CC BY
22ALT'22
B-I-4
BIOMEDICAL PHOTONICS
Study the life activity of regenerative worm Aeolosoma Viride using Raman spectroscopy and Two-Photon Fluorescence Lifetime Imaging Microscopy
Pooja Manik Badgujar1, Jia-Hua Wu1, Pei-Yang Huang1, Wrenit Gem Pearl1, Artashes V. Karmenyan1, Elena V. Perevedentseva2, Jiun-Hong Chen3, Chia-Liang Cheng1*
'Department of Physics, National Dong Hwa University, Hualien 97401, Taiwan 2P. N. Lebedev Physics Institute of Russian Academy of Sciences, Moscow, 119991, Russia 3Department of Life Sciences, National Taiwan nNiversity, Taipei, Taiwan * [email protected]
Abstract: Many regenerative studies have been conducted to understand Regeneration's mechanism and which molecular signal is responsible for Regeneration. Freshwater annelid Aeolosoma Viride is considered one of the most advanced worms because of its strong regenerative properties, both anterior and posteriorly [1]. Many researchers have been trying to understand the regenerative ability in annelids, but the mechanism is not fully understood. This study investigates the molecular signature present at the sight of regeneration in these annelids using Raman spectroscopy, confocal fluorescence microscopy and FLIM. We observe a strong Raman signal of carotenoids at wound healing, blastema formation, and blastema proliferation of the regenerated worm. This gives a clear understanding that the carotenoid molecule plays the role of a potent antioxidant and is accumulated in the wound healing region. At blastema formation, a strong accumulation of Reactive oxygen species (ROS) is confirmed by observation of strong fluorescence from the DCF dye. At blastema formation, the appearance of carotenoids can be assumed as an antioxidant as a large amount of ROS is generated, Carotenoids act as a scavenger for ROS. Carotenoids on one hand act as potent antioxidant enzymes, however, in other organisms such as invertebrates they are known to promote SOD activity by upregulating the expression of the ZnCuSOD gene and invertebrate immunity [2]. Confocal microscopy results clearly display that after carotenoids complete their first role, which is acting as an antioxidant for ROS production, later they play a role of over-expressing the SOD1 gene which plays a major role in worm and invertebrate immunity and in the process of neuronal motor cells. Two-photon imaging of A. viride body segments displays a lifetime of 0.65 ns and 2.5 ns. The novelty of this work is we can very clearly observe the participation of carotenoids in cell signaling, proliferation, dedifferentiation, and regeneration using the spectroscopic method.
Fig.1. (I) Optical image of anterior regeneration of A. viride at 6 hpa (i.e wound healing), (II) antibody tagged SOD1 gene, (III) merged image.
[1] Chen, C-P, Fok, SK-W, Hsieh, Y-W, et al. General characterization of regeneration in Aeolosoma viride (Annelida, Aeolosomatidae). Invertebr Biol; 139:e12277. 2020.
[2] Tan K, Zhang H, Lim LS, Ma H, Li S, Zheng H. Roles of Carotenoids in Invertebrate Immunology. Front Immunol. 17;10:3041
2020 Jan
