Abstract

Visualizing diverse anatomical and functional traits that span many spatial scales with high spatio-temporal resolution provides insights into the fundamentals of living organisms. Light-field microscopy (LFM) has recently emerged as a scanning-free, scalable method that allows for high-speed, volumetric functional brain imaging. Given those promising applications at the tissue level, at its other extreme, this highly-scalable approach holds great potential for observing structures and dynamics in single-cell specimens. However, the challenge remains for current LFM to achieve a subcellular level, near-diffraction-limited 3D spatial resolution. Here, we report high-resolution LFM (HR-LFM) for live-cell imaging with a resolution of 300-700 nm in all three dimensions, an imaging depth of several micrometers, and a volume acquisition time of milliseconds. We demonstrate the technique by imaging various cellular dynamics and structures and tracking single particles. The method may advance LFM as a particularly useful tool for understanding biological systems at multiple spatio-temporal levels.

© 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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2018 (1)

2017 (4)

K. M. Dean, P. Roudot, E. S. Welf, T. Pohlkamp, G. Garrelts, J. Herz, and R. Fiolka, “Imaging subcellular dynamics with fast and light-efficient volumetrically parallelized microscopy,” Optica 4(2), 263–271 (2017).
[Crossref] [PubMed]

P. P. Laissue, R. A. Alghamdi, P. Tomancak, E. G. Reynaud, and H. Shroff, “Assessing phototoxicity in live fluorescence imaging,” Nat. Methods 14(7), 657–661 (2017).
[Crossref] [PubMed]

G. Suarato, S. I. Lee, W. Li, S. Rao, T. Khan, Y. Meng, and M. Shelly, “Micellar nanocomplexes for biomagnetic delivery of intracellular proteins to dictate axon formation during neuronal development,” Biomaterials 112, 176–191 (2017).
[Crossref] [PubMed]

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[Crossref] [PubMed]

2016 (1)

2015 (2)

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, “SPED Light Sheet Microscopy: Fast Mapping of Biological System Structure and Function,” Cell 163(7), 1796–1806 (2015).
[Crossref] [PubMed]

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, “Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms,” Nat. Photonics 9(2), 113–119 (2015).
[Crossref] [PubMed]

2014 (2)

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

N. Cohen, S. Yang, A. Andalman, M. Broxton, L. Grosenick, K. Deisseroth, M. Horowitz, and M. Levoy, “Enhancing the performance of the light field microscope using wavefront coding,” Opt. Express 22(20), 24817–24839 (2014).
[Crossref] [PubMed]

2013 (2)

M. Broxton, L. Grosenick, S. Yang, N. Cohen, A. Andalman, K. Deisseroth, and M. Levoy, “Wave optics theory and 3-D deconvolution for the light field microscope,” Opt. Express 21(21), 25418–25439 (2013).
[Crossref] [PubMed]

T. Schrödel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, “Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light,” Nat. Methods 10(10), 1013–1020 (2013).
[Crossref] [PubMed]

2011 (1)

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(5), 417–423 (2011).
[Crossref] [PubMed]

2009 (2)

M. Levoy, Z. Zhang, and I. McDowall, “Recording and controlling the 4D light field in a microscope using microlens arrays,” J. Microsc. 235(2), 144–162 (2009).
[Crossref] [PubMed]

J. Wakabayashi, Z. Zhang, N. Wakabayashi, Y. Tamura, M. Fukaya, T. W. Kensler, M. Iijima, and H. Sesaki, “The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice,” J. Cell Biol. 186(6), 805–816 (2009).
[Crossref] [PubMed]

2008 (2)

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy,” Science 322(5904), 1065–1069 (2008).
[Crossref] [PubMed]

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science 320(5881), 1332–1336 (2008).
[Crossref] [PubMed]

2006 (1)

M. Levoy, R. Ng, A. Adams, M. Footer, and M. Horowitz, “Light field microscopy,” ACM Trans. Graph. 25(3), 924 (2006).
[Crossref]

2002 (1)

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[Crossref] [PubMed]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Adams, A.

M. Levoy, R. Ng, A. Adams, M. Footer, and M. Horowitz, “Light field microscopy,” ACM Trans. Graph. 25(3), 924 (2006).
[Crossref]

Adesnik, H.

Agard, D. A.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science 320(5881), 1332–1336 (2008).
[Crossref] [PubMed]

Alghamdi, R. A.

P. P. Laissue, R. A. Alghamdi, P. Tomancak, E. G. Reynaud, and H. Shroff, “Assessing phototoxicity in live fluorescence imaging,” Nat. Methods 14(7), 657–661 (2017).
[Crossref] [PubMed]

Andalman, A.

Antipa, N.

Aumayr, K.

T. Schrödel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, “Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light,” Nat. Methods 10(10), 1013–1020 (2013).
[Crossref] [PubMed]

Betzig, E.

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(5), 417–423 (2011).
[Crossref] [PubMed]

Bouchard, M. B.

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, “Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms,” Nat. Photonics 9(2), 113–119 (2015).
[Crossref] [PubMed]

Boyden, E. S.

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

Broxton, M.

Bruno, R. M.

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, “Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms,” Nat. Photonics 9(2), 113–119 (2015).
[Crossref] [PubMed]

Burke, B.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science 320(5881), 1332–1336 (2008).
[Crossref] [PubMed]

Burns, V. M.

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, “SPED Light Sheet Microscopy: Fast Mapping of Biological System Structure and Function,” Cell 163(7), 1796–1806 (2015).
[Crossref] [PubMed]

Cardoso, M. C.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science 320(5881), 1332–1336 (2008).
[Crossref] [PubMed]

Carlton, P. M.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science 320(5881), 1332–1336 (2008).
[Crossref] [PubMed]

Cohen, N.

Davidson, M. W.

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(5), 417–423 (2011).
[Crossref] [PubMed]

Dean, K. M.

Deisseroth, K.

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Fiolka, R.

Footer, M.

M. Levoy, R. Ng, A. Adams, M. Footer, and M. Horowitz, “Light field microscopy,” ACM Trans. Graph. 25(3), 924 (2006).
[Crossref]

Fukaya, M.

J. Wakabayashi, Z. Zhang, N. Wakabayashi, Y. Tamura, M. Fukaya, T. W. Kensler, M. Iijima, and H. Sesaki, “The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice,” J. Cell Biol. 186(6), 805–816 (2009).
[Crossref] [PubMed]

Galbraith, C. G.

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(5), 417–423 (2011).
[Crossref] [PubMed]

Galbraith, J. A.

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(5), 417–423 (2011).
[Crossref] [PubMed]

Gao, L.

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(5), 417–423 (2011).
[Crossref] [PubMed]

Garrelts, G.

Georgiev, T.

A. Lumsdaine and T. Georgiev, “The focused plenoptic camera,” in 2009 IEEE International Conference on Computational Photography, ICCP 09 (2009).
[Crossref]

Gerlock, M.

Grosenick, L.

Grueber, W. B.

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, “Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms,” Nat. Photonics 9(2), 113–119 (2015).
[Crossref] [PubMed]

Gustafsson, M. G. L.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science 320(5881), 1332–1336 (2008).
[Crossref] [PubMed]

Haase, S.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science 320(5881), 1332–1336 (2008).
[Crossref] [PubMed]

Herz, J.

Hillman, E. M. C.

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, “Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms,” Nat. Photonics 9(2), 113–119 (2015).
[Crossref] [PubMed]

Hoffmann, M.

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

Horowitz, M.

Iijima, M.

J. Wakabayashi, Z. Zhang, N. Wakabayashi, Y. Tamura, M. Fukaya, T. W. Kensler, M. Iijima, and H. Sesaki, “The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice,” J. Cell Biol. 186(6), 805–816 (2009).
[Crossref] [PubMed]

Kato, S.

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

Kauvar, I.

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, “SPED Light Sheet Microscopy: Fast Mapping of Biological System Structure and Function,” Cell 163(7), 1796–1806 (2015).
[Crossref] [PubMed]

Keller, P. J.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy,” Science 322(5904), 1065–1069 (2008).
[Crossref] [PubMed]

Kensler, T. W.

J. Wakabayashi, Z. Zhang, N. Wakabayashi, Y. Tamura, M. Fukaya, T. W. Kensler, M. Iijima, and H. Sesaki, “The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice,” J. Cell Biol. 186(6), 805–816 (2009).
[Crossref] [PubMed]

Khan, T.

G. Suarato, S. I. Lee, W. Li, S. Rao, T. Khan, Y. Meng, and M. Shelly, “Micellar nanocomplexes for biomagnetic delivery of intracellular proteins to dictate axon formation during neuronal development,” Biomaterials 112, 176–191 (2017).
[Crossref] [PubMed]

Kner, P.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science 320(5881), 1332–1336 (2008).
[Crossref] [PubMed]

Lacefield, C.

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, “Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms,” Nat. Photonics 9(2), 113–119 (2015).
[Crossref] [PubMed]

Laissue, P. P.

P. P. Laissue, R. A. Alghamdi, P. Tomancak, E. G. Reynaud, and H. Shroff, “Assessing phototoxicity in live fluorescence imaging,” Nat. Methods 14(7), 657–661 (2017).
[Crossref] [PubMed]

Larson, D. R.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[Crossref] [PubMed]

Lee, S. I.

G. Suarato, S. I. Lee, W. Li, S. Rao, T. Khan, Y. Meng, and M. Shelly, “Micellar nanocomplexes for biomagnetic delivery of intracellular proteins to dictate axon formation during neuronal development,” Biomaterials 112, 176–191 (2017).
[Crossref] [PubMed]

Leonhardt, H.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science 320(5881), 1332–1336 (2008).
[Crossref] [PubMed]

Levoy, M.

Li, W.

G. Suarato, S. I. Lee, W. Li, S. Rao, T. Khan, Y. Meng, and M. Shelly, “Micellar nanocomplexes for biomagnetic delivery of intracellular proteins to dictate axon formation during neuronal development,” Biomaterials 112, 176–191 (2017).
[Crossref] [PubMed]

Liu, H.-Y.

Lovett-Barron, M.

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, “SPED Light Sheet Microscopy: Fast Mapping of Biological System Structure and Function,” Cell 163(7), 1796–1806 (2015).
[Crossref] [PubMed]

Lumsdaine, A.

A. Lumsdaine and T. Georgiev, “The focused plenoptic camera,” in 2009 IEEE International Conference on Computational Photography, ICCP 09 (2009).
[Crossref]

Mann, R. S.

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, “Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms,” Nat. Photonics 9(2), 113–119 (2015).
[Crossref] [PubMed]

McDowall, I.

M. Levoy, Z. Zhang, and I. McDowall, “Recording and controlling the 4D light field in a microscope using microlens arrays,” J. Microsc. 235(2), 144–162 (2009).
[Crossref] [PubMed]

Mendes, C. S.

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, “Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms,” Nat. Photonics 9(2), 113–119 (2015).
[Crossref] [PubMed]

Meng, Y.

G. Suarato, S. I. Lee, W. Li, S. Rao, T. Khan, Y. Meng, and M. Shelly, “Micellar nanocomplexes for biomagnetic delivery of intracellular proteins to dictate axon formation during neuronal development,” Biomaterials 112, 176–191 (2017).
[Crossref] [PubMed]

Milkie, D. E.

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(5), 417–423 (2011).
[Crossref] [PubMed]

Molodtsov, M. I.

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[Crossref] [PubMed]

Ng, R.

M. Levoy, R. Ng, A. Adams, M. Footer, and M. Horowitz, “Light field microscopy,” ACM Trans. Graph. 25(3), 924 (2006).
[Crossref]

Nöbauer, T.

M. A. Taylor, T. Nöbauer, A. Pernia-Andrade, F. Schlumm, and A. Vaziri, “Brain-wide 3D light-field imaging of neuronal activity with speckle-enhanced resolution,” Optica 5(4), 345 (2018).
[Crossref]

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[Crossref] [PubMed]

Pak, N.

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

Pégard, N. C.

Pernia-Andrade, A.

Pernía-Andrade, A. J.

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[Crossref] [PubMed]

Planchon, T. A.

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(5), 417–423 (2011).
[Crossref] [PubMed]

Pohlkamp, T.

Prevedel, R.

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

T. Schrödel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, “Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light,” Nat. Methods 10(10), 1013–1020 (2013).
[Crossref] [PubMed]

Rao, S.

G. Suarato, S. I. Lee, W. Li, S. Rao, T. Khan, Y. Meng, and M. Shelly, “Micellar nanocomplexes for biomagnetic delivery of intracellular proteins to dictate axon formation during neuronal development,” Biomaterials 112, 176–191 (2017).
[Crossref] [PubMed]

Raskar, R.

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

Reynaud, E. G.

P. P. Laissue, R. A. Alghamdi, P. Tomancak, E. G. Reynaud, and H. Shroff, “Assessing phototoxicity in live fluorescence imaging,” Nat. Methods 14(7), 657–661 (2017).
[Crossref] [PubMed]

Roudot, P.

Sankaran, S.

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, “SPED Light Sheet Microscopy: Fast Mapping of Biological System Structure and Function,” Cell 163(7), 1796–1806 (2015).
[Crossref] [PubMed]

Schermelleh, L.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science 320(5881), 1332–1336 (2008).
[Crossref] [PubMed]

Schlumm, F.

Schmidt, A. D.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy,” Science 322(5904), 1065–1069 (2008).
[Crossref] [PubMed]

Schrödel, T.

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

T. Schrödel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, “Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light,” Nat. Methods 10(10), 1013–1020 (2013).
[Crossref] [PubMed]

Sedat, J. W.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science 320(5881), 1332–1336 (2008).
[Crossref] [PubMed]

Sesaki, H.

J. Wakabayashi, Z. Zhang, N. Wakabayashi, Y. Tamura, M. Fukaya, T. W. Kensler, M. Iijima, and H. Sesaki, “The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice,” J. Cell Biol. 186(6), 805–816 (2009).
[Crossref] [PubMed]

Shao, L.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science 320(5881), 1332–1336 (2008).
[Crossref] [PubMed]

Shelly, M.

G. Suarato, S. I. Lee, W. Li, S. Rao, T. Khan, Y. Meng, and M. Shelly, “Micellar nanocomplexes for biomagnetic delivery of intracellular proteins to dictate axon formation during neuronal development,” Biomaterials 112, 176–191 (2017).
[Crossref] [PubMed]

Shroff, H.

P. P. Laissue, R. A. Alghamdi, P. Tomancak, E. G. Reynaud, and H. Shroff, “Assessing phototoxicity in live fluorescence imaging,” Nat. Methods 14(7), 657–661 (2017).
[Crossref] [PubMed]

Skocek, O.

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[Crossref] [PubMed]

Stelzer, E. H. K.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy,” Science 322(5904), 1065–1069 (2008).
[Crossref] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Suarato, G.

G. Suarato, S. I. Lee, W. Li, S. Rao, T. Khan, Y. Meng, and M. Shelly, “Micellar nanocomplexes for biomagnetic delivery of intracellular proteins to dictate axon formation during neuronal development,” Biomaterials 112, 176–191 (2017).
[Crossref] [PubMed]

Tamura, Y.

J. Wakabayashi, Z. Zhang, N. Wakabayashi, Y. Tamura, M. Fukaya, T. W. Kensler, M. Iijima, and H. Sesaki, “The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice,” J. Cell Biol. 186(6), 805–816 (2009).
[Crossref] [PubMed]

Taylor, M. A.

Thompson, R. E.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[Crossref] [PubMed]

Tomancak, P.

P. P. Laissue, R. A. Alghamdi, P. Tomancak, E. G. Reynaud, and H. Shroff, “Assessing phototoxicity in live fluorescence imaging,” Nat. Methods 14(7), 657–661 (2017).
[Crossref] [PubMed]

Tomer, R.

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, “SPED Light Sheet Microscopy: Fast Mapping of Biological System Structure and Function,” Cell 163(7), 1796–1806 (2015).
[Crossref] [PubMed]

Traub, F. M.

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[Crossref] [PubMed]

Vaziri, A.

M. A. Taylor, T. Nöbauer, A. Pernia-Andrade, F. Schlumm, and A. Vaziri, “Brain-wide 3D light-field imaging of neuronal activity with speckle-enhanced resolution,” Optica 5(4), 345 (2018).
[Crossref]

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[Crossref] [PubMed]

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

T. Schrödel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, “Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light,” Nat. Methods 10(10), 1013–1020 (2013).
[Crossref] [PubMed]

Voleti, V.

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, “Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms,” Nat. Photonics 9(2), 113–119 (2015).
[Crossref] [PubMed]

Wakabayashi, J.

J. Wakabayashi, Z. Zhang, N. Wakabayashi, Y. Tamura, M. Fukaya, T. W. Kensler, M. Iijima, and H. Sesaki, “The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice,” J. Cell Biol. 186(6), 805–816 (2009).
[Crossref] [PubMed]

Wakabayashi, N.

J. Wakabayashi, Z. Zhang, N. Wakabayashi, Y. Tamura, M. Fukaya, T. W. Kensler, M. Iijima, and H. Sesaki, “The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice,” J. Cell Biol. 186(6), 805–816 (2009).
[Crossref] [PubMed]

Waller, L.

Webb, W. W.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[Crossref] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Weilguny, L.

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[Crossref] [PubMed]

Welf, E. S.

Wetzstein, G.

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

Winoto, L.

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science 320(5881), 1332–1336 (2008).
[Crossref] [PubMed]

Wittbrodt, J.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy,” Science 322(5904), 1065–1069 (2008).
[Crossref] [PubMed]

Yang, S.

Yoon, Y.-G.

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

Zhang, Z.

M. Levoy, Z. Zhang, and I. McDowall, “Recording and controlling the 4D light field in a microscope using microlens arrays,” J. Microsc. 235(2), 144–162 (2009).
[Crossref] [PubMed]

J. Wakabayashi, Z. Zhang, N. Wakabayashi, Y. Tamura, M. Fukaya, T. W. Kensler, M. Iijima, and H. Sesaki, “The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice,” J. Cell Biol. 186(6), 805–816 (2009).
[Crossref] [PubMed]

Zimmer, M.

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[Crossref] [PubMed]

T. Schrödel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, “Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light,” Nat. Methods 10(10), 1013–1020 (2013).
[Crossref] [PubMed]

ACM Trans. Graph. (1)

M. Levoy, R. Ng, A. Adams, M. Footer, and M. Horowitz, “Light field microscopy,” ACM Trans. Graph. 25(3), 924 (2006).
[Crossref]

Biomaterials (1)

G. Suarato, S. I. Lee, W. Li, S. Rao, T. Khan, Y. Meng, and M. Shelly, “Micellar nanocomplexes for biomagnetic delivery of intracellular proteins to dictate axon formation during neuronal development,” Biomaterials 112, 176–191 (2017).
[Crossref] [PubMed]

Biophys. J. (1)

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[Crossref] [PubMed]

Cell (1)

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, “SPED Light Sheet Microscopy: Fast Mapping of Biological System Structure and Function,” Cell 163(7), 1796–1806 (2015).
[Crossref] [PubMed]

J. Cell Biol. (1)

J. Wakabayashi, Z. Zhang, N. Wakabayashi, Y. Tamura, M. Fukaya, T. W. Kensler, M. Iijima, and H. Sesaki, “The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice,” J. Cell Biol. 186(6), 805–816 (2009).
[Crossref] [PubMed]

J. Microsc. (1)

M. Levoy, Z. Zhang, and I. McDowall, “Recording and controlling the 4D light field in a microscope using microlens arrays,” J. Microsc. 235(2), 144–162 (2009).
[Crossref] [PubMed]

Nat. Methods (5)

T. Schrödel, R. Prevedel, K. Aumayr, M. Zimmer, and A. Vaziri, “Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light,” Nat. Methods 10(10), 1013–1020 (2013).
[Crossref] [PubMed]

R. Prevedel, Y.-G. Yoon, M. Hoffmann, N. Pak, G. Wetzstein, S. Kato, T. Schrödel, R. Raskar, M. Zimmer, E. S. Boyden, and A. Vaziri, “Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy,” Nat. Methods 11(7), 727–730 (2014).
[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(5), 417–423 (2011).
[Crossref] [PubMed]

P. P. Laissue, R. A. Alghamdi, P. Tomancak, E. G. Reynaud, and H. Shroff, “Assessing phototoxicity in live fluorescence imaging,” Nat. Methods 14(7), 657–661 (2017).
[Crossref] [PubMed]

T. Nöbauer, O. Skocek, A. J. Pernía-Andrade, L. Weilguny, F. M. Traub, M. I. Molodtsov, and A. Vaziri, “Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy,” Nat. Methods 14(8), 811–818 (2017).
[Crossref] [PubMed]

Nat. Photonics (1)

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, “Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms,” Nat. Photonics 9(2), 113–119 (2015).
[Crossref] [PubMed]

Opt. Express (2)

Optica (3)

Science (3)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy,” Science 322(5904), 1065–1069 (2008).
[Crossref] [PubMed]

L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. L. Gustafsson, H. Leonhardt, and J. W. Sedat, “Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy,” Science 320(5881), 1332–1336 (2008).
[Crossref] [PubMed]

Other (4)

A. Lumsdaine and T. Georgiev, “The focused plenoptic camera,” in 2009 IEEE International Conference on Computational Photography, ICCP 09 (2009).
[Crossref]

M. Gu, Advanced Optical Imaging Theory (Springer, 2000).

A. C. Kak and M. Stanley, “Algorithms for Reconstruction with Nondiffracting Sources,” Princ. Comput. Tomogr. Imaging 49–112 (1999).

Supplementary code used for HR-LFM reconstruction, https://doi.org/10.6084/m9.figshare.6850409 .

Supplementary Material (6)

NameDescription
» Code 1       Supplementary code used in for HR-LFM reconstruction.
» Visualization 1       Supplementary Video 1. Time-lapse video of reconstructed mitochondria in living Drp1-/- MEFs (FOV = 133 x 133 µm, volume acquisition rate = 10 Hz). Scale bars, 10 µm (main), 1 µm (insets). Color scale bar, z = -1.0 µm (blue) to 1.5 µm (red).
» Visualization 2       Supplementary Video 2. Zoomed-in, 3D view of the corresponding region (marked with ‘Triangle’) in Supplementary Video 1 (volume acquisition rate = 10 Hz).
» Visualization 3       Supplementary Video 3. Zoomed-in, 3D view of the corresponding region (marked with ‘Cross’) in Supplementary Video 1 (volume acquisition rate = 10 Hz).
» Visualization 4       Supplementary Video 4. Time-lapse video of Golgi-derived vesicles in living COS-7 cells (FOV = 133 x 133 µm, volume acquisition rate = 100 Hz). Scale bars, 10 µm (main), 1 µm (insets). Color scale bar, z = -1.5 µm (blue) to 1.0 µm (red).
» Visualization 5       Supplementary Video 5. Time-lapse video of a reconstructed diffusing nanoparticle suspended in water (FOV = 8.3 x 133 µm, volume acquisition rate = 1000 Hz).

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Figures (13)

Fig. 1
Fig. 1 High-resolution light-field microscopy (HR-LFM). (a) A schematic of the experimental setup for HR-LFM. The objective lens (OL) and tube lens (TL) form an image at the native image plane (NIP, dashed plane). The microlens array (MLA) is situated at a and b to the NIP and the sCMOS camera, respectively. RL: relay lenses; DM: dichroic mirror; EF: emission filter; M: mirror. (b,c) Left panel, simulated light propagation from a point emitter located at the native object plane (NOP) in conventional LFM (b) and HR-LFM (c). Right panel, x-y views of the corresponding light-field information captured at the camera plane (CP). fobj, the effective focal length of the objective lens; ftl, the focal length of the tube lens; fml, the focal length of the MLA. (d) x-z views of the propagation of the optical signals in conventional LFM (top) and HR-LFM (bottom) from z = −2.0 µm to 2.5 µm. The dashed lines represent the NIPs. (e) Reconstruction of aggregated fluorescent beads placed at the NOP, using conventional LFM (left) and HR-LFM (right). Reconstruction artifacts were observed in conventional LFM. The profile across the dashed line exhibits FWHM of 387 nm. Scale bars, 2 µm (b-d), 5 µm (e).
Fig. 2
Fig. 2 Characterization of HR-LFM. (a) The reconstructed cross-sectional images (top panel) and profiles (bottom panel) of 100-nm fluorescent beads in x-y (left), y-z (middle) and x-z (right) at z = 1.1 µm. The profiles along the dashed lines exhibit isotropic FWHMs of 332 nm, 336 nm, and 334 nm in x, y, and z, respectively. (b) The 3D FWHM values of the reconstructed PSFs at various axial positions over a >3-µm range. (c) A reconstructed z-stack image at z = 1.3 µm of a surface-stained, 1-µm fluorescent microsphere. The hollow structure was observed. Inset, raw HR-LFM data of the boxed microsphere taken at a volume acquisition time of 0.2 s. (d) Lateral (x-y) and axial (x-z and y-z) cross-sectional views of the reconstructed microsphere in (c). (e) The corresponding z-stack images of the microsphere at an axial step size of 100 nm. (f) Corresponding lateral and axial cross-sectional profiles along colored dashed lines in (d). The profiles exhibited the sub-micrometer hollow structure resolved by HR-LFM and the FWHMs of the stained surface of ~400 nm at z = 1.3 µm in all three dimensions. Scale bars, 300 nm (a), 5 µm (c), 2 µm ((c) inset), 500 nm (d,e).
Fig. 3
Fig. 3 Imaging mitochondria in fixed HeLa cells using HR-LFM. (a-d) Raw light-field (a), reconstructed 3D HR-LFM (b), conventional wide-field (c) and conventional LFM (d) images of immuno-labeled mitochondria in HeLa cells. The depth-information is color-coded according to the color scale bars in (b,d). Boxed regions (1-4) in (b,c) show that HR-LFM captured mitochondria that were out-of-focus and hence poorly detected by conventional wide-field microscopy due to its limited DOF. (e,f) Zoomed-in images (leftmost panel) of the boxed regions in (b,d), respectively, and their corresponding selected z-stack images (right panel), showing both sensitive axial discrimination and suppression of reconstruction artifacts using HR-LFM. Scale bars, 10 µm (a-d), 2 µm (e,f).
Fig. 4
Fig. 4 Imaging mitochondria in living Drp1−/− mouse embryo fibroblasts (MEFs) using HR-LFM. (a) Reconstructed 3D image of mitochondria labeled with MitoTracker in living Drp1−/− MEFs at time-point t = 0.1 s, taken at a volume acquisition time of 0.1 s. The depth-information is color-coded according to the color scale bar. (b) Left, zoomed-in image of the corresponding boxed region in (a) and cross-sectional x-z and y-z views along the dashed lines. Right, the corresponding axial cross-sectional profiles at time-points t = 0.1 s, 0.2 s, 5.0 s, and 10.0 s, showing resolved axial structures separated by ~500 nm. (c) Volume rendering of the same region in (b) at time-points t = 0.1 s, 5.0 s, and 10.0 s, respectively. (d) Zoomed-in images of the corresponding boxed region in (a) at time-points t = 0.1 s, 0.2 s, 5.0 s, and 10.0 s, respectively. White arrows indicate mitochondria undergoing structural reorganization. (e) The z-stack images of (d) from z = 0.6 μm to 2.1 μm at t = 0.1 s, showing axial structural changes captured at an axial step size of 100 nm. (f) The z-stack image of (d) at z = 1.2 μm at time-points t = 0.1 s, 0.2 s, 5.0 s, and 10.0 s, respectively. (g) The cross-sectional profiles along the dashed lines in (f) exhibit sub-micrometer structural changes resolved in x (left) and y (right) at time-points t = 0.1 s, 0.2 s, 5.0 s, and 10.0 s, respectively. Scale bars: 10 μm (a), 1 μm (b-f).
Fig. 5
Fig. 5 Imaging Golgi-derived membrane vesicles in living COS-7 cells using HR-LFM. (a) Reconstructed 3D images of Golgi-derived membrane vesicles labeled with mEmerald-Golgi-7 in living COS-7 cells at time-points t = 0.01 s (left) and 5.00 s (right), taken at a volume acquisition time of 0.01 s. The depth-information is color-coded according to the color scale bar. White arrows indicate multiple moving vesicles. (b) Zoomed-in images of the corresponding boxed region in (a) at time-points t = 0.01 s, 1.00 s, 2.00 s, 3.00 s, 4.00 s, and 5.00 s. White arrows indicate multiple moving vesicles. (c) Zoomed-in image (left panel) of the boxed region in (b) and its z-stack images (right panel) from z = −0.2 μm to 0.9 μm at an axial step size of 100 nm, showing axially-resolved structural variations of nearby vesicles. (d) Zoomed-in images of the corresponding boxed region in (a) at time-points t = 0.01 s, 1.00 s, 2.00 s, 3.00 s, 4.00 s, and 5.00 s. (e) Zoomed-in image of the boxed region in (d). The inset magnifies the boxed region in (e) spanning several vesicles. (f) Each y-t image (1-3) respectively shows the intensity variations of individual columns of pixels (1-3) in the inset of (e) over five consecutive time-points t = 0.01 s, 0.02 s, 0.03 s, 0.04 s, and 0.05 s. (g) 3D (top), x-y (bottom left) and y-z (bottom right) views of the moving vesicles in (e) at time-points t = 0.01 s (triangle) and 4.00 s (star), resolving vesicles separated as close as 300-500 nm in all three dimensions. Scale bars: 10 μm (a), 2 μm (b), 1 μm (c, d, g), 500 nm (e), 100 nm ((e) inset, f).
Fig. 6
Fig. 6 Tracking nanoparticles at a volume acquisition time of 1 ms using HR-LFM. (a) 3D reconstruction of a representative HGC nanoparticle suspended in water, labeled with Cyanine3. The 3D view was processed by a tricubic-smooth function. (b) Cross-sectional x-y, y-z, and x-z views at t = 1 ms across the center (x, y, z) = (0.875 µm, 0.875 µm, 1.000 µm) of the reconstructed volume in (a). The intensity-information is color-coded according to the color scale bar. (c) Time series (t = 1 to 10 ms) of the lateral (x-y) cross-sectional views of the nanoparticle. The depth-information is color-coded according to the color scale bar. (d) 3D trajectory of the nanoparticle moving below the diffraction limit (<300 nm). Different time-points (t = 1 to 10 ms) are color-coded according to the color scale bar. Scale bars, 500 nm (b, c).
Fig. 7
Fig. 7 Design schematics of conventional LFM (a), focused plenoptic camera (b), and HR-LFM (c). (a) For conventional LFM, the MLA is placed at the NIP of a wide-field microscope (a = 0 and b = fml) [1,2]. Near the NIP, the sampling pattern of the spatial information becomes redundant (i.e. restrained to one microlens). As a result, the wave-optics based model produces prohibitive reconstruction artifacts across the NIP. (b) For the focused plenoptic camera, the MLA forms an imaging relationship between the NIP and the camera (1/a + 1/b = 1/fml) [17]. The sampling geometry of the angular information becomes less widely distributed, inherently impairing the refocusing (or volumetric imaging) capability. (c) For HR-LFM, the MLA is placed at a defocused position (1/a + 1/b > 1/fml) to facilitate simultaneous, dense sampling of both spatial and angular information. fml, the focal length of the MLA; ftl, the focal length of the tube lens; fobj, the effective focal length of the objective lens; a and b denote the distances from the MLA to the NIP and the camera, respectively; TL, tube lens; OL, objective lens; NOP, native object plane; CP, camera plane.
Fig. 8
Fig. 8 Full-width at half-maximum (FWHM) values of the reconstructed point-spread functions (PSFs) at varying depths. (a) The reconstructed cross-sectional images (left columns) and their corresponding profiles along the dashed lines (right columns) of sub-diffraction-limited 100-nm fluorescent beads in lateral (x-y) and axial (x-z and y-z) dimensions at depths (z = 2.1, 1.6, 1.1, 0.8, 0.3, −0.1, −0.5, and −1.0 µm), respectively. (b) The 3D FWHM values of the profiles at varying depths using Gaussian fitting. The reconstructed PSFs exhibited a near-diffraction-limited 300-700 nm resolution in all three dimensions over a >3 μm range. Detailed descriptions of the axial-resolving capability are demonstrated in Appendix 5. (c) Numbers of iterations taken for the reconstruction at varying depths. The numbers were determined based on the distribution of the optical signals across the MLA and the corresponding signal-to-noise ratio (SNR). These numbers were consistently used in this work for other samples. Scale bars, 300 nm.
Fig. 9
Fig. 9 Improved axial-resolving capability of HR-LFM. (a,b) Patterns of the optical signals entering the MLA (a) and at the camera sensor (b) for PSFs at varying depths. Notably, the MLA can segmentally reveal the angular information in the wavefront (i.e. spatial frequencies) of the optical signals, which provides higher sensitivity against fine axial variations. As seen in (b), the PSF of HR-LFM changes significantly for an axial displacement of 300 nm (e.g. at z = 0.7 µm and 1.0 µm), which distribution varies from covering primarily 5 × 5 to 3 × 3 microlenses, respectively. However, such variation was less remarkable in the standard Gaussian PSF (a) over the same displacement of 300 nm. (c) The cross-sectional profiles of the standard Gaussian (left) and the HR-LFM (middle) PSFs at z = 0.7 µm and 1.0 µm, respectively. As seen, the standard Gaussian PSF was moderately expanded by a distance (~250 nm), which is below the diameter of a single microlens (effectively 125 µm / 100 = 1.25 µm in the object domain). In contrast, such an expansion was better recognized by HR-LFM, resulting in a significantly changed intensity pattern on the camera (e.g. at z = 0.7 µm and 1.0 µm, the number of major intensity peaks reduces from 5 to 3, respectively). In addition, at each depth, the corresponding PSF of HR-LFM exhibited distinct ratios between its major intensity peaks. When there are multiple PSFs situated at different axial positions, the camera captures the sum of these PSFs. For example, the right figure in (c) shows the summed intensity pattern of two PSFs located at the same lateral position but different axial positions at z = 0.7 µm and 1.0 µm. While overlapping in the raw data, individual PSFs can be decoupled with the wave-optics based model which convergence considers the prior knowledge of individual PSF patterns at varying depths through iterative deconvolution. (d) Experimental results for imaging two nearby 100-nm fluorescent beads using HR-LFM. The top panel shows the reconstructed lateral cross-sectional images of two resolved beads (1 and 2) at varying depths (left to right, z = 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, and 1.2 µm, respectively). The bottom panel shows the reconstructed axial cross-sectional images in y-z of the two resolved beads at varying x-positions (left to right, x = 0.25, 0.33, 0.41, 0.50, 0.58, 0.67, and 0.75 µm, respectively). (e) 3D rendering of the reconstructed beads. (f,g) Cross-sectional profiles and positions of the two beads in three dimensions. The 3D positions of the beads and the FWHM values of their profiles were determined using Gaussian fitting. As measured, the distance between the two beads was 329 nm, slightly below the diffraction limit in the axial dimension (~ nλ N A 2 = 490 nm, where the refractive index of immersion oil n = 1.515, the emission wavelength λ = 680 nm, and NA = 1.45). This resolving capability was achieved due to the sensitive angular detection using the MLA in HR-LFM. Scale bars, 5 µm (a,b), 500 nm (d).
Fig. 10
Fig. 10 Imaging mitochondria in Drp1−/− mouse embryo fibroblasts (MEFs) using HR-LFM. (a-c) Conventional wide-field image (a), light-field raw image (b), and reconstructed 3D image (c) of mitochondria in Drp1−/− MEFs labeled with MitoTracker acquired with a volume acquisition time of 0.1s and at an axial step size of 100 nm. The depth-information is color-coded according to the color scale bar in (c). (d,e) Zoomed-in images of the boxed regions in (a) (top) and the corresponding boxed regions in (c) (bottom), respectively, showing the high spatial resolution and volumetric imaging capability of HR-LFM. Scale bars, 5 µm (a-c), 2 µm (d,e).
Fig. 11
Fig. 11 Imaging Golgi complex in HeLa cells using HR-LFM. (a) 3D reconstruction of the Golgi complex in HeLa cells. The Golgi structures were visualized using immunofluorescence staining for the Golgi marker GM130 with DyLight 549. The depth-information is color-coded according to the color scale bar. The insets show the cross-sectional images along the dashed lines in x-z and y-z. (b) Left, the zoomed-in image of the boxed region in (a), showing axially-resolved inner structures in the Golgi complex. Right, the cross-sectional profile along the dashed line, showing that axial structures as close as ~400 nm can be resolved. (c) Reconstructed z-stack images at an axial step size of 100 nm, showing fine structural variations captured at various axial positions. (d) Top, zoomed-in image of the boxed region in (c) at z = 1.6 µm. Bottom, the cross-sectional profile along the dashed line shows that lateral structures as close as ~400 nm can be resolved. The measurements are consistent with the FWHM values in Appendix 4. Scale bars, 1 µm (a), 500 nm (b,d), 2 µm (c).
Fig. 12
Fig. 12 Imaging Golgi-derived membrane vesicles in living COS-7 cells using HR-LFM. (a) Reconstructed 3D image of vesicles in living COS-7 cells labeled with mEmerald-Golgi-7 acquired with a volume acquisition time of 0.01 s. The depth-information (over a >3 µm range) is color-coded according to the color scale bar. (b) Zoomed-in image of the corresponding boxed region in (a). (c) 3D view of (b) processed by a tricubic-smooth function. (d) Zoomed-in image of the corresponding boxed region in (a). (e) z-stack images of (d) at an axial step size of 100 nm from z = −2.0 µm to + 1.3 µm. (b-e) show that several vesicles separated less than 1 µm were resolved in all three dimensions. Scale bars: 10 µm (a), 1 µm (b,d,e).
Fig. 13
Fig. 13 Imaging Golgi-derived membrane vesicles in living COS-7 cells at a volume acquisition time of 5 ms using HR-LFM. (a) Reconstructed 3D images of vesicles in living COS-7 cells labeled with mEmerald-Golgi-7 acquired at a volume acquisition time of 5 ms. The left and right are reconstructed 3D images at t = 0.005s and 5.000s of a 1000-time-point series, respectively. The depth-information is color-coded according to the color scale bar. (b) Zoomed-in images of the corresponding boxed region in (a) at t = 0.005s, 3.000s, 3.500s, 4.000s, 4.500s and 5.000s, respectively. White arrows indicate vesicles moving during time-points. (c) Zoomed-in image (leftmost) of the boxed region in (b) at t = 0.005s and its z-stack images from z = 0.2 µm to + 1.3 µm at an axial step size of 100 nm, resolving several nearby vesicles. (d) Zoomed-in images of the corresponding boxed region in (a) at t = 0.005s, 0.500s, 1.000s, 1.500s, 2.000s, 2.500s, 3.000s and 3.500s, respectively. Scale bars: 10 µm (a), 1 µm (b-d).

Equations (5)

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U i ( x,p )= M f obj 2 λ 2 exp[ iu 4 sin 2 ( α/2 ) ]× 0 α P( θ )exp[ iu sin 2 ( θ/2 ) 2 sin 2 ( α/2 ) ] J 0 [ sin( θ ) sin( α ) v ]sin( θ )dθ
U o ( x ' ,p )= i e i 2πa λ λa U i ( x,p )× e iπ λa [ ( x x ' ) 2 + ( y y ' ) 2 ] dxdy
ϕ( x ' )=rect( x ' /d )exp( ik 2 f ml x ' 2 2 )
h( x '' ,p )= F 1 { F[ U o ( x ' ,p )×Φ( x ' ) ]×exp[ i2πλb 1( f x 2 + f y 2 ) ] }
O( x '' )= | h( x '' ,p ) | 2 g( p )dp