Abstract

High-speed 3D microscopic imaging methods have led to numerous biological discoveries. In this Letter, we present a sync-free light sheet microscope (LSM) based on a static phase mask that eliminates the need for coupling the detection plane, thereby enabling high-speed volumetric imaging that is only limited by the speed of cameras, e.g., 1000 cross sections/s. In the sync-free design, the emission signals are first guided to the back of a galvanometric mirror, which laterally scans the emissions across a 2D phase mask, converting it to axial scanning that automatically compensates the focal shifts in the detection optics. Parametric models are developed to guide the phase mask design as well as to relate the axial scanning depth and magnification to design parameters. To quickly evaluate the different mask designs, a liquid-crystal-based spatial light modulator (LC-SLM) is used in the system. In the experiments, we scanned pollen and tissue samples via both the 2D phase mask and a piezoelectric objective scanner. The results show that the new method can generate clear images with comparable quality throughout the scanning range. The overall efficiency of the LSM is 10%. It is worthwhile to note that the efficiency will be significantly improved by replacing the LC-SLM with a custom-made lens. The new method realizes a compact LSM for high-speed 3D imaging that may find important applications in in vivo biological imaging.

© 2017 Optical Society of America

Full Article  |  PDF Article
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References

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2015 (4)

W. C. Lemon, S. R. Pulver, B. Höckendorf, K. McDole, K. Branson, J. Freeman, and P. J. Keller, Nat. Commun. 6, 7924 (2015).
[Crossref]

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, Cell 163, 1796 (2015).
[Crossref]

M. B. Bouchard, V. Voleti, C. S. Mendes, C. Lacefield, W. B. Grueber, R. S. Mann, R. M. Bruno, and E. M. C. Hillman, Nat. Photonics 9, 113 (2015).
[Crossref]

O. E. Olarte, J. Andilla, D. Artigas, and P. Loza-Alvarez, Optica 2, 702 (2015).
[Crossref]

2013 (2)

F. O. Fahrbach, F. F. Voigt, B. Schmid, F. Helmchen, and J. Huisken, Opt. Express 21, 21010 (2013).
[Crossref]

M. B. Ahrens, M. B. Orger, D. N. Robson, J. M. Li, and P. J. Keller, Nat. Methods 10, 413 (2013).
[Crossref]

2011 (1)

R. Tomer, K. Khairy, and P. J. Keller, Curr. Opin. Genet. Dev. 21, 558 (2011).
[Crossref]

2010 (1)

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, Nat. Methods 7, 637 (2010).
[Crossref]

2008 (4)

E. G. Reynaud, U. Krzic, K. Greger, and E. H. K. Stelzer, HFSP J. 2, 266 (2008).
[Crossref]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, Science 322, 1065 (2008).
[Crossref]

T. F. Holekamp, D. Turaga, and T. E. Holy, Neuron 57, 661 (2008).
[Crossref]

K. Jaqaman, D. Loerke, M. Mettlen, H. Kuwata, S. Grinstein, S. L. Schmid, and G. Danuser, Nat. Methods 5, 695 (2008).
[Crossref]

2006 (1)

P. J. Keller, F. Pampaloni, and E. H. Stelzer, Curr. Opin. Cell Biol. 18, 117 (2006).
[Crossref]

2004 (1)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[Crossref]

Ahrens, M. B.

M. B. Ahrens, M. B. Orger, D. N. Robson, J. M. Li, and P. J. Keller, Nat. Methods 10, 413 (2013).
[Crossref]

Andalman, A.

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, Cell 163, 1796 (2015).
[Crossref]

Andilla, J.

Artigas, D.

Bao, Z.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, Nat. Methods 7, 637 (2010).
[Crossref]

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, Nat. Photonics 9, 113 (2015).
[Crossref]

Branson, K.

W. C. Lemon, S. R. Pulver, B. Höckendorf, K. McDole, K. Branson, J. Freeman, and P. J. Keller, Nat. Commun. 6, 7924 (2015).
[Crossref]

Broxton, M.

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, Cell 163, 1796 (2015).
[Crossref]

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, Nat. Photonics 9, 113 (2015).
[Crossref]

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, Cell 163, 1796 (2015).
[Crossref]

Danuser, G.

K. Jaqaman, D. Loerke, M. Mettlen, H. Kuwata, S. Grinstein, S. L. Schmid, and G. Danuser, Nat. Methods 5, 695 (2008).
[Crossref]

Deisseroth, K.

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, Cell 163, 1796 (2015).
[Crossref]

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[Crossref]

Fahrbach, F. O.

Freeman, J.

W. C. Lemon, S. R. Pulver, B. Höckendorf, K. McDole, K. Branson, J. Freeman, and P. J. Keller, Nat. Commun. 6, 7924 (2015).
[Crossref]

Greger, K.

E. G. Reynaud, U. Krzic, K. Greger, and E. H. K. Stelzer, HFSP J. 2, 266 (2008).
[Crossref]

Grinstein, S.

K. Jaqaman, D. Loerke, M. Mettlen, H. Kuwata, S. Grinstein, S. L. Schmid, and G. Danuser, Nat. Methods 5, 695 (2008).
[Crossref]

Grosenick, L.

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, Cell 163, 1796 (2015).
[Crossref]

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, Nat. Photonics 9, 113 (2015).
[Crossref]

Helmchen, F.

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, Nat. Photonics 9, 113 (2015).
[Crossref]

Höckendorf, B.

W. C. Lemon, S. R. Pulver, B. Höckendorf, K. McDole, K. Branson, J. Freeman, and P. J. Keller, Nat. Commun. 6, 7924 (2015).
[Crossref]

Holekamp, T. F.

T. F. Holekamp, D. Turaga, and T. E. Holy, Neuron 57, 661 (2008).
[Crossref]

Holy, T. E.

T. F. Holekamp, D. Turaga, and T. E. Holy, Neuron 57, 661 (2008).
[Crossref]

Huisken, J.

F. O. Fahrbach, F. F. Voigt, B. Schmid, F. Helmchen, and J. Huisken, Opt. Express 21, 21010 (2013).
[Crossref]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[Crossref]

Jaqaman, K.

K. Jaqaman, D. Loerke, M. Mettlen, H. Kuwata, S. Grinstein, S. L. Schmid, and G. Danuser, Nat. Methods 5, 695 (2008).
[Crossref]

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, Cell 163, 1796 (2015).
[Crossref]

Keller, P. J.

W. C. Lemon, S. R. Pulver, B. Höckendorf, K. McDole, K. Branson, J. Freeman, and P. J. Keller, Nat. Commun. 6, 7924 (2015).
[Crossref]

M. B. Ahrens, M. B. Orger, D. N. Robson, J. M. Li, and P. J. Keller, Nat. Methods 10, 413 (2013).
[Crossref]

R. Tomer, K. Khairy, and P. J. Keller, Curr. Opin. Genet. Dev. 21, 558 (2011).
[Crossref]

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, Nat. Methods 7, 637 (2010).
[Crossref]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, Science 322, 1065 (2008).
[Crossref]

P. J. Keller, F. Pampaloni, and E. H. Stelzer, Curr. Opin. Cell Biol. 18, 117 (2006).
[Crossref]

Khairy, K.

R. Tomer, K. Khairy, and P. J. Keller, Curr. Opin. Genet. Dev. 21, 558 (2011).
[Crossref]

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, Nat. Methods 7, 637 (2010).
[Crossref]

Krzic, U.

E. G. Reynaud, U. Krzic, K. Greger, and E. H. K. Stelzer, HFSP J. 2, 266 (2008).
[Crossref]

Kuwata, H.

K. Jaqaman, D. Loerke, M. Mettlen, H. Kuwata, S. Grinstein, S. L. Schmid, and G. Danuser, Nat. Methods 5, 695 (2008).
[Crossref]

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, Nat. Photonics 9, 113 (2015).
[Crossref]

Lemon, W. C.

W. C. Lemon, S. R. Pulver, B. Höckendorf, K. McDole, K. Branson, J. Freeman, and P. J. Keller, Nat. Commun. 6, 7924 (2015).
[Crossref]

Li, J. M.

M. B. Ahrens, M. B. Orger, D. N. Robson, J. M. Li, and P. J. Keller, Nat. Methods 10, 413 (2013).
[Crossref]

Loerke, D.

K. Jaqaman, D. Loerke, M. Mettlen, H. Kuwata, S. Grinstein, S. L. Schmid, and G. Danuser, Nat. Methods 5, 695 (2008).
[Crossref]

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, Cell 163, 1796 (2015).
[Crossref]

Loza-Alvarez, P.

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, Nat. Photonics 9, 113 (2015).
[Crossref]

McDole, K.

W. C. Lemon, S. R. Pulver, B. Höckendorf, K. McDole, K. Branson, J. Freeman, and P. J. Keller, Nat. Commun. 6, 7924 (2015).
[Crossref]

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, Nat. Photonics 9, 113 (2015).
[Crossref]

Mettlen, M.

K. Jaqaman, D. Loerke, M. Mettlen, H. Kuwata, S. Grinstein, S. L. Schmid, and G. Danuser, Nat. Methods 5, 695 (2008).
[Crossref]

Olarte, O. E.

Orger, M. B.

M. B. Ahrens, M. B. Orger, D. N. Robson, J. M. Li, and P. J. Keller, Nat. Methods 10, 413 (2013).
[Crossref]

Pampaloni, F.

P. J. Keller, F. Pampaloni, and E. H. Stelzer, Curr. Opin. Cell Biol. 18, 117 (2006).
[Crossref]

Pulver, S. R.

W. C. Lemon, S. R. Pulver, B. Höckendorf, K. McDole, K. Branson, J. Freeman, and P. J. Keller, Nat. Commun. 6, 7924 (2015).
[Crossref]

Reynaud, E. G.

E. G. Reynaud, U. Krzic, K. Greger, and E. H. K. Stelzer, HFSP J. 2, 266 (2008).
[Crossref]

Robson, D. N.

M. B. Ahrens, M. B. Orger, D. N. Robson, J. M. Li, and P. J. Keller, Nat. Methods 10, 413 (2013).
[Crossref]

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, Cell 163, 1796 (2015).
[Crossref]

Santella, A.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, Nat. Methods 7, 637 (2010).
[Crossref]

Schmid, B.

Schmid, S. L.

K. Jaqaman, D. Loerke, M. Mettlen, H. Kuwata, S. Grinstein, S. L. Schmid, and G. Danuser, Nat. Methods 5, 695 (2008).
[Crossref]

Schmidt, A. D.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, Nat. Methods 7, 637 (2010).
[Crossref]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, Science 322, 1065 (2008).
[Crossref]

Stelzer, E. H.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, Nat. Methods 7, 637 (2010).
[Crossref]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, Science 322, 1065 (2008).
[Crossref]

P. J. Keller, F. Pampaloni, and E. H. Stelzer, Curr. Opin. Cell Biol. 18, 117 (2006).
[Crossref]

Stelzer, E. H. K.

E. G. Reynaud, U. Krzic, K. Greger, and E. H. K. Stelzer, HFSP J. 2, 266 (2008).
[Crossref]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[Crossref]

Swoger, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[Crossref]

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, Cell 163, 1796 (2015).
[Crossref]

R. Tomer, K. Khairy, and P. J. Keller, Curr. Opin. Genet. Dev. 21, 558 (2011).
[Crossref]

Turaga, D.

T. F. Holekamp, D. Turaga, and T. E. Holy, Neuron 57, 661 (2008).
[Crossref]

Voigt, F. F.

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, Nat. Photonics 9, 113 (2015).
[Crossref]

Wittbrodt, J.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, Nat. Methods 7, 637 (2010).
[Crossref]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, Science 322, 1065 (2008).
[Crossref]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[Crossref]

Yang, S.

R. Tomer, M. Lovett-Barron, I. Kauvar, A. Andalman, V. M. Burns, S. Sankaran, L. Grosenick, M. Broxton, S. Yang, and K. Deisseroth, Cell 163, 1796 (2015).
[Crossref]

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, Cell 163, 1796 (2015).
[Crossref]

Curr. Opin. Cell Biol. (1)

P. J. Keller, F. Pampaloni, and E. H. Stelzer, Curr. Opin. Cell Biol. 18, 117 (2006).
[Crossref]

Curr. Opin. Genet. Dev. (1)

R. Tomer, K. Khairy, and P. J. Keller, Curr. Opin. Genet. Dev. 21, 558 (2011).
[Crossref]

HFSP J. (1)

E. G. Reynaud, U. Krzic, K. Greger, and E. H. K. Stelzer, HFSP J. 2, 266 (2008).
[Crossref]

Nat. Commun. (1)

W. C. Lemon, S. R. Pulver, B. Höckendorf, K. McDole, K. Branson, J. Freeman, and P. J. Keller, Nat. Commun. 6, 7924 (2015).
[Crossref]

Nat. Methods (3)

K. Jaqaman, D. Loerke, M. Mettlen, H. Kuwata, S. Grinstein, S. L. Schmid, and G. Danuser, Nat. Methods 5, 695 (2008).
[Crossref]

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, Nat. Methods 7, 637 (2010).
[Crossref]

M. B. Ahrens, M. B. Orger, D. N. Robson, J. M. Li, and P. J. Keller, Nat. Methods 10, 413 (2013).
[Crossref]

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, Nat. Photonics 9, 113 (2015).
[Crossref]

Neuron (1)

T. F. Holekamp, D. Turaga, and T. E. Holy, Neuron 57, 661 (2008).
[Crossref]

Opt. Express (1)

Optica (1)

Science (2)

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, Science 322, 1065 (2008).
[Crossref]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004).
[Crossref]

Supplementary Material (3)

NameDescription
» Supplement 1       Supplemental material
» Visualization 1       Slice-by-slice axial scanning sequence of pollen samples, comparing the 2-D phase mask (top) and the piezoelectric scanner (bottom), over a depth of 25 microns.
» Visualization 2       Slice-by-slice axial scanning sequence of bovine pulmonary artery endothelial cells (F36924, Invitrogen, USA), comparing the 2-D phase mask (top) and the piezoelectric scanner (bottom), over a depth of 25 microns.

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Figures (6)

Fig. 1.
Fig. 1.

Optical configuration of the light sheet microscope (LSM). CL, cylindrical lens; GS, galvanometric scanner; IO, illumination objective; DO, detection objective; P, polarizer; EMCCD, electron multiplying charge coupled device.

Fig. 2.
Fig. 2.

(a) Illustration of spherical aberration induced axial focal shifts. (b) Illustration of tangent slope calculation.

Fig. 3.
Fig. 3.

Calculated phase patterns programmed to the LC-SLM.

Fig. 4.
Fig. 4.

(a) Relationship between the fSLM and the axial scan distance (Δz). Note that the LC-SLM adds no optical power when fSLM=, i.e., white screen on LC-SLM. (b) Corresponding magnification (M) versus axial scan distance (Δz).

Fig. 5.
Fig. 5.

Images of pollens at different depths from the LSM based on (a) 2D phase mask and (b) piezoelectric objective scanner. The scale bar is 10 μm at z=0.

Fig. 6.
Fig. 6.

Measured PSF at four different depths, i.e., 15/0/30/60  μm, where the 0 position corresponds to the inherent focal plane of the DO.

Tables (1)

Tables Icon

Table 1. Algorithms to Obtain the Phase Patterns

Equations (6)

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

Δz=fIO·fL1fL2·tan(2θ),
fSLM=fDO2d12f32Δzd1,
ϕxy,x=k·(n2n1)·p(x).
ϕxy,y=ky22q(x).
ϕxy=ϕxy,x+ϕxy,y.
M=f4d1+d2f4·d1fDOf3d1fDO2+f32Δz,

Metrics