Abstract

We demonstrate a confocal optical microscope that combines cw two-photon-excited fluorescence microscopy with confocal Raman microscopy. With this microscope fast image acquisition with fluorescence imaging can be used to select areas of interest for subsequent chemical analysis with spontaneous Raman imaging. The distribution of the UV-absorbing fluorophore Hoechst 33342 in the apoptotic HeLa cells is measured in the combined cw two-photon-excited fluorescence and Raman microscopy modes. The 647-nm line of a Kr-ion laser is used to excite both the Raman scattering and the two-photon-excited fluorescence emission. The lateral and axial resolutions in the two imaging modes are compared by use of the Gaussian beam approximation and backprojection of the focal volume through the confocal pinhole.

© 2003 Optical Society of America

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  1. J.-X. Cheng, Y. Jia, G. Zheng, and X. Xie, Biophys. J. 83, 502 (2002).
    [CrossRef] [PubMed]
  2. G. Puppels, H. Garritsen, J. Kummer, and J. Greve, Cytometry 14, 251 (1993).
    [CrossRef]
  3. R. Ramanauskaite, G. Segers-Nolten, K. de Grauw, N. Sijtsema, L. van der Maas, J. Greve, and C. Otto, C. Figdor, Pure Appl. Chem. 69, 2131 (1997).
    [CrossRef]
  4. S. Arikan, H. Sands, R. Rodway, and D. Batchelder, Anim. Reprod. Sci. 71, 249 (2002).
    [CrossRef] [PubMed]
  5. N. Uzunbajakava, A. Lenferink, Y. Kraan, B. Willekens, G. Vrensen, J. Greve, and C. Otto, Biopolymers 72, 1 (2003).
    [CrossRef]
  6. N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, G. Vrensen, J. Greve, and C. Otto, Biophys. J. 84, 3968 (2003).
    [CrossRef] [PubMed]
  7. W. Denk, J. Strickler, and W. Webb, Science 248, 73 (1990).
    [CrossRef] [PubMed]
  8. S. Hell, M. Booth, and S. Wilms, Opt. Lett. 23, 1238 (1998).
    [CrossRef]
  9. M. Booth and S. Hell, J. Microsc. 190, 298 (1997).
    [CrossRef]
  10. A. Kirsch, V. Subramaniam, G. Striker, C. Schnetter, D. Arndt-Jovin, and T. Jovin, Biophys. J. 75, 1513 (1998).
    [CrossRef] [PubMed]
  11. Y. Liu, G. Sonek, M. W. Berns, K. Konig, and B. J. Tromberg, Opt. Lett. 20, 2246 (1995).
    [CrossRef]
  12. J. Kerr, A. Wyllie, and A. Curie, Br. J. Cancer 26, 239 (1972).
    [CrossRef] [PubMed]
  13. F. Zimmermann, B. Zimmermann, J. Panitz, and A. Wokaun, J. Raman Spectrosc. 26, 435 (1995).
    [CrossRef]
  14. H. Deng, V. Bloomfield, J. Benevides, and G. Thomas, Biopolymers 50, 656 (1999).
    [CrossRef] [PubMed]
  15. W. Peticolas, W. Kubasek, G. Thomas, and M. Tsuboi, in Biological Applications of Raman Spectroscopy, T. Spiro, ed. (Wiley, New York, 1987), Vol. 1, pp. 81–134.
  16. F. van der Heijden, Image Based Measurement Systems: Object Recognition and Parameter Estimation (Wiley, New York, 1994).
  17. Z. Darzynkiewicz, in Flow Cytometry and Sorting, M. Melamed, T. Lindmo, and M. Mendelsohn, eds. (Wiley, New York, 1990), pp. 315–340.
  18. C. de Grauw, N. Sijtsema, C. Otto, and J. Greve, J. Microsc. 188, 273 (1997).
    [CrossRef]
  19. C. Xu W. Webb, in Topics in Fluorescence Spectroscopy, J. Lakowicz, ed. (Plenum, New York, 1997), Vol. 5, pp. 472–540.
  20. C. Sheppard and M. Gu, Optik 86, 104 (1990).
  21. S. Hell and E. Stelzer, Opt. Commun. 93, 277 (1992).
    [CrossRef]

2003

N. Uzunbajakava, A. Lenferink, Y. Kraan, B. Willekens, G. Vrensen, J. Greve, and C. Otto, Biopolymers 72, 1 (2003).
[CrossRef]

N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, G. Vrensen, J. Greve, and C. Otto, Biophys. J. 84, 3968 (2003).
[CrossRef] [PubMed]

2002

J.-X. Cheng, Y. Jia, G. Zheng, and X. Xie, Biophys. J. 83, 502 (2002).
[CrossRef] [PubMed]

S. Arikan, H. Sands, R. Rodway, and D. Batchelder, Anim. Reprod. Sci. 71, 249 (2002).
[CrossRef] [PubMed]

1999

H. Deng, V. Bloomfield, J. Benevides, and G. Thomas, Biopolymers 50, 656 (1999).
[CrossRef] [PubMed]

1998

A. Kirsch, V. Subramaniam, G. Striker, C. Schnetter, D. Arndt-Jovin, and T. Jovin, Biophys. J. 75, 1513 (1998).
[CrossRef] [PubMed]

S. Hell, M. Booth, and S. Wilms, Opt. Lett. 23, 1238 (1998).
[CrossRef]

1997

M. Booth and S. Hell, J. Microsc. 190, 298 (1997).
[CrossRef]

C. de Grauw, N. Sijtsema, C. Otto, and J. Greve, J. Microsc. 188, 273 (1997).
[CrossRef]

R. Ramanauskaite, G. Segers-Nolten, K. de Grauw, N. Sijtsema, L. van der Maas, J. Greve, and C. Otto, C. Figdor, Pure Appl. Chem. 69, 2131 (1997).
[CrossRef]

1995

F. Zimmermann, B. Zimmermann, J. Panitz, and A. Wokaun, J. Raman Spectrosc. 26, 435 (1995).
[CrossRef]

Y. Liu, G. Sonek, M. W. Berns, K. Konig, and B. J. Tromberg, Opt. Lett. 20, 2246 (1995).
[CrossRef]

1993

G. Puppels, H. Garritsen, J. Kummer, and J. Greve, Cytometry 14, 251 (1993).
[CrossRef]

1992

S. Hell and E. Stelzer, Opt. Commun. 93, 277 (1992).
[CrossRef]

1990

C. Sheppard and M. Gu, Optik 86, 104 (1990).

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

1972

J. Kerr, A. Wyllie, and A. Curie, Br. J. Cancer 26, 239 (1972).
[CrossRef] [PubMed]

Arikan, S.

S. Arikan, H. Sands, R. Rodway, and D. Batchelder, Anim. Reprod. Sci. 71, 249 (2002).
[CrossRef] [PubMed]

Arndt-Jovin, D.

A. Kirsch, V. Subramaniam, G. Striker, C. Schnetter, D. Arndt-Jovin, and T. Jovin, Biophys. J. 75, 1513 (1998).
[CrossRef] [PubMed]

Batchelder, D.

S. Arikan, H. Sands, R. Rodway, and D. Batchelder, Anim. Reprod. Sci. 71, 249 (2002).
[CrossRef] [PubMed]

Benevides, J.

H. Deng, V. Bloomfield, J. Benevides, and G. Thomas, Biopolymers 50, 656 (1999).
[CrossRef] [PubMed]

Berns, M. W.

Bloomfield, V.

H. Deng, V. Bloomfield, J. Benevides, and G. Thomas, Biopolymers 50, 656 (1999).
[CrossRef] [PubMed]

Booth, M.

Cheng, J.-X.

J.-X. Cheng, Y. Jia, G. Zheng, and X. Xie, Biophys. J. 83, 502 (2002).
[CrossRef] [PubMed]

Curie, A.

J. Kerr, A. Wyllie, and A. Curie, Br. J. Cancer 26, 239 (1972).
[CrossRef] [PubMed]

Darzynkiewicz, Z.

Z. Darzynkiewicz, in Flow Cytometry and Sorting, M. Melamed, T. Lindmo, and M. Mendelsohn, eds. (Wiley, New York, 1990), pp. 315–340.

de Grauw, C.

C. de Grauw, N. Sijtsema, C. Otto, and J. Greve, J. Microsc. 188, 273 (1997).
[CrossRef]

de Grauw, K.

R. Ramanauskaite, G. Segers-Nolten, K. de Grauw, N. Sijtsema, L. van der Maas, J. Greve, and C. Otto, C. Figdor, Pure Appl. Chem. 69, 2131 (1997).
[CrossRef]

Deng, H.

H. Deng, V. Bloomfield, J. Benevides, and G. Thomas, Biopolymers 50, 656 (1999).
[CrossRef] [PubMed]

Denk, W.

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

Figdor, C.

R. Ramanauskaite, G. Segers-Nolten, K. de Grauw, N. Sijtsema, L. van der Maas, J. Greve, and C. Otto, C. Figdor, Pure Appl. Chem. 69, 2131 (1997).
[CrossRef]

Garritsen, H.

G. Puppels, H. Garritsen, J. Kummer, and J. Greve, Cytometry 14, 251 (1993).
[CrossRef]

Greve, J.

N. Uzunbajakava, A. Lenferink, Y. Kraan, B. Willekens, G. Vrensen, J. Greve, and C. Otto, Biopolymers 72, 1 (2003).
[CrossRef]

N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, G. Vrensen, J. Greve, and C. Otto, Biophys. J. 84, 3968 (2003).
[CrossRef] [PubMed]

R. Ramanauskaite, G. Segers-Nolten, K. de Grauw, N. Sijtsema, L. van der Maas, J. Greve, and C. Otto, C. Figdor, Pure Appl. Chem. 69, 2131 (1997).
[CrossRef]

C. de Grauw, N. Sijtsema, C. Otto, and J. Greve, J. Microsc. 188, 273 (1997).
[CrossRef]

G. Puppels, H. Garritsen, J. Kummer, and J. Greve, Cytometry 14, 251 (1993).
[CrossRef]

Gu, M.

C. Sheppard and M. Gu, Optik 86, 104 (1990).

Hell, S.

S. Hell, M. Booth, and S. Wilms, Opt. Lett. 23, 1238 (1998).
[CrossRef]

M. Booth and S. Hell, J. Microsc. 190, 298 (1997).
[CrossRef]

S. Hell and E. Stelzer, Opt. Commun. 93, 277 (1992).
[CrossRef]

Jia, Y.

J.-X. Cheng, Y. Jia, G. Zheng, and X. Xie, Biophys. J. 83, 502 (2002).
[CrossRef] [PubMed]

Jovin, T.

A. Kirsch, V. Subramaniam, G. Striker, C. Schnetter, D. Arndt-Jovin, and T. Jovin, Biophys. J. 75, 1513 (1998).
[CrossRef] [PubMed]

Kerr, J.

J. Kerr, A. Wyllie, and A. Curie, Br. J. Cancer 26, 239 (1972).
[CrossRef] [PubMed]

Kirsch, A.

A. Kirsch, V. Subramaniam, G. Striker, C. Schnetter, D. Arndt-Jovin, and T. Jovin, Biophys. J. 75, 1513 (1998).
[CrossRef] [PubMed]

Konig, K.

Kraan, Y.

N. Uzunbajakava, A. Lenferink, Y. Kraan, B. Willekens, G. Vrensen, J. Greve, and C. Otto, Biopolymers 72, 1 (2003).
[CrossRef]

N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, G. Vrensen, J. Greve, and C. Otto, Biophys. J. 84, 3968 (2003).
[CrossRef] [PubMed]

Kubasek, W.

W. Peticolas, W. Kubasek, G. Thomas, and M. Tsuboi, in Biological Applications of Raman Spectroscopy, T. Spiro, ed. (Wiley, New York, 1987), Vol. 1, pp. 81–134.

Kummer, J.

G. Puppels, H. Garritsen, J. Kummer, and J. Greve, Cytometry 14, 251 (1993).
[CrossRef]

Lenferink, A.

N. Uzunbajakava, A. Lenferink, Y. Kraan, B. Willekens, G. Vrensen, J. Greve, and C. Otto, Biopolymers 72, 1 (2003).
[CrossRef]

N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, G. Vrensen, J. Greve, and C. Otto, Biophys. J. 84, 3968 (2003).
[CrossRef] [PubMed]

Liu, Y.

Otto, C.

N. Uzunbajakava, A. Lenferink, Y. Kraan, B. Willekens, G. Vrensen, J. Greve, and C. Otto, Biopolymers 72, 1 (2003).
[CrossRef]

N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, G. Vrensen, J. Greve, and C. Otto, Biophys. J. 84, 3968 (2003).
[CrossRef] [PubMed]

R. Ramanauskaite, G. Segers-Nolten, K. de Grauw, N. Sijtsema, L. van der Maas, J. Greve, and C. Otto, C. Figdor, Pure Appl. Chem. 69, 2131 (1997).
[CrossRef]

C. de Grauw, N. Sijtsema, C. Otto, and J. Greve, J. Microsc. 188, 273 (1997).
[CrossRef]

Panitz, J.

F. Zimmermann, B. Zimmermann, J. Panitz, and A. Wokaun, J. Raman Spectrosc. 26, 435 (1995).
[CrossRef]

Peticolas, W.

W. Peticolas, W. Kubasek, G. Thomas, and M. Tsuboi, in Biological Applications of Raman Spectroscopy, T. Spiro, ed. (Wiley, New York, 1987), Vol. 1, pp. 81–134.

Puppels, G.

G. Puppels, H. Garritsen, J. Kummer, and J. Greve, Cytometry 14, 251 (1993).
[CrossRef]

Ramanauskaite, R.

R. Ramanauskaite, G. Segers-Nolten, K. de Grauw, N. Sijtsema, L. van der Maas, J. Greve, and C. Otto, C. Figdor, Pure Appl. Chem. 69, 2131 (1997).
[CrossRef]

Rodway, R.

S. Arikan, H. Sands, R. Rodway, and D. Batchelder, Anim. Reprod. Sci. 71, 249 (2002).
[CrossRef] [PubMed]

Sands, H.

S. Arikan, H. Sands, R. Rodway, and D. Batchelder, Anim. Reprod. Sci. 71, 249 (2002).
[CrossRef] [PubMed]

Schnetter, C.

A. Kirsch, V. Subramaniam, G. Striker, C. Schnetter, D. Arndt-Jovin, and T. Jovin, Biophys. J. 75, 1513 (1998).
[CrossRef] [PubMed]

Segers-Nolten, G.

R. Ramanauskaite, G. Segers-Nolten, K. de Grauw, N. Sijtsema, L. van der Maas, J. Greve, and C. Otto, C. Figdor, Pure Appl. Chem. 69, 2131 (1997).
[CrossRef]

Sheppard, C.

C. Sheppard and M. Gu, Optik 86, 104 (1990).

Sijtsema, N.

R. Ramanauskaite, G. Segers-Nolten, K. de Grauw, N. Sijtsema, L. van der Maas, J. Greve, and C. Otto, C. Figdor, Pure Appl. Chem. 69, 2131 (1997).
[CrossRef]

C. de Grauw, N. Sijtsema, C. Otto, and J. Greve, J. Microsc. 188, 273 (1997).
[CrossRef]

Sonek, G.

Stelzer, E.

S. Hell and E. Stelzer, Opt. Commun. 93, 277 (1992).
[CrossRef]

Strickler, J.

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

Striker, G.

A. Kirsch, V. Subramaniam, G. Striker, C. Schnetter, D. Arndt-Jovin, and T. Jovin, Biophys. J. 75, 1513 (1998).
[CrossRef] [PubMed]

Subramaniam, V.

A. Kirsch, V. Subramaniam, G. Striker, C. Schnetter, D. Arndt-Jovin, and T. Jovin, Biophys. J. 75, 1513 (1998).
[CrossRef] [PubMed]

Thomas, G.

H. Deng, V. Bloomfield, J. Benevides, and G. Thomas, Biopolymers 50, 656 (1999).
[CrossRef] [PubMed]

W. Peticolas, W. Kubasek, G. Thomas, and M. Tsuboi, in Biological Applications of Raman Spectroscopy, T. Spiro, ed. (Wiley, New York, 1987), Vol. 1, pp. 81–134.

Tromberg, B. J.

Tsuboi, M.

W. Peticolas, W. Kubasek, G. Thomas, and M. Tsuboi, in Biological Applications of Raman Spectroscopy, T. Spiro, ed. (Wiley, New York, 1987), Vol. 1, pp. 81–134.

Uzunbajakava, N.

N. Uzunbajakava, A. Lenferink, Y. Kraan, B. Willekens, G. Vrensen, J. Greve, and C. Otto, Biopolymers 72, 1 (2003).
[CrossRef]

N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, G. Vrensen, J. Greve, and C. Otto, Biophys. J. 84, 3968 (2003).
[CrossRef] [PubMed]

van der Heijden, F.

F. van der Heijden, Image Based Measurement Systems: Object Recognition and Parameter Estimation (Wiley, New York, 1994).

van der Maas, L.

R. Ramanauskaite, G. Segers-Nolten, K. de Grauw, N. Sijtsema, L. van der Maas, J. Greve, and C. Otto, C. Figdor, Pure Appl. Chem. 69, 2131 (1997).
[CrossRef]

Volokhina, E.

N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, G. Vrensen, J. Greve, and C. Otto, Biophys. J. 84, 3968 (2003).
[CrossRef] [PubMed]

Vrensen, G.

N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, G. Vrensen, J. Greve, and C. Otto, Biophys. J. 84, 3968 (2003).
[CrossRef] [PubMed]

N. Uzunbajakava, A. Lenferink, Y. Kraan, B. Willekens, G. Vrensen, J. Greve, and C. Otto, Biopolymers 72, 1 (2003).
[CrossRef]

Webb, W.

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

C. Xu W. Webb, in Topics in Fluorescence Spectroscopy, J. Lakowicz, ed. (Plenum, New York, 1997), Vol. 5, pp. 472–540.

Willekens, B.

N. Uzunbajakava, A. Lenferink, Y. Kraan, B. Willekens, G. Vrensen, J. Greve, and C. Otto, Biopolymers 72, 1 (2003).
[CrossRef]

Wilms, S.

Wokaun, A.

F. Zimmermann, B. Zimmermann, J. Panitz, and A. Wokaun, J. Raman Spectrosc. 26, 435 (1995).
[CrossRef]

Wyllie, A.

J. Kerr, A. Wyllie, and A. Curie, Br. J. Cancer 26, 239 (1972).
[CrossRef] [PubMed]

Xie, X.

J.-X. Cheng, Y. Jia, G. Zheng, and X. Xie, Biophys. J. 83, 502 (2002).
[CrossRef] [PubMed]

Xu, C.

C. Xu W. Webb, in Topics in Fluorescence Spectroscopy, J. Lakowicz, ed. (Plenum, New York, 1997), Vol. 5, pp. 472–540.

Zheng, G.

J.-X. Cheng, Y. Jia, G. Zheng, and X. Xie, Biophys. J. 83, 502 (2002).
[CrossRef] [PubMed]

Zimmermann, B.

F. Zimmermann, B. Zimmermann, J. Panitz, and A. Wokaun, J. Raman Spectrosc. 26, 435 (1995).
[CrossRef]

Zimmermann, F.

F. Zimmermann, B. Zimmermann, J. Panitz, and A. Wokaun, J. Raman Spectrosc. 26, 435 (1995).
[CrossRef]

Anim. Reprod. Sci.

S. Arikan, H. Sands, R. Rodway, and D. Batchelder, Anim. Reprod. Sci. 71, 249 (2002).
[CrossRef] [PubMed]

Biophys. J.

J.-X. Cheng, Y. Jia, G. Zheng, and X. Xie, Biophys. J. 83, 502 (2002).
[CrossRef] [PubMed]

N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, G. Vrensen, J. Greve, and C. Otto, Biophys. J. 84, 3968 (2003).
[CrossRef] [PubMed]

A. Kirsch, V. Subramaniam, G. Striker, C. Schnetter, D. Arndt-Jovin, and T. Jovin, Biophys. J. 75, 1513 (1998).
[CrossRef] [PubMed]

Biopolymers

N. Uzunbajakava, A. Lenferink, Y. Kraan, B. Willekens, G. Vrensen, J. Greve, and C. Otto, Biopolymers 72, 1 (2003).
[CrossRef]

H. Deng, V. Bloomfield, J. Benevides, and G. Thomas, Biopolymers 50, 656 (1999).
[CrossRef] [PubMed]

Br. J. Cancer

J. Kerr, A. Wyllie, and A. Curie, Br. J. Cancer 26, 239 (1972).
[CrossRef] [PubMed]

Cytometry

G. Puppels, H. Garritsen, J. Kummer, and J. Greve, Cytometry 14, 251 (1993).
[CrossRef]

J. Microsc.

C. de Grauw, N. Sijtsema, C. Otto, and J. Greve, J. Microsc. 188, 273 (1997).
[CrossRef]

M. Booth and S. Hell, J. Microsc. 190, 298 (1997).
[CrossRef]

J. Raman Spectrosc.

F. Zimmermann, B. Zimmermann, J. Panitz, and A. Wokaun, J. Raman Spectrosc. 26, 435 (1995).
[CrossRef]

Opt. Commun.

S. Hell and E. Stelzer, Opt. Commun. 93, 277 (1992).
[CrossRef]

Opt. Lett.

Optik

C. Sheppard and M. Gu, Optik 86, 104 (1990).

Pure Appl. Chem.

R. Ramanauskaite, G. Segers-Nolten, K. de Grauw, N. Sijtsema, L. van der Maas, J. Greve, and C. Otto, C. Figdor, Pure Appl. Chem. 69, 2131 (1997).
[CrossRef]

Science

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

Other

C. Xu W. Webb, in Topics in Fluorescence Spectroscopy, J. Lakowicz, ed. (Plenum, New York, 1997), Vol. 5, pp. 472–540.

W. Peticolas, W. Kubasek, G. Thomas, and M. Tsuboi, in Biological Applications of Raman Spectroscopy, T. Spiro, ed. (Wiley, New York, 1987), Vol. 1, pp. 81–134.

F. van der Heijden, Image Based Measurement Systems: Object Recognition and Parameter Estimation (Wiley, New York, 1994).

Z. Darzynkiewicz, in Flow Cytometry and Sorting, M. Melamed, T. Lindmo, and M. Mendelsohn, eds. (Wiley, New York, 1990), pp. 315–340.

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Figures (4)

Fig. 1
Fig. 1

Confocal Raman and TPE fluorescent microscope. The Raman and fluorescent signals were excited with the 647.1-nm line of a Kr-ion laser (Coherent, Innova 90-K). SM, scanning mirror (Leica Lasertechnique); BS1, dichroic beam splitter; M1, foldable mirror; NF, notch filter; ph1, ph2, confocal pinholes; L1, L2, L3, L4, lenses with focal lengths of 100, 35, 30, and 100 mm, respectively; F, colored glass filter; CCD, CCD camera; PD, photodiode used to monitor the long-term laser power stability; BS2, pellicle beam splitter used to monitor the bright-light image of the cell and the position of the laser beam in the cell on the video camera.

Fig. 2
Fig. 2

Absorption (dotted curve) and emission (dashed curve) spectra of the Hoechst 33342 dye bound to DNA. The transmission curve of the filter BG12 (Melles Griot) used to discriminate for the fluorescent signal is shown as a solid curve.

Fig. 3
Fig. 3

A, Raman spectrum of 3-mM Hoechst 33342 in water measured at the excitation power of 96 mW during 60 s in a square glass capillary with a 40×, 0.6-N.A. objective. B, Raman spectrum obtained at the single position in the nucleus of the apoptotic HeLa cell. C, Raman spectrum obtained at the single position of the apoptotic HeLa cell incubated with 20µM Hoechst 33342. Data were acquired with the excitation power of 100 mW during 1 s with a 63×, 1.2-N.A. water immersion objective.

Fig. 4
Fig. 4

A, Transmission bright-light image. B, Nucleotide distribution in the apoptotic HeLa cells as obtained by TPE fluorescence of the Hoechst. C, D, Raman imaging of the nucleotide on the basis of the 788cm-1 band and on the basis of the 1558cm-1 band of Hoechst. The Raman and TPE images were acquired by scanning an area of 12 µm×12 µm in 64×64 steps. The imaging time of the cw TPE fluorescence image was 4.1 s.

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