## 3 Results  
Fucaxanthin Has Fluorescent Properties Regardless of the Presence or Absence of A\(\beta\) Monomers or Aggregates  
To specify the fluorescent spectrum of fucoxanthin extracted from microalgae _P. tricor-nutum_, we performed a fluorescent spectral scan of fucoxanthin at various concentrations (2 mM, 1 mM, 500 \(\upmu\)M, and 250 \(\upmu\)M). First, the absorbance scans were conducted to decide the excitation wavelength of fucoxanthin. Due to the high concentration, the absorbance exceeded the measurable range at 2 mM, but the absorbance decreased in a concentration-dependent manner at the concentrations of 1 mM, 500 \(\upmu\)M, and 250 \(\upmu\)M, and had maximum values at 430, 442, and 444 nm, respectively (Figure 2a). Figure 2: The fluorescent properties of fucoxanthin. Excitation and emission wavelengths of fucoxanthin at various concentrations were determined through absorbance (**a**) and emission scan (**b**); excitation, 444 nm; emission, 686 nm. Fluorescent spectra of each fucoxanthin solution with A\(\beta\)40 monomers or aggregates in a 3:1 ratio were recorded with excitation and emission wavelengths as above (**c**–**f**). A.U. = arbitrary unit. Then, the emission scans of each fucoxanthin solution were measured using the excitation wavelength (444 nm) determined through an absorbance scan. In the emission spectrum, the fluorescence intensities of fucoxanthin reduced with decreasing concentration, and it had the narrowest peak at the same wavelength of 686 nm (Figure 2b). These results show that the fucoxanthin used in this study has fluorescent spectrum with an excitation point of 430-440 nm and an emission point of 686 nm, and thus can be monitored using a fluorescence microscope laser corresponding to the range. It has been previously reported that fucoxanthin directly binds to A\(\beta\) peptide through hydrophobic interaction [14]. We conducted additional experiments to determine whether a fluorescence shift occurs when fucoxanthin binds to A\(\beta\) in brain tissue. Four concentrations of fucoxanthin, 2 mM, 1 mM, 500 uM, and 250 uM, were added to A\(\beta\) monomers (20 uM, 0-day incubation) or aggregates (20 uM, 6-day incubation) respectively. The emission scans of each mixed solution were measured by applying the wavelength as above. Consequently, fucoxanthin in the presence of A\(\beta\) monomers or aggregates showed a decrease when compared to the native fluorescence intensity of fucoxanthin itself (Figure 2c-f). However, it was observed that fucoxanthin has sufficient fluorescence intensity without losing its fluorescence properties, indicating that fucoxanthin could be applied as a fluorescence probe targeting A\(\beta\). Fucoxanthin Detected Amyloid Aggregates in AD Transgenic Mouse Models Using a Water-Based Washing Method  
The popularly used AD transgenic mouse models, APP/PS1 and 5\(\times\)FAD, possess mutations in APP and presenilin (PSEN1) genes, which are known to be associated with A\(\beta\) production. Thus, these mouse models show rapidly increased A\(\beta\) production and result in extensive plaque deposition and behavioral deficits [18]. As APP/PS1 and 5\(\times\)FAD mouse models were originally generated only for the development of A\(\beta\) deposits, ThS single staining for A\(\beta\) plaque detection is widely used [19; 20]. However, in the 5\(\times\)FAD mouse model, it was found that ThS-positive tau aggregates make difficult the quantification of amyloid plaque. This suggests the necessity to develop new fluorescent dyes to support verification of A\(\beta\) plaque detection. To investigate whether fucoxanthin can detect A\(\beta\) aggregates, we examined the histological staining in brain tissue from APP/PS1 and 5\(\times\)FAD mice, with water- or organic solvent-based washing solutions during staining. We applied different concentrations of fucoxanthin in the brain for optimization during the staining process. Interaction between fucoxanthin and A\(\beta\) aggregates can be monitored by the comparison of ThS- and fucoxanthin-stained regions in the brains.