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Intraocular multiphoton microscopy with subcellular spatial resolution by infrared femtosecond lasers

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Abstract

We reported on the in situ nonlinear optical sectioning of the corneal and retinal tissues based on the multiphoton microscopy (MPM) with different excitation wavelengths of infrared femtosecond (fs) lasers. The multiphoton nonlinear processing including two-photon fluorescence (2PF) and second harmonic generation (SHG) was induced under condition of high light intensities on an order of MW-GW/cm2. The laser beams emitted from the solid-state Ti: sapphire systems were focused in a 0.1 femtoliter focus volume of a high numerous aperture diffraction-limited objective (40 × 1.3 N.A., oil). The corneal layers have been visualized using nonlinear optical tomography. In particular, corneal Bowman’s layer was optically determined in situ. The cellular and collagen components of tissues were selectively displayed with submicron spatial resolution and high efficiency without any assistance of staining or slicing. The preliminary study on retinal optical tomography is here also reported. MPM is a promising and convenient non-invasive technique by which the tissue layers can be visualized and the selective displaying of the tissue microstructures be realized. The optical biopsy based on intrinsic emission of MPM yields details that provide three-dimensional displaying of the tissue component and even have the potential to be used in clinical diagnostics.

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Acknowledgments

We acknowledge Dr. Dietrich Schweitzer and Dr. Martin Hammer from Eye Hospital, University of Jena for their outstanding support with retina study, Professor Chris P. Lohmann from Eye Hospital Rechts der Isar, Technical University of Munich for his helpful discussion. We thank Shuping Song from University of Jena for careful viewing of this manuscript. The authors also wish to thank Isa Lemke, Helmut Hoerig, Michael Szabó and Sabine Hitschke from Institute of Anatomy II, University of Jena for their skilful technical assistance.

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Authors and Affiliations

  1. Lasermicroscopy Research, Institute of Microscopic Anatomy (Anatomy II), University of Jena, Teichgraben str. 7, 07743, Jena, Germany

    Bao-Gui Wang, Reimar Krieg & Karl-Juergen Halbhuber

  2. Fraunhofer Institute of Biomedical Technology (IBMT), Ensheimer Straße 48, 66386, St. Ingbert, Germany

    Karsten Koenig & Iris Riemann

  3. Faculty of Physics and Mechatronics, University of Saarland, Ensheimer Straße 48, 66386, St. Ingbert, Germany

    Karsten Koenig

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  1. Bao-Gui Wang
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  2. Karsten Koenig
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  3. Iris Riemann
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  4. Reimar Krieg
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  5. Karl-Juergen Halbhuber
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Correspondence to Karl-Juergen Halbhuber.

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Dedicated on the occasion of the 66th birthday of Professor Dr. Karl-Juergen Halbhuber

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Wang, BG., Koenig, K., Riemann, I. et al. Intraocular multiphoton microscopy with subcellular spatial resolution by infrared femtosecond lasers. Histochem Cell Biol 126, 507–515 (2006). https://doi.org/10.1007/s00418-006-0187-0

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