24 May 2018 Endogenous two- and three-photon fluorescence of a biological substance with a picosecond ultrawide band laser source
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Multiphoton microscopy (MPM) is a method for characterizing biological samples, becoming more and more established in life sciences labs thanks to its label-free imaging ability. In MPM, few biological substances have been highlighted as endogenously fluorescent, such as elastin, myosin, keratin, redox indicators, collagen or amino acids. Tryptophan, an amino acid fundamental in the synthesis of proteins, is known for its endogenous fluorescence. In this article, we propose to show an original solution specifically dedicated to multiphoton microscopy with an ultrawide band laser system. The specificity stands into the filtering system based on a prisms-line allowing spatial shaping of the spectrum. Our custom-designed multiphoton microscope, coupled with a picosecond ultrawide band laser correctly filtered spectrally, is adapted for charactering the two- and three-photon absorption ranges and imaging of tryptophan. This highlights in one hand that the use of a picosecond ultrawide band laser spectrally filtered does allow to reach both the two- and three-photon abortion (TPA and ThPA) ranges of this substance. In another hand, a quantitative comparison of the resulting images shows high differences in the image quality where the three-photon image looks better contrasted and better resolved than the two-photon one. An explanation of this highly interesting phenomena can be proposed with a study of the probability of presence of multiphoton processes involved and the cross section values of ThPA and TPA. This initiating work, cumulating an innovative multiphoton setup and interesting results, plays a crucial role for extending the label-free imaging ability of MPM.
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Claire Lefort, Claire Lefort, Laetitia Magnol, Laetitia Magnol, Philippe Lévêque, Philippe Lévêque, Véronique Blanquet, Véronique Blanquet, "Endogenous two- and three-photon fluorescence of a biological substance with a picosecond ultrawide band laser source", Proc. SPIE 10679, Optics, Photonics, and Digital Technologies for Imaging Applications V, 106791N (24 May 2018); doi: 10.1117/12.2306681; https://doi.org/10.1117/12.2306681

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