Abstract

We report on the application of a novel all-solid-state violet laser diode source to confocal microscopy. The source has the potential to replace argon ion lasers in a range of fluorescence based imaging systems. Improvements in system performance and image quality through the use of anamorphic prisms to modify the beam profile have been characterised. These modifications have permitted high quality, optically sectioned images to be obtained from laser diodes operating around 406nm. Living mammalian cells stained with a range of biologically significant fluorophores have been imaged. In addition, it has been shown that at this wavelength it is possible to image dyes that normally require excitation with UV argon laser lines.

©2000 Optical Society of America

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References

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  1. J. B. Pawley (Ed.), “Handbook of Biological Confocal Microscopy,” 2nd Edition, Plenum (1995).
  2. R. P. Haugland, “Handbook of Fluorescent Probes and Research Chemicals, 6th Edition,” Molecular Probes Inc.
  3. S. Nakamura, M. Senoh, S. Nagahama, N. Iwasa, T. Yamada, T. Matsushita, Y. Sugimoto, and H. Kiyoku, “Optical gain and carrier lifetime of InGaN multi-quantum well structure laser diodes,” Appl. Phys Lett 691568–1572 (1996).
    [Crossref]
  4. S. Nakamura “Developments in blue and UV optical semiconductor sources,” Proc. SPIE 3621-01 125–135 (1999).
  5. J. Bewersdorf and S. W. Hell, “Picosecond pulsed two-photon imaging with repetition rates of 200 and 400 MHz,” Journal of Microscopy 19128–38 (1998).
    [Crossref]
  6. P. E. Hockerber, T. A. Skimina, V. E. Centonze, C. Lavin, S. Chu, S. Dadras, J. K. Reddy, and J. G. White, “Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells,” Proc. Natl. Sci. 966255–6260 (1999)
    [Crossref]
  7. A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent protein and calmodulin,” Nature 388882–887 (1997).
    [Crossref] [PubMed]

1999 (1)

P. E. Hockerber, T. A. Skimina, V. E. Centonze, C. Lavin, S. Chu, S. Dadras, J. K. Reddy, and J. G. White, “Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells,” Proc. Natl. Sci. 966255–6260 (1999)
[Crossref]

1998 (1)

J. Bewersdorf and S. W. Hell, “Picosecond pulsed two-photon imaging with repetition rates of 200 and 400 MHz,” Journal of Microscopy 19128–38 (1998).
[Crossref]

1997 (1)

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent protein and calmodulin,” Nature 388882–887 (1997).
[Crossref] [PubMed]

1996 (1)

S. Nakamura, M. Senoh, S. Nagahama, N. Iwasa, T. Yamada, T. Matsushita, Y. Sugimoto, and H. Kiyoku, “Optical gain and carrier lifetime of InGaN multi-quantum well structure laser diodes,” Appl. Phys Lett 691568–1572 (1996).
[Crossref]

Adams, J. A.

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent protein and calmodulin,” Nature 388882–887 (1997).
[Crossref] [PubMed]

Bewersdorf, J.

J. Bewersdorf and S. W. Hell, “Picosecond pulsed two-photon imaging with repetition rates of 200 and 400 MHz,” Journal of Microscopy 19128–38 (1998).
[Crossref]

Centonze, V. E.

P. E. Hockerber, T. A. Skimina, V. E. Centonze, C. Lavin, S. Chu, S. Dadras, J. K. Reddy, and J. G. White, “Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells,” Proc. Natl. Sci. 966255–6260 (1999)
[Crossref]

Chu, S.

P. E. Hockerber, T. A. Skimina, V. E. Centonze, C. Lavin, S. Chu, S. Dadras, J. K. Reddy, and J. G. White, “Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells,” Proc. Natl. Sci. 966255–6260 (1999)
[Crossref]

Dadras, S.

P. E. Hockerber, T. A. Skimina, V. E. Centonze, C. Lavin, S. Chu, S. Dadras, J. K. Reddy, and J. G. White, “Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells,” Proc. Natl. Sci. 966255–6260 (1999)
[Crossref]

Haugland, R. P.

R. P. Haugland, “Handbook of Fluorescent Probes and Research Chemicals, 6th Edition,” Molecular Probes Inc.

Heim, R.

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent protein and calmodulin,” Nature 388882–887 (1997).
[Crossref] [PubMed]

Hell, S. W.

J. Bewersdorf and S. W. Hell, “Picosecond pulsed two-photon imaging with repetition rates of 200 and 400 MHz,” Journal of Microscopy 19128–38 (1998).
[Crossref]

Hockerber, P. E.

P. E. Hockerber, T. A. Skimina, V. E. Centonze, C. Lavin, S. Chu, S. Dadras, J. K. Reddy, and J. G. White, “Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells,” Proc. Natl. Sci. 966255–6260 (1999)
[Crossref]

Ikura, M.

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent protein and calmodulin,” Nature 388882–887 (1997).
[Crossref] [PubMed]

Iwasa, N.

S. Nakamura, M. Senoh, S. Nagahama, N. Iwasa, T. Yamada, T. Matsushita, Y. Sugimoto, and H. Kiyoku, “Optical gain and carrier lifetime of InGaN multi-quantum well structure laser diodes,” Appl. Phys Lett 691568–1572 (1996).
[Crossref]

Kiyoku, H.

S. Nakamura, M. Senoh, S. Nagahama, N. Iwasa, T. Yamada, T. Matsushita, Y. Sugimoto, and H. Kiyoku, “Optical gain and carrier lifetime of InGaN multi-quantum well structure laser diodes,” Appl. Phys Lett 691568–1572 (1996).
[Crossref]

Lavin, C.

P. E. Hockerber, T. A. Skimina, V. E. Centonze, C. Lavin, S. Chu, S. Dadras, J. K. Reddy, and J. G. White, “Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells,” Proc. Natl. Sci. 966255–6260 (1999)
[Crossref]

Llopis, J.

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent protein and calmodulin,” Nature 388882–887 (1997).
[Crossref] [PubMed]

Matsushita, T.

S. Nakamura, M. Senoh, S. Nagahama, N. Iwasa, T. Yamada, T. Matsushita, Y. Sugimoto, and H. Kiyoku, “Optical gain and carrier lifetime of InGaN multi-quantum well structure laser diodes,” Appl. Phys Lett 691568–1572 (1996).
[Crossref]

McCaffery, J. M.

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent protein and calmodulin,” Nature 388882–887 (1997).
[Crossref] [PubMed]

Miyawaki, A.

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent protein and calmodulin,” Nature 388882–887 (1997).
[Crossref] [PubMed]

Nagahama, S.

S. Nakamura, M. Senoh, S. Nagahama, N. Iwasa, T. Yamada, T. Matsushita, Y. Sugimoto, and H. Kiyoku, “Optical gain and carrier lifetime of InGaN multi-quantum well structure laser diodes,” Appl. Phys Lett 691568–1572 (1996).
[Crossref]

Nakamura, S.

S. Nakamura, M. Senoh, S. Nagahama, N. Iwasa, T. Yamada, T. Matsushita, Y. Sugimoto, and H. Kiyoku, “Optical gain and carrier lifetime of InGaN multi-quantum well structure laser diodes,” Appl. Phys Lett 691568–1572 (1996).
[Crossref]

S. Nakamura “Developments in blue and UV optical semiconductor sources,” Proc. SPIE 3621-01 125–135 (1999).

Reddy, J. K.

P. E. Hockerber, T. A. Skimina, V. E. Centonze, C. Lavin, S. Chu, S. Dadras, J. K. Reddy, and J. G. White, “Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells,” Proc. Natl. Sci. 966255–6260 (1999)
[Crossref]

Senoh, M.

S. Nakamura, M. Senoh, S. Nagahama, N. Iwasa, T. Yamada, T. Matsushita, Y. Sugimoto, and H. Kiyoku, “Optical gain and carrier lifetime of InGaN multi-quantum well structure laser diodes,” Appl. Phys Lett 691568–1572 (1996).
[Crossref]

Skimina, T. A.

P. E. Hockerber, T. A. Skimina, V. E. Centonze, C. Lavin, S. Chu, S. Dadras, J. K. Reddy, and J. G. White, “Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells,” Proc. Natl. Sci. 966255–6260 (1999)
[Crossref]

Sugimoto, Y.

S. Nakamura, M. Senoh, S. Nagahama, N. Iwasa, T. Yamada, T. Matsushita, Y. Sugimoto, and H. Kiyoku, “Optical gain and carrier lifetime of InGaN multi-quantum well structure laser diodes,” Appl. Phys Lett 691568–1572 (1996).
[Crossref]

Tsien, R.

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent protein and calmodulin,” Nature 388882–887 (1997).
[Crossref] [PubMed]

White, J. G.

P. E. Hockerber, T. A. Skimina, V. E. Centonze, C. Lavin, S. Chu, S. Dadras, J. K. Reddy, and J. G. White, “Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells,” Proc. Natl. Sci. 966255–6260 (1999)
[Crossref]

Yamada, T.

S. Nakamura, M. Senoh, S. Nagahama, N. Iwasa, T. Yamada, T. Matsushita, Y. Sugimoto, and H. Kiyoku, “Optical gain and carrier lifetime of InGaN multi-quantum well structure laser diodes,” Appl. Phys Lett 691568–1572 (1996).
[Crossref]

Appl. Phys Lett (1)

S. Nakamura, M. Senoh, S. Nagahama, N. Iwasa, T. Yamada, T. Matsushita, Y. Sugimoto, and H. Kiyoku, “Optical gain and carrier lifetime of InGaN multi-quantum well structure laser diodes,” Appl. Phys Lett 691568–1572 (1996).
[Crossref]

Journal of Microscopy (1)

J. Bewersdorf and S. W. Hell, “Picosecond pulsed two-photon imaging with repetition rates of 200 and 400 MHz,” Journal of Microscopy 19128–38 (1998).
[Crossref]

Nature (1)

A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Tsien, “Fluorescent indicators for Ca2+ based on green fluorescent protein and calmodulin,” Nature 388882–887 (1997).
[Crossref] [PubMed]

Proc. Natl. Sci. (1)

P. E. Hockerber, T. A. Skimina, V. E. Centonze, C. Lavin, S. Chu, S. Dadras, J. K. Reddy, and J. G. White, “Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells,” Proc. Natl. Sci. 966255–6260 (1999)
[Crossref]

Other (3)

S. Nakamura “Developments in blue and UV optical semiconductor sources,” Proc. SPIE 3621-01 125–135 (1999).

J. B. Pawley (Ed.), “Handbook of Biological Confocal Microscopy,” 2nd Edition, Plenum (1995).

R. P. Haugland, “Handbook of Fluorescent Probes and Research Chemicals, 6th Edition,” Molecular Probes Inc.

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Figures (2)

Figure 1.
Figure 1. Axial profile x63 1.35NA objective, laser diode with and without anamorphic beam shaping

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Figure 2.
Figure 2. Live vascular smooth muscle cell stained with FITC imaged directly, 2a directly with laser diode, 2b laser diode with anamorphic beam shaping, 2c Cos-7 cell with eGFP transfection

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Tables (2)

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Table 1. Measured optical performance

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Table 2. Summary of flurophores imaged with 406nm

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