Multidimensional Characterization of the Physiological State of Hematococcuspluvialis Using Scanning Structured Illumination Super-Resolution Microscopy

<i>Haematococcus pluvialis</i> (HP) is a freshwater alga known for its ability to accumulate the potent antioxidant astaxanthin, which has extensive applications in aquaculture, pharmaceuticals, and cosmetics. Astaxanthin rapidly accumulates under unfavorable environmental conditions. However, the mechanisms of astaxanthin accumulation under various stress conditions remain unclear. This mainly stems from the limitations of current imaging techniques, which lack super-resolution, label-free, and three-dimensional (3D) imaging capabilities. In this study, we employed scanning structured illumination microscopy (SSIM) to achieve dynamic 3D ultrastructural reconstructions of HP cells under various stress conditions. This advanced imaging approach allowed us to closely observe the stress responses of HP cells, revealing significant morphological changes induced by different stressors. Additionally, we examined alterations in the HP cell wall under these conditions and explored the relationship between these morphological changes and the rate of astaxanthin accumulation during identical stress durations. The results clearly demonstrate that light stress, which induces a more comprehensive disruption of the entire cell, leads to a faster rate of astaxanthin accumulation compared to salt stress, which exerts its effects from the exterior inward. The rate of astaxanthin accumulation under light stress is approximately twice that observed under salt stress conditions. Our findings offer new insights into the subcellular dynamics of astaxanthin accumulation in HP, underscoring the effectiveness of super-resolution techniques in clarifying these processes.