Nothing to Hide: A three-dimensional view into Aquatic Anatomy via Optical Clearing

Authors:

Lelde Hermane1,2,3, Sandra C. Silva1,4, Rute C. Felix2,4, Christina Egger5,6, Naghmeh Jafari Pastaki4, Hamed Abdollahpour4, Aschwin H. Engelen7, Márcio A. G. Coelho5, Adelino V. M. Canário4, Deborah M. Power4, Teresa M. Correia1

Affiliations:

1 Quantitative Bio-Imaging Lab, Centro de Ciências do Mar do Algarve (CCMAR/CIMAR LA), Campus de Gambelas, Universidade do Algarve, Faro, Portugal

2 GreenCoLab – Associação Oceano Verde, Campus de Gambelas, Universidade do Algarve, Faro, Portugal

3 iNOVA4Health, NOVA Medical School, Universidade Nova de Lisboa, Faculdade de Ciências Médicas, Lisbon, Portugal

4 Comparative Endocrinology and Integrative Biology Group, Centro de Ciências do Mar do Algarve (CCMAR/CIMAR LA), Campus de Gambelas, Universidade do Algarve, Faro, Portugal

5 Biogeography, Ecology and Evolution Group, Centro de Ciências do Mar do Algarve (CCMAR/CIMAR LA), Campus de Gambelas, Universidade do Algarve, Faro, Portugal

6 SNSB - Bavarian State Collection of Zoology, Münchhausenstraße 21, 81247 Munich, Bavaria, Germany

7 Marine Microbial Ecology & Biotechnology Group, Centro de Ciências do Mar do Algarve (CCMAR/CIMAR LA), Campus de Gambelas, Universidade do Algarve, Faro, Portugal

Abstract:

Advances in 3D microscopy have driven the development of tissue clearing techniques. While they have been optimized for model organisms, protocols for aquatic organisms face wide variety of challenges, including calcified structures and strong pigmentation, remaining underdeveloped and largely underexplored. We optimised clearing protocols for general use with aquatic organisms, including freshwater rainbow trout (Oncorhynchus mykiss), Senegalese sole (Solea senegalensis), sterlet sturgeon (Acipenser ruthenus) and octocoral (Paramuricea sp.), to determine the most effective approach for clearly visualizing the internal structures of intact whole organisms.

Aquatic samples (n=3) were cleared with CUBIC (Matsumoto et al., 2019), iDISCO (Habart et al., 2023), 2ECI (Masselink et al., 2019) and BABB (Foster et al., 2019), propidium iodide labelled and imaged with ZEISS Lightsheet Z.1. Protocols were assessed based on size changes, attenuation rate, image quality and other metrics, including sample rigidity, handling and time.

The different clearing methods resulted in various levels of area preservation (from 101% [CUBIC] to 87% [2ECI]), fluorescence attenuation rate (from 28.00 [iDISCO and 2ECI] to 55.00 [CUBIC]) and visualisation efficacy across different organs, e.g. CUBIC was the best at revealing cardiac structures, iDISCO and BABB at anatomical overview (Fig.1).

The optimized protocols can be used for selecting the clearing technique depending on the sample and the research question.

References: Foster et al. SciRep 2019.; Habart et al. STAR 2023.; Masselink et al. Development 2019.; Matsumoto et al. NatProtoc 2019.

Acknowledgements: UID/04326/2025 (DOI: 10.54499/UID/04326/2025), UID/PRR/04326/2025 (DOI: 10.54499/UID/PRR/04326/2025), LA/P/0101/2020 (DOI: 10.54499/LA/P/0101/2020), DivRestore/0013/2020, 2024.03331.BDANA (DOI: 10.54499/2024.03331.BDANA), 2020.08486.BD, CRESC Algarve 2020 / COMPETE 2020: EMBRC.PT ALG-01-0145-FEDER-022121, Horizon Europe IMAGINE: GA#101094250 (HORIZON-INFRA-2022-TECH01-01), H2020 MSCA OPTIMAR: GA#867450, BiodivRestore ERA-NET Cofund: GA#101003777, EMBRC JDA: ID 3193, Fundação Belmiro de Azevedo: CORALFORESTS