Abstract

Maximizing the amount of spatiotemporal information retrieved in confocal laser scanning microscopy is crucial to understand fundamental three-dimensional (3D) dynamic processes in life sciences. However, current 3D confocal microscopy is based on an inherently slow stepwise process that consists of acquiring multiple 2D sections at different focal planes by mechanical or optical z-focus translation. Here, we show that by using an acoustically-driven optofluidic lens integrated in a commercial confocal system we can capture an entire 3D image in a single step. Our method is based on continuous axial scanning at speeds as high as 140 kHz combined with fast readout. In this way, one or more focus sweeps are produced on a pixel by pixel basis and the detected photons can be assigned to their corresponding focal plane enabling simultaneous multiplane imaging. We exemplify this method by imaging calibration and biological fluorescence samples. These results open the door to exploring new fundamental processes in science with an unprecedented time resolution.

© 2014 Optical Society of America

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References

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    [CrossRef] [PubMed]

2014 (2)

2013 (3)

2012 (3)

W. Song, A. E. Vasdekis, and D. Psaltis, “Elastomer based tunable optofluidic devices,” Lab Chip 12(19), 3590–3597 (2012).
[CrossRef] [PubMed]

M. Duocastella, B. Sun, and C. B. Arnold, “Simultaneous imaging of multiple focal planes for three-dimensional microscopy using ultra-high-speed adaptive optics,” J. Biomed. Opt. 17(5), 050505 (2012).
[CrossRef] [PubMed]

F. P. Martial and N. A. Hartell, “Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror,” PLoS ONE 7(8), e43942 (2012).
[CrossRef] [PubMed]

2011 (2)

B. F. Grewe, F. F. Voigt, M. van ’t Hoff, and F. Helmchen, “Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens,” Biomed. Opt. Express 2(7), 2035–2046 (2011).
[CrossRef] [PubMed]

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8(2), 139–142 (2011).
[CrossRef] [PubMed]

2010 (2)

N.-T. Nguyen, “Micro-optofluidic lenses: a review,” Biomicrofluidics 4(3), 031501 (2010).
[CrossRef] [PubMed]

B. Schmid, J. Schindelin, A. Cardona, M. Longair, and M. Heisenberg, “A high-level 3D visualization API for Java and ImageJ,” BMC Bioinformatics 11(1), 274 (2010).
[CrossRef] [PubMed]

2009 (2)

2008 (4)

A. Mermillod-Blondin, E. McLeod, and C. B. Arnold, “High-speed varifocal imaging with a tunable acoustic gradient index of refraction lens,” Opt. Lett. 33(18), 2146–2148 (2008).
[CrossRef] [PubMed]

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[CrossRef] [PubMed]

E. J. Botcherby, M. J. Booth, R. Juskaitis, and T. Wilson, “Real-time extended depth of field microscopy,” Opt. Express 16(26), 21843–21848 (2008).
[CrossRef] [PubMed]

U. Levy and R. Shamai, “Tunable optofluidic devices,” Microfluid. Nanofluidics 4(1–2), 97–105 (2008).
[CrossRef]

2007 (2)

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[CrossRef] [PubMed]

W. Amir, R. Carriles, E. E. Hoover, T. A. Planchon, C. G. Durfee, and J. A. Squier, “Simultaneous imaging of multiple focal planes using a two-photon scanning microscope,” Opt. Lett. 32(12), 1731–1733 (2007).
[CrossRef] [PubMed]

1999 (1)

N. Callamaras and I. Parker, “Construction of a confocal microscope for real-time x-y and x-z imaging,” Cell Calcium 26(6), 271–279 (1999).
[CrossRef] [PubMed]

1996 (1)

A. Diaspro, F. Beltrame, M. Fato, and P. Ramoino, “Characterizing biostructures and cellular events in 2D/3D,” IEE Eng. Med. Biol. 15(1), 92–100 (1996).
[CrossRef]

1995 (1)

Amir, W.

Arisaka, K.

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8(2), 139–142 (2011).
[CrossRef] [PubMed]

Arnold, C. B.

M. Duocastella and C. B. Arnold, “Enhanced depth of field laser processing using an ultra-high-speed axial scanner,” Appl. Phys. Lett. 102(6), 061113 (2013).
[CrossRef]

M. Duocastella, B. Sun, and C. B. Arnold, “Simultaneous imaging of multiple focal planes for three-dimensional microscopy using ultra-high-speed adaptive optics,” J. Biomed. Opt. 17(5), 050505 (2012).
[CrossRef] [PubMed]

N. Olivier, A. Mermillod-Blondin, C. B. Arnold, and E. Beaurepaire, “Two-photon microscopy with simultaneous standard and extended depth of field using a tunable acoustic gradient-index lens,” Opt. Lett. 34(11), 1684–1686 (2009).
[CrossRef] [PubMed]

A. Mermillod-Blondin, E. McLeod, and C. B. Arnold, “High-speed varifocal imaging with a tunable acoustic gradient index of refraction lens,” Opt. Lett. 33(18), 2146–2148 (2008).
[CrossRef] [PubMed]

Basiji, D. A.

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[CrossRef] [PubMed]

Beaurepaire, E.

Beltrame, F.

A. Diaspro, F. Beltrame, M. Fato, and P. Ramoino, “Characterizing biostructures and cellular events in 2D/3D,” IEE Eng. Med. Biol. 15(1), 92–100 (1996).
[CrossRef]

Booth, M. J.

Botcherby, E. J.

Callamaras, N.

N. Callamaras and I. Parker, “Construction of a confocal microscope for real-time x-y and x-z imaging,” Cell Calcium 26(6), 271–279 (1999).
[CrossRef] [PubMed]

Cardona, A.

B. Schmid, J. Schindelin, A. Cardona, M. Longair, and M. Heisenberg, “A high-level 3D visualization API for Java and ImageJ,” BMC Bioinformatics 11(1), 274 (2010).
[CrossRef] [PubMed]

Carriles, R.

Cathey, W. T.

Chen, H. S.

Cheng, A.

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8(2), 139–142 (2011).
[CrossRef] [PubMed]

Cheng, S.

Cheng, Y.-S. L.

Cuenca, R.

Czarske, J. W.

Diaspro, A.

A. Diaspro, F. Beltrame, M. Fato, and P. Ramoino, “Characterizing biostructures and cellular events in 2D/3D,” IEE Eng. Med. Biol. 15(1), 92–100 (1996).
[CrossRef]

Dowski, E. R.

Duemani Reddy, G.

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[CrossRef] [PubMed]

Duocastella, M.

M. Duocastella and C. B. Arnold, “Enhanced depth of field laser processing using an ultra-high-speed axial scanner,” Appl. Phys. Lett. 102(6), 061113 (2013).
[CrossRef]

M. Duocastella, B. Sun, and C. B. Arnold, “Simultaneous imaging of multiple focal planes for three-dimensional microscopy using ultra-high-speed adaptive optics,” J. Biomed. Opt. 17(5), 050505 (2012).
[CrossRef] [PubMed]

Durfee, C. G.

Fahrbach, F. O.

Fato, M.

A. Diaspro, F. Beltrame, M. Fato, and P. Ramoino, “Characterizing biostructures and cellular events in 2D/3D,” IEE Eng. Med. Biol. 15(1), 92–100 (1996).
[CrossRef]

Fink, R.

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[CrossRef] [PubMed]

Finkeldey, M.

Fischer, A.

Frost, K.

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[CrossRef] [PubMed]

Gerhardt, N. C.

Golshani, P.

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8(2), 139–142 (2011).
[CrossRef] [PubMed]

Gonçalves, J. T.

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8(2), 139–142 (2011).
[CrossRef] [PubMed]

Grewe, B. F.

Hall, B. E.

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[CrossRef] [PubMed]

Hartell, N. A.

F. P. Martial and N. A. Hartell, “Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror,” PLoS ONE 7(8), e43942 (2012).
[CrossRef] [PubMed]

Heisenberg, M.

B. Schmid, J. Schindelin, A. Cardona, M. Longair, and M. Heisenberg, “A high-level 3D visualization API for Java and ImageJ,” BMC Bioinformatics 11(1), 274 (2010).
[CrossRef] [PubMed]

Helmchen, F.

Hofmann, M. R.

Hoover, E. E.

Huisken, J.

Ishikawa, M.

H. Oku and M. Ishikawa, “High-speed liquid lens with 2 ms response and 80.3 nm root-mean-square wavefront error,” Appl. Phys. Lett. 94(22), 221108 (2009).
[CrossRef]

Jabbour, J. M.

Jo, J. A.

Juskaitis, R.

Kelleher, K.

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[CrossRef] [PubMed]

Koukourakis, N.

Leithold, C.

Levy, U.

U. Levy and R. Shamai, “Tunable optofluidic devices,” Microfluid. Nanofluidics 4(1–2), 97–105 (2008).
[CrossRef]

Liang, L.

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[CrossRef] [PubMed]

Lin, Y. H.

Longair, M.

B. Schmid, J. Schindelin, A. Cardona, M. Longair, and M. Heisenberg, “A high-level 3D visualization API for Java and ImageJ,” BMC Bioinformatics 11(1), 274 (2010).
[CrossRef] [PubMed]

Maitland, K. C.

Malik, B. H.

Martial, F. P.

F. P. Martial and N. A. Hartell, “Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror,” PLoS ONE 7(8), e43942 (2012).
[CrossRef] [PubMed]

McLeod, E.

Mermillod-Blondin, A.

Nguyen, N.-T.

N.-T. Nguyen, “Micro-optofluidic lenses: a review,” Biomicrofluidics 4(3), 031501 (2010).
[CrossRef] [PubMed]

Oku, H.

H. Oku and M. Ishikawa, “High-speed liquid lens with 2 ms response and 80.3 nm root-mean-square wavefront error,” Appl. Phys. Lett. 94(22), 221108 (2009).
[CrossRef]

Olivier, N.

Olsovsky, C.

Ortyn, W. E.

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[CrossRef] [PubMed]

Parker, I.

N. Callamaras and I. Parker, “Construction of a confocal microscope for real-time x-y and x-z imaging,” Cell Calcium 26(6), 271–279 (1999).
[CrossRef] [PubMed]

Perry, D. J.

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[CrossRef] [PubMed]

Planchon, T. A.

Portera-Cailliau, C.

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8(2), 139–142 (2011).
[CrossRef] [PubMed]

Psaltis, D.

W. Song, A. E. Vasdekis, and D. Psaltis, “Elastomer based tunable optofluidic devices,” Lab Chip 12(19), 3590–3597 (2012).
[CrossRef] [PubMed]

Ramoino, P.

A. Diaspro, F. Beltrame, M. Fato, and P. Ramoino, “Characterizing biostructures and cellular events in 2D/3D,” IEE Eng. Med. Biol. 15(1), 92–100 (1996).
[CrossRef]

Saggau, P.

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[CrossRef] [PubMed]

Schindelin, J.

B. Schmid, J. Schindelin, A. Cardona, M. Longair, and M. Heisenberg, “A high-level 3D visualization API for Java and ImageJ,” BMC Bioinformatics 11(1), 274 (2010).
[CrossRef] [PubMed]

Schmid, B.

F. O. Fahrbach, F. F. Voigt, B. Schmid, F. Helmchen, and J. Huisken, “Rapid 3D light-sheet microscopy with a tunable lens,” Opt. Express 21(18), 21010–21026 (2013).
[CrossRef] [PubMed]

B. Schmid, J. Schindelin, A. Cardona, M. Longair, and M. Heisenberg, “A high-level 3D visualization API for Java and ImageJ,” BMC Bioinformatics 11(1), 274 (2010).
[CrossRef] [PubMed]

Shamai, R.

U. Levy and R. Shamai, “Tunable optofluidic devices,” Microfluid. Nanofluidics 4(1–2), 97–105 (2008).
[CrossRef]

Song, W.

W. Song, A. E. Vasdekis, and D. Psaltis, “Elastomer based tunable optofluidic devices,” Lab Chip 12(19), 3590–3597 (2012).
[CrossRef] [PubMed]

Squier, J. A.

Stürmer, M.

Sun, B.

M. Duocastella, B. Sun, and C. B. Arnold, “Simultaneous imaging of multiple focal planes for three-dimensional microscopy using ultra-high-speed adaptive optics,” J. Biomed. Opt. 17(5), 050505 (2012).
[CrossRef] [PubMed]

van ’t Hoff, M.

Vasdekis, A. E.

W. Song, A. E. Vasdekis, and D. Psaltis, “Elastomer based tunable optofluidic devices,” Lab Chip 12(19), 3590–3597 (2012).
[CrossRef] [PubMed]

Venkatachalam, V.

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[CrossRef] [PubMed]

Voigt, F. F.

Wallrabe, U.

Wilson, T.

Wright, J. M.

Appl. Opt. (1)

Appl. Phys. Lett. (2)

M. Duocastella and C. B. Arnold, “Enhanced depth of field laser processing using an ultra-high-speed axial scanner,” Appl. Phys. Lett. 102(6), 061113 (2013).
[CrossRef]

H. Oku and M. Ishikawa, “High-speed liquid lens with 2 ms response and 80.3 nm root-mean-square wavefront error,” Appl. Phys. Lett. 94(22), 221108 (2009).
[CrossRef]

Biomed. Opt. Express (2)

Biomicrofluidics (1)

N.-T. Nguyen, “Micro-optofluidic lenses: a review,” Biomicrofluidics 4(3), 031501 (2010).
[CrossRef] [PubMed]

BMC Bioinformatics (1)

B. Schmid, J. Schindelin, A. Cardona, M. Longair, and M. Heisenberg, “A high-level 3D visualization API for Java and ImageJ,” BMC Bioinformatics 11(1), 274 (2010).
[CrossRef] [PubMed]

Cell Calcium (1)

N. Callamaras and I. Parker, “Construction of a confocal microscope for real-time x-y and x-z imaging,” Cell Calcium 26(6), 271–279 (1999).
[CrossRef] [PubMed]

Cytometry A (1)

W. E. Ortyn, D. J. Perry, V. Venkatachalam, L. Liang, B. E. Hall, K. Frost, and D. A. Basiji, “Extended depth of field imaging for high speed cell analysis,” Cytometry A 71(4), 215–231 (2007).
[CrossRef] [PubMed]

IEE Eng. Med. Biol. (1)

A. Diaspro, F. Beltrame, M. Fato, and P. Ramoino, “Characterizing biostructures and cellular events in 2D/3D,” IEE Eng. Med. Biol. 15(1), 92–100 (1996).
[CrossRef]

J. Biomed. Opt. (1)

M. Duocastella, B. Sun, and C. B. Arnold, “Simultaneous imaging of multiple focal planes for three-dimensional microscopy using ultra-high-speed adaptive optics,” J. Biomed. Opt. 17(5), 050505 (2012).
[CrossRef] [PubMed]

Lab Chip (1)

W. Song, A. E. Vasdekis, and D. Psaltis, “Elastomer based tunable optofluidic devices,” Lab Chip 12(19), 3590–3597 (2012).
[CrossRef] [PubMed]

Microfluid. Nanofluidics (1)

U. Levy and R. Shamai, “Tunable optofluidic devices,” Microfluid. Nanofluidics 4(1–2), 97–105 (2008).
[CrossRef]

Nat. Methods (1)

A. Cheng, J. T. Gonçalves, P. Golshani, K. Arisaka, and C. Portera-Cailliau, “Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing,” Nat. Methods 8(2), 139–142 (2011).
[CrossRef] [PubMed]

Nat. Neurosci. (1)

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[CrossRef] [PubMed]

Opt. Express (4)

Opt. Lett. (3)

PLoS ONE (1)

F. P. Martial and N. A. Hartell, “Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror,” PLoS ONE 7(8), e43942 (2012).
[CrossRef] [PubMed]

Other (3)

A. Diaspro, Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances (Wiley-Liss, 2002).

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

H. Oku and M. Ishikawa, “A variable-focus lens with 1kHz bandwidth applied to axial-scan of a confocal scanning microscope,” in Lasers Electro-Optics Soc. 2003. LEOS 2003 (2003), Vol. 1, pp. 309–310.

Supplementary Material (1)

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Fig. 1
Fig. 1

Principle of operation of the high-speed z-scanning confocal microscope. a) Due to the high axial scanning speed enabled by the optofluidic lens, several axial scans are recorded within a pixel exposure. b) The sum of all photons providing from different focal planes forms an image with an extended depth of field (EDOF). c) By using a fast acquisition card and appropriate synchronization, photons arriving at different times can be sorted according to their corresponding focal plane. Such spatiotemporal multiplexing results in a user-selectable number of focal planes.

Fig. 2
Fig. 2

Scheme of the high-speed z-scanning confocal microscope. The acoustically driven optofluidic lens (TAG lens) is placed at the back focal aperture of the objective of a commercial microscope, enabling light modulation in both excitation and detection paths.

Fig. 3
Fig. 3

Optical performance of the high-speed z-scanning confocal microscope. a) Plot of the normalized intensity of a thin fluorescence sample at different axial positions for different driving voltages. By changing the voltage amplitude applied to the TAG lens, the full width at half maximum (FWHM) of the axial intensity can be modified, demonstrating a user-controllable EDOF. b) Plot of the FWHM versus TAG lens driving voltage. c) Multi-plane imaging by spatiotemporal multiplexing. The information of the photons arrival time with respect to the TAG lens oscillation is used to group the collected photons (thick line) into arbitrarily defined time intervals (thin lines). Such intervals correspond to different slices or focal planes. d) Plot of the different slices with respect to their axial position. Since the TAG lens produces a sinusoidal scanning, we first select 20 time intervals (dots) that we then regroup according to their axial position into 8 different slices (colored bars) in order to enhance the SNR.

Fig. 4
Fig. 4

EDOF confocal microscopy of a Convallaria majalis sample. a) When the TAG lens is off, only an optical section is captured. b-c) As the voltage applied to the TAG lens increases, the DOF increases. In this way, details that were initially at different focal planes and thereby remained hidden, become visible. d) Plot of the intensity across the dotted line in Figs. 3(a)3(c). The arrow indicates the increase in intensity for EDOF microscopy for an object initially situated out of focus.

Fig. 5
Fig. 5

Multiplane imaging confocal microscopy of a Convallaria majalis sample. a) Conventional confocal image of a thin optical section of a biological sample. This image is obtained by mechanically scanning the laser beam in the XY plane using galvanometric mirrors. b) By high-speed z-scanning confocal microscopy (TAG lens at 10 Vpp) and appropriate time sorting, a single XY scan results in 8 different images each corresponding to a different focal plane. c) Comparison of the images obtained with a traditional z-stage and those of high-speed z-scanning confocal microscopy. d) Cross-correlation coefficient of the images obtained using the two methods. A comparison between the different axial planes and slice 5 is also included as a reference to show how rapidly the structure decorrelates with depth. The difference between both curves has been highlighted in gray. e) Video frame of the 3D image reconstructed using the high-speed z-scanning confocal microscope (Media 1). Scale bars are 20 µm.

Equations (1)

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n photons I×tim e pixel × n pixe l x × n pixe l y

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