Abstract

A conventional microscope produces a sharp image from just a single object-plane. This is often a limitation, notably in cell biology. We present a microscope attachment which records sharp images from several object-planes simultaneously. The key concept is to introduce a distorted diffraction grating into the optical system, establishing an order-dependent focussing power in order to generate several images, each arising from a different object-plane. We exploit this multiplane imaging not just for bio-imaging but also for nano-particle tracking, achieving ~10 nm z position resolution by parameterising the images with an image sharpness metric.

© 2010 OSA

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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2008 (4)

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

J. Rosen and G. Brooker, “Non-scanning motionless fluorescence three-dimensional holographic microscopy,” Nat. Photonics 2(3), 190–195 (2008).
[CrossRef]

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

I. Scott and D. C. Logan, “Mitochondrial morphology transition is an early indicator of subsequent cell death in Arabidopsis,” New Phytol. 177(1), 90–101 (2008).

2007 (3)

L. Holtzer, T. Meckel, and T. Schmidt, “Nanometric three-dimensional tracking of individual quantum dots in cells,” Appl. Phys. Lett. 90(5), 053902–053904 (2007).
[CrossRef]

G. A. Lessard, P. M. Goodwin, and J. H. Werner, “Three-dimensional tracking of individual quantum dots,” Appl. Phys. Lett. 91(22), 224106–224103 (2007).
[CrossRef]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

2006 (2)

2005 (1)

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

2004 (2)

X. Qu, D. Wu, L. Mets, and N. F. Scherer, “Nanometer-localized multiple single-molecule fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 101(31), 11298–11303 (2004).
[CrossRef] [PubMed]

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, “Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions,” IEEE Trans. Nanobioscience 3(4), 237–242 (2004).
[CrossRef]

2003 (3)

2001 (1)

G. Seisenberger, M. U. Ried, T. Endress, H. Büning, M. Hallek, and C. Bräuchle, “Real-time single-molecule imaging of the infection pathway of an adeno-associated virus,” Science 294(5548), 1929–1932 (2001).
[CrossRef] [PubMed]

2000 (2)

1999 (1)

1974 (1)

Allan, V. J.

D. J. Stephens and V. J. Allan, “Light microscopy techniques for live cell imaging,” Science 300(5616), 82–86 (2003).
[CrossRef] [PubMed]

Bennett, B. T.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

Bewersdorf, J.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

Blanchard, P. M.

P. M. Blanchard and A. H. Greenaway, “Broadband simultaneous multiplane imaging,” Opt. Commun. 183(1-4), 29–36 (2000).
[CrossRef]

P. M. Blanchard and A. H. Greenaway, “Simultaneous multiplane imaging with a distorted diffraction grating,” Appl. Opt. 38(32), 6692–6699 (1999).
[CrossRef]

Bräuchle, C.

G. Seisenberger, M. U. Ried, T. Endress, H. Büning, M. Hallek, and C. Bräuchle, “Real-time single-molecule imaging of the infection pathway of an adeno-associated virus,” Science 294(5548), 1929–1932 (2001).
[CrossRef] [PubMed]

Brooker, G.

J. Rosen and G. Brooker, “Non-scanning motionless fluorescence three-dimensional holographic microscopy,” Nat. Photonics 2(3), 190–195 (2008).
[CrossRef]

Buffington, A.

Büning, H.

G. Seisenberger, M. U. Ried, T. Endress, H. Büning, M. Hallek, and C. Bräuchle, “Real-time single-molecule imaging of the infection pathway of an adeno-associated virus,” Science 294(5548), 1929–1932 (2001).
[CrossRef] [PubMed]

Campbell, H. I.

Cole, M. J.

Djidel, S.

Dowling, K.

Eisenstein, M.

M. Eisenstein, “Something to see,” Nature 443(7114), 1017–1021 (2006).
[PubMed]

Endress, T.

G. Seisenberger, M. U. Ried, T. Endress, H. Büning, M. Hallek, and C. Bräuchle, “Real-time single-molecule imaging of the infection pathway of an adeno-associated virus,” Science 294(5548), 1929–1932 (2001).
[CrossRef] [PubMed]

Florin, E. L.

French, P. M. W.

Gansel, J. K.

Goodwin, P. M.

G. A. Lessard, P. M. Goodwin, and J. H. Werner, “Three-dimensional tracking of individual quantum dots,” Appl. Phys. Lett. 91(22), 224106–224103 (2007).
[CrossRef]

Gould, T. J.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

Greenaway, A. H.

Greger, K.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Gustafsson, M. G. L.

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

Hallek, M.

G. Seisenberger, M. U. Ried, T. Endress, H. Büning, M. Hallek, and C. Bräuchle, “Real-time single-molecule imaging of the infection pathway of an adeno-associated virus,” Science 294(5548), 1929–1932 (2001).
[CrossRef] [PubMed]

Hell, S. W.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Hess, S. T.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

Holtzer, L.

L. Holtzer, T. Meckel, and T. Schmidt, “Nanometric three-dimensional tracking of individual quantum dots in cells,” Appl. Phys. Lett. 90(5), 053902–053904 (2007).
[CrossRef]

Jahn, R.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Jericho, M. H.

Jonás, A.

Jones, R.

Juette, M. F.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

Juskaitis, R.

Kamin, D.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Kreuzer, H. J.

Lauterbach, M. A.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Lessard, G. A.

G. A. Lessard, P. M. Goodwin, and J. H. Werner, “Three-dimensional tracking of individual quantum dots,” Appl. Phys. Lett. 91(22), 224106–224103 (2007).
[CrossRef]

Lessard, M. D.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

Lever, M. J.

Logan, D. C.

I. Scott and D. C. Logan, “Mitochondrial morphology transition is an early indicator of subsequent cell death in Arabidopsis,” New Phytol. 177(1), 90–101 (2008).

Marcello, M.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Meckel, T.

L. Holtzer, T. Meckel, and T. Schmidt, “Nanometric three-dimensional tracking of individual quantum dots in cells,” Appl. Phys. Lett. 90(5), 053902–053904 (2007).
[CrossRef]

Meinertzhagen, I. A.

Mets, L.

X. Qu, D. Wu, L. Mets, and N. F. Scherer, “Nanometer-localized multiple single-molecule fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 101(31), 11298–11303 (2004).
[CrossRef] [PubMed]

Mlodzianoski, M. J.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

Muller, R. A.

Nagpure, B. S.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

Neil, M. A. A.

Ober, R. J.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, “Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions,” IEEE Trans. Nanobioscience 3(4), 237–242 (2004).
[CrossRef]

Pampaloni, F.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Prabhat, P.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, “Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions,” IEEE Trans. Nanobioscience 3(4), 237–242 (2004).
[CrossRef]

Qu, X.

X. Qu, D. Wu, L. Mets, and N. F. Scherer, “Nanometer-localized multiple single-molecule fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 101(31), 11298–11303 (2004).
[CrossRef] [PubMed]

Ram, S.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, “Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions,” IEEE Trans. Nanobioscience 3(4), 237–242 (2004).
[CrossRef]

Ried, M. U.

G. Seisenberger, M. U. Ried, T. Endress, H. Büning, M. Hallek, and C. Bräuchle, “Real-time single-molecule imaging of the infection pathway of an adeno-associated virus,” Science 294(5548), 1929–1932 (2001).
[CrossRef] [PubMed]

Rizzoli, S. O.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Rosen, J.

J. Rosen and G. Brooker, “Non-scanning motionless fluorescence three-dimensional holographic microscopy,” Nat. Photonics 2(3), 190–195 (2008).
[CrossRef]

Scherer, N. F.

X. Qu, D. Wu, L. Mets, and N. F. Scherer, “Nanometer-localized multiple single-molecule fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 101(31), 11298–11303 (2004).
[CrossRef] [PubMed]

Schmidt, T.

L. Holtzer, T. Meckel, and T. Schmidt, “Nanometric three-dimensional tracking of individual quantum dots in cells,” Appl. Phys. Lett. 90(5), 053902–053904 (2007).
[CrossRef]

Scott, I.

I. Scott and D. C. Logan, “Mitochondrial morphology transition is an early indicator of subsequent cell death in Arabidopsis,” New Phytol. 177(1), 90–101 (2008).

Seisenberger, G.

G. Seisenberger, M. U. Ried, T. Endress, H. Büning, M. Hallek, and C. Bräuchle, “Real-time single-molecule imaging of the infection pathway of an adeno-associated virus,” Science 294(5548), 1929–1932 (2001).
[CrossRef] [PubMed]

Siegel, J.

Speidel, M.

Stelzer, E. H. K.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Stephens, D. J.

D. J. Stephens and V. J. Allan, “Light microscopy techniques for live cell imaging,” Science 300(5616), 82–86 (2003).
[CrossRef] [PubMed]

Sucharov, L. O. D.

Swoger, J.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Verveer, P. J.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Ward, E. S.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, “Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions,” IEEE Trans. Nanobioscience 3(4), 237–242 (2004).
[CrossRef]

Webb, S. E. D.

Werner, J. H.

G. A. Lessard, P. M. Goodwin, and J. H. Werner, “Three-dimensional tracking of individual quantum dots,” Appl. Phys. Lett. 91(22), 224106–224103 (2007).
[CrossRef]

Westphal, V.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Wilson, T.

Wu, D.

X. Qu, D. Wu, L. Mets, and N. F. Scherer, “Nanometer-localized multiple single-molecule fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 101(31), 11298–11303 (2004).
[CrossRef] [PubMed]

Xu, W.

Appl. Opt. (1)

Appl. Phys. Lett. (2)

L. Holtzer, T. Meckel, and T. Schmidt, “Nanometric three-dimensional tracking of individual quantum dots in cells,” Appl. Phys. Lett. 90(5), 053902–053904 (2007).
[CrossRef]

G. A. Lessard, P. M. Goodwin, and J. H. Werner, “Three-dimensional tracking of individual quantum dots,” Appl. Phys. Lett. 91(22), 224106–224103 (2007).
[CrossRef]

IEEE Trans. Nanobioscience (1)

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, “Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions,” IEEE Trans. Nanobioscience 3(4), 237–242 (2004).
[CrossRef]

J. Opt. Soc. Am. (1)

Nat. Methods (2)

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007).
[PubMed]

Nat. Photonics (1)

J. Rosen and G. Brooker, “Non-scanning motionless fluorescence three-dimensional holographic microscopy,” Nat. Photonics 2(3), 190–195 (2008).
[CrossRef]

Nature (1)

M. Eisenstein, “Something to see,” Nature 443(7114), 1017–1021 (2006).
[PubMed]

New Phytol. (1)

I. Scott and D. C. Logan, “Mitochondrial morphology transition is an early indicator of subsequent cell death in Arabidopsis,” New Phytol. 177(1), 90–101 (2008).

Opt. Commun. (1)

P. M. Blanchard and A. H. Greenaway, “Broadband simultaneous multiplane imaging,” Opt. Commun. 183(1-4), 29–36 (2000).
[CrossRef]

Opt. Express (1)

Opt. Lett. (3)

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

X. Qu, D. Wu, L. Mets, and N. F. Scherer, “Nanometer-localized multiple single-molecule fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 101(31), 11298–11303 (2004).
[CrossRef] [PubMed]

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

Science (3)

D. J. Stephens and V. J. Allan, “Light microscopy techniques for live cell imaging,” Science 300(5616), 82–86 (2003).
[CrossRef] [PubMed]

G. Seisenberger, M. U. Ried, T. Endress, H. Büning, M. Hallek, and C. Bräuchle, “Real-time single-molecule imaging of the infection pathway of an adeno-associated virus,” Science 294(5548), 1929–1932 (2001).
[CrossRef] [PubMed]

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

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Figures (4)

Fig. 1
Fig. 1

Schematic of the microscope and grating operation. (A) The custom built wide-field 100x fluorescence microscope. Sample illumination is either from an interchangeable source below the sample or for fluorescence studies, fibre-launched excitation light from above. (B) A 1:1 relay system allows the QD grating to be positioned in the telecentric position. (C) Operational schematic of the QD grating. Multiple object-planes, separated by Δz, are simultaneously imaged onto a single image-plane, spatially separated by Δd.

Fig. 2
Fig. 2

(A) A single nano-hole imaged as a function of defocus position using the QD grating system and 633 nm illumination. Each image corresponds to an area of 6x6 μm2 (0.63x0.63 mm2) in the object (image) plane. (B), (C) and (D) mark the focal points for the −1, 0th and + 1 orders, respectively. (E), (F) and (G) show cross-sectional slices through each of these respective images along with Gaussian fits to the data.

Fig. 3
Fig. 3

(A) A single Arabidopsis protoplast imaged in bright field using 510 nm wavelength LED illumination. (B) Artificially colored image showing 510 nm wavelength fluorescence from mitochondrial-targeted GFP. The illumination is provided by a 470 nm wavelength laser. The grating gives an object-plane separation of 0.9 μm at 510 nm wavelength.

Fig. 4
Fig. 4

(A) The image sharpness from each diffraction order as a function of defocus. At each defocus position 50 separate images (exposure time 0.5 ms) were taken at 100 msec intervals. Plotted is the average sharpness. (B) The standard deviation of the maximum likelihood estimate analysis of the sharpness data from 50 images at each z position.

Metrics