Research Papers: Imaging

Two-photon spectral fluorescence lifetime and second-harmonic generation imaging of the porcine cornea with a 12-femtosecond laser microscope

[+] Author Affiliations
Ana Batista

Saarland University, Faculty of Physics and Mechatronics, Department of Biophotonics and Laser Technology, Campus A5.1, Saarbrücken 66123, Germany

University of Coimbra, Faculty of Medicine, Institute for Biomedical Imaging and Life Sciences, Azinhaga Santa Comba—Celas, Coimbra 3000-548, Portugal

Hans Georg Breunig, Karsten König

Saarland University, Faculty of Physics and Mechatronics, Department of Biophotonics and Laser Technology, Campus A5.1, Saarbrücken 66123, Germany

JenLab GmbH, Schillerstr. 1, Jena 07745, Germany

Aisada Uchugonova

Saarland University, Faculty of Physics and Mechatronics, Department of Biophotonics and Laser Technology, Campus A5.1, Saarbrücken 66123, Germany

António Miguel Morgado

University of Coimbra, Faculty of Medicine, Institute for Biomedical Imaging and Life Sciences, Azinhaga Santa Comba—Celas, Coimbra 3000-548, Portugal

University of Coimbra, Faculty of Sciences and Technology, Department of Physics, Rua Larga, Coimbra 3004-516, Portugal

J. Biomed. Opt. 21(3), 036002 (Mar 08, 2016). doi:10.1117/1.JBO.21.3.036002
History: Received October 27, 2015; Accepted February 12, 2016
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Abstract.  Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD(P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions.

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© 2016 Society of Photo-Optical Instrumentation Engineers

Citation

Ana Batista ; Hans Georg Breunig ; Aisada Uchugonova ; António Miguel Morgado and Karsten König
"Two-photon spectral fluorescence lifetime and second-harmonic generation imaging of the porcine cornea with a 12-femtosecond laser microscope", J. Biomed. Opt. 21(3), 036002 (Mar 08, 2016). ; http://dx.doi.org/10.1117/1.JBO.21.3.036002


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