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0Detecting the Raman signature responsible for the life activity of regenerating worm A. Viride using Raman and two-photon fluorescence lifetime imaging spectroscopy
P.M. Badgujar1, P.-Y. Huang1, A.V. Karmenyan1, V.V. Nikolaev2, Y.V. Kistenev2, J.-H. Chen3, C.-L. Cheng1*
1-Department of Physics, National Dong Hwa University, Hualien 97401, Taiwan 2- Department of Physics, Tomsk State University, Tomsk, 634050, Russia 3- Department of Life Sciences, National Taiwan University, Taipei, Taiwan
* clcheng@gms.ndhu.edu.tw
In regenerative organisms, regeneration arises with the help of repatterning co-existing tissues after a wound or trauma has occurred in a regenerative organism. Understanding these mechanisms is crucial as critical metabolic functions are involved during the process of regeneration. With the emerging field of regenerative medicine, understanding the "molecular signature" of what determines and triggers the process of regeneration is of utmost importance. In this study, we uncover the molecular signature responsible for the process of regeneration; in one of the simpler and advanced freshwater regenerative annelid, Aeolosoma Viride (Annelida, Aeolosomatidae). A. viride is tasked with building entire body segments out of their single starting cell at their amputated region and undergoing epimorphic regeneration; therefore these annelids are the most suitable for the study of regeneration [1]. In this work, we carefully address the participation of primary and secondary Carotenoids in the life activity of the regenerating anterior region of A. viride. At the site of regeneration, primary antioxidant carotenoids were detected with the Raman spectral band of zeaxanthin at 1525-1527 cm-1, 1159 cm-1, and 1005-1008 cm-1. The secondary role of carotenoids in the process of cell signaling was confirmed with the upregulation of the Cu/Zn superoxide (SOD1) gene at the regenerated area at the cell patterning state. The intracellular participation and distribution of the proliferated blastemal cells in the anterior regeneration segments were monitored by applying a phasor approach to the TP-FLIM label-free without any specific staining. The application of micro Raman spectroscopy with the combination of Two-photon fluorescence lifetime imaging can be a good technique in the study of regenerative medicine. Furthermore, we demonstrated that a high amount of reactive oxygen species is produced at the wound healing state and the blastema budding state during the anterior regeneration process. In the field of regenerative medicine; the approach of the Raman spectroscopic technique in combination with the TP-FLIM could be an advancement in the regenerative studies.
Fig. 1. (A) The Raman spectroscopic signature at wound healing (6 hpa) and budding blastema cells at 24 (hpa). (B) Detection of Reactive Oxygen Species at the wound healing at the anterior regenerated segment. (C) Two-Photon Fluorescence Lifetime Imaging label-free direct screening of the Blastema bud at 24 hpa.
[1] C.-P. Chen, S.K.-W. Fok, Y.-W. Hsieh, et al, General characterization of regeneration in Aeolosoma viride (Annelida, Aeolosomatidae).
Invertebr Biol. 139:e12277 (2020).
