Light-sheet fluorescent microscopy in tardigrade anoxybiosis

Tardigrades are invertebrates that are known for their tolerance to extreme conditions. This research focuses on anoxia, the total lack of oxygen, and the adaptation and survival of anoxiainduced experiments. Cryptobiosis is a form of dormancy that enables the survival of the animals, however, many forms of cryptobiosis are still poorly understood and the mechanisms and physiological responses are not entirely explained. This study aimed to create a protocol that utilizes fluorescent dyes to visualize the transition into anoxybiosis and morphometric changes at the cellular level involved in the phenomenon. Lightsheet fluorescent microscopy enabled fast 3D volumetric scanning of the transition and revealed what happened to the animal when anoxia was chemically applied. Results were aligned with the expectations: during anoxybiosis, tardigrades became immobilized and swollen leading to the relocation and reorganization of cells. Importantly it was observed that variation throughout the experiments was quite significant and in further studies, this should be outlined. In this research, two specimens of M. ripperi were used and obtained data were compared including cell number, volume, and displacement over time. The most fundamental issue is how to gain stable and reproducible results. Tardigrade cuticle and overall variation of body state of the animals create sources of error, particularly dyeing by soaking. By soaking, there are very few possibilities to control, how the dye is distributed and attached to the cellular compartments, therefore in this study we did not pay so close attention to the statistical significance of the results. Animals chosen for the experiments were random and therefore variables such as sex, age, and fasting were not included. Fluorescent microscopy is a widely used method in biological studies and this study showed that it can be used in live imaging of tardigrades. However, as a pilot experiment, this led to many open questions and features to improve, especially in the image analysis part. Large datasets need a lighter pipeline to gain a higher throughput method. In the future, the (light sheet) fluorescent imaging can be a beneficial tool for similar cellular studies; however, the individual sources of variation need to be minimized. Tardigrades can be a promising model organism for studies including cell survival, cancer research, and storage and storing solutions for various drug components when understanding the mechanisms that enable the animal stress tolerance and survival