hu, Q. Zhu, K. Elkins, K. Tse, Y. Li, J. A. J. Fitzpatrick, I. M. Verma, and H. Cang, “Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells,” Opt. Nanosc. 2, 1–12 (2013).
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C. a. Konopka and S. Y. Bednarek, “Variable-angle epifluorescence microscopy: A new way to look at protein dynamics in the plant cell cortex,” Plant J. 53, 186–196 (2008).
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PLoS One (2)

C. C. Valley, S. Liu, D. S. Lidke, and K. A. Lidke, “Sequential Superresolution Imaging of Multiple Targets Using a Single Fluorophore,” PLoS One 10, e0123941 (2015).
[Crossref] [PubMed]

Y. Lin, J. J. Long, F. Huang, W. C. Duim, S. Kirschbaum, Y. Zhang, L. K. Schroeder, A. A. Rebane, M. G. M. Velasco, A. Virrueta, D. W. Moonan, J. Jiao, S. Y. Hernandez, Y. Zhang, and J. Bewersdorf, “Quantifying and optimizing single-molecule switching nanoscopy at high speeds,” PLoS One 10, e0128135 (2015).
[Crossref] [PubMed]

Science (1)

B.-C. Chen, W. R. Legant, K. Wang, L. Shao, D. E. Milkie, M. W. Davidson, C. Janetopoulos, X. S. Wu, J. A. Hammer, Z. Liu, B. P. English, Y. Mimori-Kiyosue, D. P. Romero, A. T. Ritter, J. Lippincott-Schwartz, L. Fritz-Laylin, R. D. Mullins, D. M. Mitchell, J. N. Bembenek, A.-C. Reymann, R. Bohme, S. W. Grill, J. T. Wang, G. Seydoux, U. S. Tulu, D. P. Kiehart, and E. Betzig, “Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution,” Science 3461257998 (2014).
[Crossref]

Sens. Actuators, A (1)

Y. W. Xu, A. Michael, and C. Y. Kwok, “Formation of ultra-smooth 45 micromirror on (100) silicon with low concentration TMAH and surfactant: Techniques for enlarging the truly 45 portion,” Sens. Actuators, A 166, 164–171 (2011).
[Crossref]

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P. J. Keller and E. H. K. Stelzer, “Digital scanned laser light sheet fluorescence microscopy.” Cold Spring Harbor protocols2010, pdb.top78 (2010).

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Figures (12)

Fig. 1
Fig. 1

Principle of reflected beam light-sheet microscopy. A laser line is focused through the objective and is reflected from a 45° mirror side wall of a microfluidic channel. The light-sheet is reflected at the focal plane of the objective, thereby illuminating only the in-focus plane of the cell. 3D optical sectioning is achieved by moving the channel and cell up and downwards and repositioning of the light-sheet such that it is reflected at the focal plane.

Fig. 2
Fig. 2

Single objective light-sheet microscope setup. (a) Schematic overview of microscope setup. The image shows the horizontal plane of the optical table. Note that the objective and microfluidic channel are drawn horizontally for visualization purposes, but they actually point upwards. (b) The laser line generating light paths (yellow highlighted part in (a) are shown for the x–z and y–z planes.

Fig. 3
Fig. 3

Positioning the beam waist. The beam path in the x–z dimension is shown starting from CY2 (top). The optical components are labeled the same as in Fig. 2. I1 is the virtual object of I2, I2 is the real object of I3, which is the beam waist, and I4 is both the virtual image of I2 and the virtual object of I3. If I3 is at the designed focal position of the objective lens, S1 is equal to the focal length of L1(S1 = f1). D1, D2 and D3 are fixed distances, which are equal to f1 + f2, f2 + f3 and f3, respectively (D3 is approximately equal to f3, because the focal length of the objective lens is usually much smaller than the focal length of the tube lens TL1). In the bottom drawing, CY2 is moved by a distance d1, and thereby the beam waist I3 is moved away from the objective lens.

Fig. 4
Fig. 4

Light-sheet dimensions. (a) Cartoon representing the measured parameters of the beam dimensions. ω0 is the beam radius at the waist, z0 is the distance between the waist and the position along the propagation direction at which the beam radius is 2 × ω 0.×The confocal parameter is 2 × z0. (b) In order to image the beam dimension at various distances from the objective the position of the tube lens relative to the camera is changed, resulting in a shift of the focal plane. (c) At each focus an image of the beam is taken (top panel) and the profile of the image along the x dimension is fitted to a Gaussian in order to determine the width. (d) Graph showing the measured beam width (crosses) for each z-position (propagation direction), where 0 is the closest position to the objective that we could measure. The beam was measured for two different slit aperture widths (1 mm and 2 mm) and for both the profile was fit to Eq. (6).

Fig. 5
Fig. 5

Channel fabrication and characterization. (a) Schematic of channel fabrication. A silicon wafer is used to etch the channel (i). An oxide mask is grown on the silicon (ii) and a mask opening is created by photolithography (iii). The channel is etched by KOH which leaves oxide mask overhangs (iv). The mask is removed (v) and the channel surface is coated with aluminum (vi) before being anodically bonded to a glass coverslip (vii). (b) Optical microscope image of four channels. (c) SEM image of four channels after KOH etch but before HF etch showing oxide mask overhangs. (d) SEM image depicting a single channel after HF etch to remove oxide mask.

Fig. 6
Fig. 6

Chip layout. (a) Exploded view of chip packaging. Each chip is packaged into five layers of PMMA with two O-rings incorporated for each in and outlet. (b) Transparent top and side view images of chip assembled in packaging. (c) Schematic showing the fluidic connections inside the chip. (d,e) Photographs showing top (d) with tubing connections and bottom (e) with coverslip surface of packaged chip.

Fig. 7
Fig. 7

Light-sheet illumination reduces background and increases contrast in diffraction limited imaging. Images of an RBL cell illuminated in wide field (a) and light-sheet (b) mode. Shown are one x–y plane and x–z and y–z projections. (c) Intensity profiles for the lines shown in (a). Scale bar is 5 μm.

Fig. 8
Fig. 8

SO-LSM improves 2D super-resolution microscopy. HeLa cells were labeled for TOM-20 to visualize mitochondria and imaged for 200 sequences of 2000 frames each. 2D reconstructions are shown for cells imaged using wide field (a) and light-sheet illumination (b). White boxes show the position of zoomed in regions shown to the right of each image. Histograms of intensity (c), per pixel background (d), signal to noise ratio (SNR, e) and localization accuracy reported by the CRLB σx (f) of individual emitters are shown. Green dotted lines depict results for cells imaged with wide field illumination and solid purple lines depict results for light-sheet illumination. Histograms are summed over 4 cells imaged for each condition. LS = light-sheet, WF = wide field, scale bar 2 μm.

Fig. 9
Fig. 9

SO-LSM improves 3D super-resolution microscopy. HeLa cells were labeled for TOM-20 to visualize mitochondria and imaged for 150 sequences of 2000 frames each. Single frame raw data images, corrected for offset and gain, are shown for cells imaged using wide field (a) and light-sheet illumination (b). Histograms of intensity (c), per pixel background (d), signal to noise ratio (SNR, e) and localization accuracy reported by the CRLB on σz (f) of individual emitters are shown. Green dotted lines depict results for cells imaged with wide field illumination and solid purple lines depict results for light-sheet illumination. LS = light-sheet, WF = wide field, scale bar 3 μm.

Fig. 10
Fig. 10

SO-LSM speeds-up acquisition of 3D super-resolution data. HeLa cells were labeled for TOM-20 to visualize mitochondria and imaged at a single plane for 5 minutes using wide field (a) and light-sheet illumination (b). 3D reconstructions are shown for data acquired in 1, 3 and 5 minutes. Zoom of single mitochondria are shown from the white boxed regions. z–position of emitters is indicated by color coding. Scale bar 2 μm.

Fig. 11
Fig. 11

SO-LSM improves whole-cell 3D super-resolution microscopy. HeLa cells were labeled for TOM-20 to visualize mitochondria and imaged using the 3D SMSR method. Cells were imaged from bottom to top and sequences of 2000 frames were acquired at z–planes spaced 250 nm apart. The scan through the cell was repeated 12 times. x–y projections of reconstructed images with color coded z–depth are shown for cells imaged using wide field (a,c,e) and light-sheet illumination (b,d,f). Zoom of single mitochondria are shown from the white boxed regions. Z-position of emitters is indicated by color coding. Image are reconstructed from whole-cell data (a,b) and 2 μm thick slices (c,d,e,f). LS = light-sheet, WF = wide field, scale bars 1 μm

Fig. 12
Fig. 12

SO-LSM reduces bleaching during whole-cell 3D super-resolution microscopy. Relative number of accepted fits per repeat (normalized to max) are shown for the data from figure 11. LS = light-sheet, WF = wide field

Equations (6)

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

S 2 = ( f 2 f 1 ) 2 × d 1 + f 2
S 3 = f 2 + f 3 S 2
1 S 3 1 S 4 = 1 f 3
R 1 = S 4 + D 3 = S 4 + f 3
R 1 = ( f 1 × f 3 f 2 ) 2 × 1 d 1
ω z = ω 0 × ( 1 + ( z z 0 ) 2 )

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