## 3 Results and Discussion
### Microscopic Observation and NPL-Assisted IL-Mediated Extraction Process  
Figure 3 illustrates the changes observed in the cysts microscopically and provides the schematic representation of astaxanthin extraction from _H. lacustris_ cysts using \(\alpha\)-quartz NPLs and [Emim] HSO\({}_{4}\) at room temperature. When mature 90-day-old _H. lacustris_ cysts (Figure 3a) were treated with \(\alpha\)-quartz NPLs with sonication for 5 min, the intracellular circular domain-containing red astaxanthin-rich lipids appeared slightly smaller under a light microscope (Figure 3b). Furthermore, the region between the outer perimeter of the red domain and the cell wall exhibited differential interference contrast imaging, primarily because of the light scattering effects of \(\alpha\)-quartz NPLs passing through the cell wall [13]. After the subsequent introduction of [Emim] HSO\({}_{4}\), the astaxanthin-containing lipid droplets began to emerge from the cyst cell, owing to the lactating effect of \(\alpha\)-quartz NPLs (Figure 3c). Astaxanthin could be recovered from the upper ethyl acetate phase after the addition of ethyl acetate to the IL-treated algal solution, followed by centrifugation (Figure 3d). Astaxanthin-extracted and/or broken cells were observed at the interface of the two-phase solvent system (Figure 3e). IL uptake by _H. lacustris_ cyst cells was further analyzed by fluorescence microscopy based on the fluorescence of [Emim] HSO\({}_{4}\) at approximately 450 nm. Several areas of weak blue fluorescence were observed inside the IL-treated cysts not treated with \(\alpha\)-quartz NPLs. Instead, the outer layer of the cyst cell exhibited a strong blue fluorescence owing to the binding of [Emim] HSO\({}_{4}\), which indicated a significant barrier effect of [Emim] HSO\({}_{4}\) on the algal cell wall (Figure 4a). In contrast, _H. lacustris_ cyst cells subjected to NPL-assisted IL treatment were able to sufficiently absorb [Emim] HSO\({}_{4}\) and emitted stronger cytoplasmic blue fluorescence than cells treated with IL alone. The average blue fluorescence intensity of the former was approximately 4.3 times higher than that of the latter (Figure 4b). Along with the astaxanthin recovery results (Figure 2), these results demonstrate the synergistic  
Figure 3: Microscopic images and schematic illustrations of cell wall disruption and room-temperature astaxanthin (AXT) extraction from 90-day-old _Haematococcus lacustris_ cysts using \(\alpha\)-quartz NPLs and [Emim] HSO\({}_{4}\): (**a**) Initial mature cyst cell. (**b**) Intact cyst cell after 5-min NPL treatment in a sonication bath. The intracellular contrast difference indicates the presence of \(\alpha\)-quartz NPLs inside the cyst. (**c**) Cyst cells after subsequent IL treatment for 2 h. The black arrow indicates that red-AXT-containing lipid droplets were extracted from lacerations caused by \(\alpha\)-quartz NPLs in the cell wall. (**d**) AXT recovery with EA. After centrifugation, the reaction tube shows AXT in the EA phase and [Emim] HSO\({}_{4}\) and \(\alpha\)-quartz NPLs in the distilled water (DW) phase. (**e**) Broken and empty cells were collected at the interface between the EA and aqueous phases. Scale bars represent 20 \(\upmu\)m.  
Various IL solvents were used to extract astaxanthin from _H. lacustris_ cyst biomass at different temperatures. Of note, the astaxanthin extractability of IL is highly dependent on the state of the algal biomass, such as cultivation conditions, aging, and the drying methods used, as well as the extraction parameters, such as IL concentration, temperature, and processing time. [PERSON] et al. [21] reported 77% astaxanthin recovery from spray-dried _H. lacustris_ cysts using 1-butyl-3-methylimidazolium [Bmim] dibutylphosphate at 55 \({}^{\circ}\)C. [PERSON] et al. [30] reported 91% astaxanthin extraction from commercial _H. lacustris_ powder by applying [Bmim] Cl at 70 \({}^{\circ}\)C. Notably, [PERSON] et al. [17] reported almost complete astaxanthin recovery from lyophilized 18-day-old _H.