Abstract

We demonstrate simultaneous imaging by optical coherence microscopy (OCM) and two-photon-excited (TPE) fluorescence microscopy. A mode-locked Ti:sapphire laser is focused and scanned in three dimensions through a fixed sample, generating both backscattered light and fluorescence light, which are independently detected. Both imaging modes provide rapid en-face imaging with submicrometer resolution. High-power delivery into the sample yields an OCM sensitivity in excess of 130 dB at 100-kHz pixel rates. Simultaneous imaging of cell nuclei with OCM and TPE is demonstrated in live drosophila embryos.

© 1999 Optical Society of America

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    [CrossRef]

1998

1997

J. M. Schmitt and A. Knüttel, J. Opt. Soc. Am. A 14, 1231 (1997).
[CrossRef]

J. M. Schmitt, S. L. Hee, and K. M. Yung, Opt. Commun. 142, 203 (1997).
[CrossRef]

G. J. Tearney, M. E. Brezinski, B. E. Bouma, S. A. Bopart, C. Pitris, J. F. Southern, and J. G. Fujimoto, Science 276, 2037 (1997).
[CrossRef] [PubMed]

A. Gh. Podoleanu, G. M. Dobre, D. J. Webb, and D. A. Jackson, Opt. Lett. 22, 1039 (1997).
[CrossRef] [PubMed]

1996

M. Kempe, W. Rudolph, and E. Welsch, J. Opt. Soc. Am. A 13, 46 (1996).
[CrossRef]

J. A. Izatt, M. D. Kulkarni, H.-W. Wang, K. Kobayashi, and M. V. Sivak, IEEE J. Sel. Top. Quantum Electron. 2, 1017 (1996).
[CrossRef]

1994

1991

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, Science 254, 1178 (1991).
[CrossRef] [PubMed]

1990

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

Beaurepaire, E.

Blanchot, L.

Boccara, A. C.

Bopart, S. A.

G. J. Tearney, M. E. Brezinski, B. E. Bouma, S. A. Bopart, C. Pitris, J. F. Southern, and J. G. Fujimoto, Science 276, 2037 (1997).
[CrossRef] [PubMed]

Boppart, S. A.

S. A. Boppart, B. E. Bouma, C. Pitris, J. F. Southern, M. E. Brezinski, and J. G. Fujimoto, Nature Med. 4, 861 (1998).
[CrossRef]

Bouma, B. E.

S. A. Boppart, B. E. Bouma, C. Pitris, J. F. Southern, M. E. Brezinski, and J. G. Fujimoto, Nature Med. 4, 861 (1998).
[CrossRef]

G. J. Tearney, M. E. Brezinski, B. E. Bouma, S. A. Bopart, C. Pitris, J. F. Southern, and J. G. Fujimoto, Science 276, 2037 (1997).
[CrossRef] [PubMed]

Brezinski, M. E.

S. A. Boppart, B. E. Bouma, C. Pitris, J. F. Southern, M. E. Brezinski, and J. G. Fujimoto, Nature Med. 4, 861 (1998).
[CrossRef]

G. J. Tearney, M. E. Brezinski, B. E. Bouma, S. A. Bopart, C. Pitris, J. F. Southern, and J. G. Fujimoto, Science 276, 2037 (1997).
[CrossRef] [PubMed]

Chang, W.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, Science 254, 1178 (1991).
[CrossRef] [PubMed]

Colston, B. W.

Da Silva, L. B.

Denk, W.

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

W. Denk, D. Piston, and W. W. Webb, in The Handbook of Confocal Microscopy, 2nd ed., J. Pawley, ed. (Plenum, New York, 1995), p. 445.
[CrossRef]

Dobre, G. M.

Everett, M. J.

Flotte, T.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, Science 254, 1178 (1991).
[CrossRef] [PubMed]

Fujimoto, J. G.

S. A. Boppart, B. E. Bouma, C. Pitris, J. F. Southern, M. E. Brezinski, and J. G. Fujimoto, Nature Med. 4, 861 (1998).
[CrossRef]

G. J. Tearney, M. E. Brezinski, B. E. Bouma, S. A. Bopart, C. Pitris, J. F. Southern, and J. G. Fujimoto, Science 276, 2037 (1997).
[CrossRef] [PubMed]

J. A. Izatt, M. R. Hee, G. M. Owen, E. A. Swanson, and J. G. Fujimoto, Opt. Lett. 19, 590 (1994).
[CrossRef] [PubMed]

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, Science 254, 1178 (1991).
[CrossRef] [PubMed]

Gregory, K.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, Science 254, 1178 (1991).
[CrossRef] [PubMed]

Hee, M. R.

J. A. Izatt, M. R. Hee, G. M. Owen, E. A. Swanson, and J. G. Fujimoto, Opt. Lett. 19, 590 (1994).
[CrossRef] [PubMed]

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, Science 254, 1178 (1991).
[CrossRef] [PubMed]

Hee, S. L.

J. M. Schmitt, S. L. Hee, and K. M. Yung, Opt. Commun. 142, 203 (1997).
[CrossRef]

Huang, D.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, Science 254, 1178 (1991).
[CrossRef] [PubMed]

Izatt, J. A.

J. A. Izatt, M. D. Kulkarni, H.-W. Wang, K. Kobayashi, and M. V. Sivak, IEEE J. Sel. Top. Quantum Electron. 2, 1017 (1996).
[CrossRef]

J. A. Izatt, M. R. Hee, G. M. Owen, E. A. Swanson, and J. G. Fujimoto, Opt. Lett. 19, 590 (1994).
[CrossRef] [PubMed]

Jackson, D. A.

Kempe, M.

Knüttel, A.

Kobayashi, K.

J. A. Izatt, M. D. Kulkarni, H.-W. Wang, K. Kobayashi, and M. V. Sivak, IEEE J. Sel. Top. Quantum Electron. 2, 1017 (1996).
[CrossRef]

Kulkarni, M. D.

J. A. Izatt, M. D. Kulkarni, H.-W. Wang, K. Kobayashi, and M. V. Sivak, IEEE J. Sel. Top. Quantum Electron. 2, 1017 (1996).
[CrossRef]

Lebec, M.

Lin, C. P.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, Science 254, 1178 (1991).
[CrossRef] [PubMed]

Owen, G. M.

Piston, D.

W. Denk, D. Piston, and W. W. Webb, in The Handbook of Confocal Microscopy, 2nd ed., J. Pawley, ed. (Plenum, New York, 1995), p. 445.
[CrossRef]

Pitris, C.

S. A. Boppart, B. E. Bouma, C. Pitris, J. F. Southern, M. E. Brezinski, and J. G. Fujimoto, Nature Med. 4, 861 (1998).
[CrossRef]

G. J. Tearney, M. E. Brezinski, B. E. Bouma, S. A. Bopart, C. Pitris, J. F. Southern, and J. G. Fujimoto, Science 276, 2037 (1997).
[CrossRef] [PubMed]

Podoleanu, A. Gh.

Puliafito, C. A.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, Science 254, 1178 (1991).
[CrossRef] [PubMed]

Rudolph, W.

Saint-Jalmes, H.

Schmitt, J. M.

J. M. Schmitt and A. Knüttel, J. Opt. Soc. Am. A 14, 1231 (1997).
[CrossRef]

J. M. Schmitt, S. L. Hee, and K. M. Yung, Opt. Commun. 142, 203 (1997).
[CrossRef]

Schoenenberger, K.

Schuman, J. S.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, Science 254, 1178 (1991).
[CrossRef] [PubMed]

Sivak, M. V.

J. A. Izatt, M. D. Kulkarni, H.-W. Wang, K. Kobayashi, and M. V. Sivak, IEEE J. Sel. Top. Quantum Electron. 2, 1017 (1996).
[CrossRef]

Southern, J. F.

S. A. Boppart, B. E. Bouma, C. Pitris, J. F. Southern, M. E. Brezinski, and J. G. Fujimoto, Nature Med. 4, 861 (1998).
[CrossRef]

G. J. Tearney, M. E. Brezinski, B. E. Bouma, S. A. Bopart, C. Pitris, J. F. Southern, and J. G. Fujimoto, Science 276, 2037 (1997).
[CrossRef] [PubMed]

Stinson, W. G.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, Science 254, 1178 (1991).
[CrossRef] [PubMed]

Strickler, J. H.

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

Swanson, E. A.

J. A. Izatt, M. R. Hee, G. M. Owen, E. A. Swanson, and J. G. Fujimoto, Opt. Lett. 19, 590 (1994).
[CrossRef] [PubMed]

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, Science 254, 1178 (1991).
[CrossRef] [PubMed]

Tearney, G. J.

G. J. Tearney, M. E. Brezinski, B. E. Bouma, S. A. Bopart, C. Pitris, J. F. Southern, and J. G. Fujimoto, Science 276, 2037 (1997).
[CrossRef] [PubMed]

Wang, H.-W.

J. A. Izatt, M. D. Kulkarni, H.-W. Wang, K. Kobayashi, and M. V. Sivak, IEEE J. Sel. Top. Quantum Electron. 2, 1017 (1996).
[CrossRef]

Webb, D. J.

Webb, W. W.

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

W. Denk, D. Piston, and W. W. Webb, in The Handbook of Confocal Microscopy, 2nd ed., J. Pawley, ed. (Plenum, New York, 1995), p. 445.
[CrossRef]

Welsch, E.

Yung, K. M.

J. M. Schmitt, S. L. Hee, and K. M. Yung, Opt. Commun. 142, 203 (1997).
[CrossRef]

IEEE J. Sel. Top. Quantum Electron.

J. A. Izatt, M. D. Kulkarni, H.-W. Wang, K. Kobayashi, and M. V. Sivak, IEEE J. Sel. Top. Quantum Electron. 2, 1017 (1996).
[CrossRef]

J. Opt. Soc. Am. A

Nature Med.

S. A. Boppart, B. E. Bouma, C. Pitris, J. F. Southern, M. E. Brezinski, and J. G. Fujimoto, Nature Med. 4, 861 (1998).
[CrossRef]

Opt. Commun.

J. M. Schmitt, S. L. Hee, and K. M. Yung, Opt. Commun. 142, 203 (1997).
[CrossRef]

Opt. Lett.

Science

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

G. J. Tearney, M. E. Brezinski, B. E. Bouma, S. A. Bopart, C. Pitris, J. F. Southern, and J. G. Fujimoto, Science 276, 2037 (1997).
[CrossRef] [PubMed]

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, Science 254, 1178 (1991).
[CrossRef] [PubMed]

Other

W. Denk, D. Piston, and W. W. Webb, in The Handbook of Confocal Microscopy, 2nd ed., J. Pawley, ed. (Plenum, New York, 1995), p. 445.
[CrossRef]

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Figures (4)

Fig. 1
Fig. 1

Configuration of the combined optical coherence and TPE fluorescence microscope: HWP, half-wave plate; PBS, polarizing beam splitter; M’s, mirrors; QWP, quarter-wave plate; Wol., Wollaston prism; PD’s, photodiodes; PZT, piezo translator; PC, computer; PMT, photomultiplier tube. The reference mirror is mounted on the same translation stage as the objective.

Fig. 2
Fig. 2

OCM and TPE spatial resolutions measured by acquisition of three-dimensional images of a 0.3µm-diameter fluorescent polystyrene bead immobilized in a poly(dimethylsiloxane) gel. (a) The envelope of the (squared) OCM response is defined by the confocal detection and is similar to the TPE response. The OCM response oscillations of period λ/4 correspond to the rectified phase difference between the interferometer arms. Sampling is every 100 nm. (b) Radial OCM and TPE responses in the plane of focus. Axial and radial resolutions are 2 and 0.4 µm, respectively (FWHM). The back aperture of the objective is underfilled by the ratio w/a=0.85 (w is the beam waist; a is the radius of the back aperture).

Fig. 3
Fig. 3

Comparison of (a) coherent and (b) standard confocal imaging in a scattering medium. A metallic grid is imaged through six scattering mean free paths of a suspension of polystyrene spheres. Incident power, 80 mW. The mean detected confocal signal is 13 dB above the noise floor.

Fig. 4
Fig. 4

Simultaneous en-face (a) OCM and (b) TPE fluorescence imaging of a live drosophila embryo transfected to express green fluorescent protein in its cell nuclei. The nuclei appear as nonscattering structures in the OCM image. Incident power, <10 mW; pixel rate, 100 kHz; total acquisition time, 2.8 s.

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