## 4 Discussion  
Here, we report that fucoxanthin, extracted from the microalgae _P. tricornutum_, has a fluorescent spectrum with excitation and emission wavelengths (excitation: 430-440 nm, emission: 686 nm) regardless of the presence or absence of A\(\beta\) monomers or aggregates. When fucoxanthin was utilized as a fluorescent dye, it was colocalized with ThS-positive amyloid protein aggregates in the fixed brain tissue of AD transgenic mouse models, and different washing methods did not affect the histological staining of fucoxanthin. Effective and accurate detection of A\(\beta\), one of the pathological hallmarks of AD, is very important in its diagnosis and prognosis [21]. It is characterized by the cohesion of oligomers, fibrils, and plaques from monomers; therefore, specific detection substances for A\(\beta\) aggregates are used in AD diagnosis. ThS is most used to detect A\(\beta\) aggregates; however, recent studies have shown that ThS also detects tau protein aggregates in certain  
Figure 6: Fucoxanthin staining using organic solvent-based washing method detected ThS-positive A\(\beta\) plaques in 5\(\times\)FAD brain tissue. The brains of 10–15-month-old 5\(\times\)FAD mice (\(n=4\)) were visualized for A\(\beta\) plaques with fucoxanthin (Fx) and ThS. The images of ThS and Fx are merged to confirm that they are colocalized. Scale bars = 200 \(\upmu\)m. The region of brain was separated with the white dash line on the images; upper region of dash line, cortex; under region of dash line, hippocampus. AD transgenic mouse brains, such as 5\(\times\)FAD [6]. Therefore, additional reagents capable of specific or supportable detection of A\(\beta\) aggregates is needed. The use of A\(\beta\)-specific antibodies enables very specific detection of different A\(\beta\) forms, but it takes at least 16-24 h [22; 23]. Furthermore, the need for additional fluorescent secondary antibodies and chemicals significantly adds to the total cost for staining (Table 1). Fucoxanthin is a carotenoid produced by plants and algae, including _P. tricornutum_, and it is already known to have beneficial biological properties such as anticancer, anti-inflammatory, and antiobesity physiological activities [10; 12; 16; 24]. Recently, fucoxanthin has been reported to interact through binding to the amyloid protein, suggesting an association with AD [14]; therefore, the detection of A\(\beta\) aggregates via fucoxanthin could be presented as an additional imaging method that confirms A\(\beta\) detection via ThS staining. Like ThS staining, fucoxanthin is an advantageous staining procedure because it takes less than 4 h without additional chemicals. Furthermore, fucoxanthin can be mass-produced and has the potential to be applied to living animals as it is a naturally derived carotenoid, but ThS, which is a chemical material, is difficult to apply to clinical trials or AD patients since it is not proven yet. In contrast, we confirmed that fucoxanthin stained with oligomeric tau in the brains of 5\(\times\)FAD, not in the WT and APP/PS1 brains (Figures S2-S4). Although fucoxanthin may not overcome all shortcomings of other dyes, it is expected that it will help to further refine AD diagnosis by reducing false-positive misdiagnosis as much as possible by at least supplementing the dyes. A precise analysis of whether fucoxanthin interferes with other hallmarks of AD, such as tau aggregation, is required. Additional studies should investigate whether fluorescence imaging with fucoxanthin can be translated into diagnosis and prognosis in AD.