Abstract

Fast 3D microscopic imaging methods have been playing a crucial role in many biological studies. In this Letter, we present a revolutionary way to design and build a two-photon excitation (TPE) microscope for 3D and random-access imaging based on a digital micromirror device (DMD), achieving a scanning speed of 22.7 kHz. When pairing with a 40× objective lens, the maximum scanning range in the x, y, z axes are 103, 206, 524 μm, respectively. The axial and lateral scanning resolution (i.e., minimum step size) are 270 nm and 130 nm, respectively. In the system, the focal point of the femtosecond laser can be arbitrarily positioned to any random point in space by switching the binary holograms stored in the DMD. Parametric models are derived to deterministically link the DMD parameters (i.e., pixel size and aperture) with the scanner characteristics, i.e., scan range and minimum step size, in each axis. In the experiments, we demonstrate conventional raster scanning, scanning along arbitrarily programmed surfaces, and random-access scanning on a pollen grain sample via the DMD-based TPE system. We also perform experiments to demonstrate the unique capability of selective optical stimulation, where selected locations within the specimen are photobleached by extending the laser dwell time. With its versatility and high scanning rate, the TPE microscope may find important applications in brain research, realizing in vivo random-access imaging and optical stimulation with tens of microseconds temporal resolution.

© 2017 Optical Society of America

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References

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Bullen, A.

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Cheng, J.

Chiovini, B.

G. Katona, G. Szalay, P. Maak, A. Kaszas, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rozsa, Nat. Methods 9, 201 (2012).
[Crossref]

Evans, G.

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Fahrbach, F.

Griffiths, V.

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Gu, C.

Helmchen, F.

Hillier, D.

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[Crossref]

Katona, G.

G. Katona, G. Szalay, P. Maak, A. Kaszas, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rozsa, Nat. Methods 9, 201 (2012).
[Crossref]

Kirkby, P.

K. Nadella, H. Ros, C. Baragli, V. Griffiths, G. Konstantinou, T. Koimtzis, G. Evans, P. Kirkby, and R. Silver, Nat. Methods 13, 1001 (2016).
[Crossref]

Koimtzis, T.

K. Nadella, H. Ros, C. Baragli, V. Griffiths, G. Konstantinou, T. Koimtzis, G. Evans, P. Kirkby, and R. Silver, Nat. Methods 13, 1001 (2016).
[Crossref]

Konstantinou, G.

K. Nadella, H. Ros, C. Baragli, V. Griffiths, G. Konstantinou, T. Koimtzis, G. Evans, P. Kirkby, and R. Silver, Nat. Methods 13, 1001 (2016).
[Crossref]

Lee, W.

Maak, P.

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[Crossref]

Nadella, K.

K. Nadella, H. Ros, C. Baragli, V. Griffiths, G. Konstantinou, T. Koimtzis, G. Evans, P. Kirkby, and R. Silver, Nat. Methods 13, 1001 (2016).
[Crossref]

Patel, S.

A. Bullen, S. Patel, and P. Saggau, Biophys. J. 73, 477 (1997).
[Crossref]

Reddy, G.

G. Reddy and P. Saggau, J. Biomed. Opt. 10, 064038 (2005).
[Crossref]

Ros, H.

K. Nadella, H. Ros, C. Baragli, V. Griffiths, G. Konstantinou, T. Koimtzis, G. Evans, P. Kirkby, and R. Silver, Nat. Methods 13, 1001 (2016).
[Crossref]

Roska, B.

G. Katona, G. Szalay, P. Maak, A. Kaszas, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rozsa, Nat. Methods 9, 201 (2012).
[Crossref]

Rozsa, B.

G. Katona, G. Szalay, P. Maak, A. Kaszas, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rozsa, Nat. Methods 9, 201 (2012).
[Crossref]

Saggau, P.

V. Iyer, T. Hoogland, and P. Saggau, J. Neurophysiol. 95, 535 (2006).
[Crossref]

G. Reddy and P. Saggau, J. Biomed. Opt. 10, 064038 (2005).
[Crossref]

A. Bullen, S. Patel, and P. Saggau, Biophys. J. 73, 477 (1997).
[Crossref]

Schmid, B.

Silver, R.

K. Nadella, H. Ros, C. Baragli, V. Griffiths, G. Konstantinou, T. Koimtzis, G. Evans, P. Kirkby, and R. Silver, Nat. Methods 13, 1001 (2016).
[Crossref]

Szalay, G.

G. Katona, G. Szalay, P. Maak, A. Kaszas, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rozsa, Nat. Methods 9, 201 (2012).
[Crossref]

Veress, M.

G. Katona, G. Szalay, P. Maak, A. Kaszas, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rozsa, Nat. Methods 9, 201 (2012).
[Crossref]

Vizi, E.

G. Katona, G. Szalay, P. Maak, A. Kaszas, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rozsa, Nat. Methods 9, 201 (2012).
[Crossref]

Voigt, F.

Wang, D.

Zhang, D.

Appl. Opt. (2)

Biophys. J. (1)

A. Bullen, S. Patel, and P. Saggau, Biophys. J. 73, 477 (1997).
[Crossref]

J. Biomed. Opt. (1)

G. Reddy and P. Saggau, J. Biomed. Opt. 10, 064038 (2005).
[Crossref]

J. Neurophysiol. (1)

V. Iyer, T. Hoogland, and P. Saggau, J. Neurophysiol. 95, 535 (2006).
[Crossref]

Nat. Methods (2)

G. Katona, G. Szalay, P. Maak, A. Kaszas, M. Veress, D. Hillier, B. Chiovini, E. Vizi, B. Roska, and B. Rozsa, Nat. Methods 9, 201 (2012).
[Crossref]

K. Nadella, H. Ros, C. Baragli, V. Griffiths, G. Konstantinou, T. Koimtzis, G. Evans, P. Kirkby, and R. Silver, Nat. Methods 13, 1001 (2016).
[Crossref]

Opt. Express (1)

Opt. Lett. (2)

Supplementary Material (1)

NameDescription
» Visualization 1: AVI (354 KB)      Demonstration of two-photon imaging on arbitrarily programmed surfaces: (1) spherical surface and (2) sinusoidal surface.

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Figures (7)

Fig. 1.
Fig. 1.

Optical configuration of the DMD-based TPE microscope based on a single DMD; M1, M2, high-reflectivity mirrors; L1–L6, lenses (fL1, fL2, fL3, fL4, fL5, fL6=50, 200, 100, 100, 200, and 200 mm, respectively); DM, dichroic mirror; PMT, photomultiplier tube.

Fig. 2.
Fig. 2.

Lateral scanning in the focal plane of L5, where x-axis scanning is realized via varying the spatial frequency fx. θ is the diffraction angle between the 0th- and 1st-order diffraction.

Fig. 3.
Fig. 3.

(a) Visual illustration of the DMD scanner work space: the light and dark gray volume represent work spaces of the 1st- and +1st-order diffractions, respectively. The work space can be found by subtracting the cone regions (c) from the cuboidal DMD work space (b). Note that the 0th- and ±1st-order diffractions overlap in the cones.

Fig. 4.
Fig. 4.

Cross-sectional images of a pollen grain at eight different depths, scanned by DMD; the scale bar is 10 μm.

Fig. 5.
Fig. 5.

Cross-sectional images of two selected pollen grains on arbitrarily programmed spherical surfaces (a)–(c) and sinusoidal surfaces (d)–(f).

Fig. 6.
Fig. 6.

Random-access imaging experiments on a pollen grain: (a) eight imaged layers of the pollen grain, where six distant points are selected at different layers with color labels; the scale bar is 10 μm; (b) recorded voltages (i.e., fluorescence intensities) of the selected points; the color bar associates the fluorescence data to specific points in (a).

Fig. 7.
Fig. 7.

Photobleaching process of the seven selected spikes on the pollen grain for a total of (a) 0 s; (b) 3 s; and (c) 15 s; the scale bar is 10 μm.

Equations (7)

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

φ(x,y)=π(x2+y2)λf,
h(i,j)={1,q2R(x,y)T+φ(x,y)2π+kq20,otherwise,
Δx=ΔθfL5Mobj=λfL5MobjΔfx=λfL5Mobjd(1NTx,min1NTx,max).
Δy=λfL5MobjΔfy=2λfL5MobjNTy,mind.
Δz=nrfL52PmaxMobj2=2nrfL52λMobj2NTnd2,
2π·nd2·Δfx,min(πNTx)max.
δx=λL5MobjΔfx,min=λL5MobjnNTx,mind.

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