Abstract

Despite all the advances in nonlinear microscopy, all existing instruments are constrained to obtain images of one focal plane at a time. In this Letter we demonstrate a two-photon absorption fluorescence scanning microscope capable of imaging two focal planes simultaneously. This is accomplished by temporally demultiplexing the signal coming from two focal volumes at different sample depths. The scheme can be extended to three or more focal planes.

© 2007 Optical Society of America

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References

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2006

2004

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, IEEE Trans. Nanobiosci. 3, 237 (2004).
[CrossRef]

2003

2002

L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, J. Microsc. 206, 65 (2002).
[CrossRef] [PubMed]

2000

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, J. Microsc. 200, 105 (2000).
[CrossRef] [PubMed]

1999

1990

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Albert, O.

L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, J. Microsc. 206, 65 (2002).
[CrossRef] [PubMed]

Amir, W.

Booth, M. J.

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, J. Microsc. 200, 105 (2000).
[CrossRef] [PubMed]

Brakenhoff, G. J.

G. J. Brakenhoff and K. Visscher, in Handbook of Biological Confocal Microscopy, J.B.Pawley, ed. (Plenum, 1995), p. 355.

Burns, D.

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Durfee, C. G.

Gabolde, P.

Girkin, J. M.

Juskaitis, R.

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, J. Microsc. 200, 105 (2000).
[CrossRef] [PubMed]

Kawata, S.

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, J. Microsc. 200, 105 (2000).
[CrossRef] [PubMed]

Marsh, P. N.

Müller, M.

Neil, M. A. A.

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, J. Microsc. 200, 105 (2000).
[CrossRef] [PubMed]

Norris, T. B.

L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, J. Microsc. 206, 65 (2002).
[CrossRef] [PubMed]

Ober, R. J.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, IEEE Trans. Nanobiosci. 3, 237 (2004).
[CrossRef]

Planchon, T. A.

Prabhat, P.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, IEEE Trans. Nanobiosci. 3, 237 (2004).
[CrossRef]

Ram, S.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, IEEE Trans. Nanobiosci. 3, 237 (2004).
[CrossRef]

Sherman, L.

L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, J. Microsc. 206, 65 (2002).
[CrossRef] [PubMed]

Squier, J.

Squier, J. A.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Tanaka, T.

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, J. Microsc. 200, 105 (2000).
[CrossRef] [PubMed]

Trebino, R.

Visscher, K.

G. J. Brakenhoff and K. Visscher, in Handbook of Biological Confocal Microscopy, J.B.Pawley, ed. (Plenum, 1995), p. 355.

Ward, E. S.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, IEEE Trans. Nanobiosci. 3, 237 (2004).
[CrossRef]

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Wilson, T.

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, J. Microsc. 200, 105 (2000).
[CrossRef] [PubMed]

Ye, J. Y.

L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, J. Microsc. 206, 65 (2002).
[CrossRef] [PubMed]

Appl. Opt.

IEEE Trans. Nanobiosci.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, IEEE Trans. Nanobiosci. 3, 237 (2004).
[CrossRef]

J. Microsc.

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, J. Microsc. 200, 105 (2000).
[CrossRef] [PubMed]

L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, J. Microsc. 206, 65 (2002).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Science

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Other

G. J. Brakenhoff and K. Visscher, in Handbook of Biological Confocal Microscopy, J.B.Pawley, ed. (Plenum, 1995), p. 355.

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Figures (3)

Fig. 1
Fig. 1

SMARTI setup: HWP, half-wave plate; BS, polarizing cube beam splitter; QWP, quarter-wave plate; L 1 L 4 , lenses ( L 1 = 100 mm , L 2 = 200 mm , L 3 = L 4 = 100 mm ); DM, deformable mirror; PD, photodiode; SM, scanning mirrors; D, dichroic mirror; PMT, photomultiplier tube. Dotted (dashed) line pulses represent pulses coming from the delay (DM) arm. Top inset, relative timing of the master clock and PMT signal. Pulses from the master clock (PD signal) have a well-defined period, while PMT signal pulses can shift in time due to fluorescence lifetime; counters 1 and 2 represent how the values stored in each counter change with time.

Fig. 2
Fig. 2

Displacement of the DM focal plane, with respect to the delay arm, as a function of voltage applied to the DM for two different objectives ( 40 × Zeiss A-plan 0.65 NA and 40 × Zeiss water immersion IR-Achroplan 0.8 NA ). Different telescope magnifications of 1:2 (“×” marks) and 1:4 (circles) were used for the 0.8 NA objective, showing the influence of magnification on achievable depth. The results for the 0.65 NA objective, using the 1:2 telescope, are shown by the “+” marks. Voltage is applied to all the DM actuators to provide only the focus term.

Fig. 3
Fig. 3

Images of 15 μ m microspheres on Lucifer yellow dye and suspended in agarose: (a) white-light microphotograph of the scanned area, (b) and (c) TPA fluorescence images obtained by blocking the delay (DM) arm of the microscope and using conventional acquisition scheme, (d) and (e) TPA fluorescence images obtained simultaneously using the SMARTI technique. All TPA images are 101 × 101 pixels REF, images from the delayed arm.

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