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Updated: Apr 4, 2026

Simultaneous Label-Free Autofluorescence Multi-Harmonic Microscopy
Published on: August 29, 2025
Spectral behavior of second harmonic signals from organic and non-organic materials in multiphoton microscopy
Tobias Ehmke1, Andreas Knebl1, Stephan Reiss
1Institute of Applied Optics, Friedrich-Schiller-University Jena , Froebelstieg 1, 07743 Jena, Germany.
Abstract:
Multimodal nonlinear microscopy allows imaging of highly ordered biological tissue due to spectral separation of nonlinear signals. This requires certain knowledge about the spectral distribution of the different nonlinear signals. In contrast to several publications we demonstrate a factor of [Formula: see text] relating the full width at half maximum of a gaussian laser pulse spectrum to the corresponding second harmonic pulse spectrum in the spatial domain by using a simple theoretical model. Experiments on monopotassium phosphate crystals (KDP-crystals) and on porcine corneal tissue support our theoretical predictions. Furthermore, no differences in spectral width were found for epi- and trans-detection of the second harmonic signal. Overall, these results may help to build an optimized multiphoton setup for spectral separation of nonlinear signals.
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