# Fluorescence Correlation Spectroscopy Measurement Based on Fiber Optics for Biological Materials
## 4 Conclusions  
In this study, we developed a new FCS system called FB-FCS by introducing a water immersion objective lens to focus the excitation laser. Measurements of the concentration and molecular size were successfully performed.  
The fluorescence sensitivity was still lower than conventional FCS instruments because of fluorescence loss by fiber optics. However, that was sufficient to measure the solutions of the fluorescent dye monomer. This is a very important improvement for biological and biomedical applications. The fluorescence sensitivity (CPM per excitation laser power, CPM/LP) of FB-FCS was improved 1168 times higher than it of the FF-FCS system (Figure A2 and Table A1 in Appendix B). Furthermore, the sensitivity of FB-FCS reached 62.6% of a conventional FCS system (307-15471, Wako, Osaka, Japan) as shown in Figure A3 and Table A1.  
The concentration measurement showed good linearity in the region above 63 nM. To measure a lower-concentration sample, the measurement duration should be extended. The molecular diameters measured by FB-FCS and the corresponding values reported in the literature also showed good linearity.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Sample**} & \multicolumn{2}{c}{**FB-FCS**} & \multicolumn{2}{c}{**Literature Value**} \\ \cline{2-4}  & **Diametera [nm]** & **Diameter [nm]** & **Ref.** \\ \hline Fluorescein & 1.01 \(\pm\) 0.07 & 1.00 & [19] \\ AcGFP & 5.63 \(\pm\) 0.19 & 5.64 & [20] \\ BSA & 7.39 \(\pm\) 0.33 & 6.96 & [21] \\ HSA & 6.65 \(\pm\) 0.44 & 7.0 & [22] \\ IgG & 10.50 \(\pm\) 0.23 & 10.9 & [23] \\ \hline \hline \end{tabular}
\end{table}
Table 1: Measurement Results of Various Samples. The diameter of FB-FCS shows mean \(\pm\) standard error (\(u\) = 5–8).  
Figure 4: FB-FCS measurements on fluorescent beads on different days. (**a**) Normalized ACFs. (**b**) Diffusion time. (**c**) CPM.  
Finally, the robustness of FB-FCS was validated. Both the diffusion time and CPM were stable for two weeks, and the coefficients of variation were less than 5% without any mechanical adjustment.  
These results indicate that FB-FCS can be used instead of the conventional FCS system to measure the concentration and size of target molecules. However, this system cannot take microscopic images, unlike a conventional FCS system based on the frame of a microscope. Therefore, it is difficult to determine the measurement position in the cell for FB-FCS. The conventional FCS system might be suitable for such in vivo FCS measurements.  
The FB-FCS system does not require any mechanical adjustment of the optical setup. Furthermore, FB-FCS is low-cost and compact compared to conventional FCS instruments. For these reasons, the availability of FB-FCS may be higher than that of high-spec FCS systems. We expect that the FB-FCS system will be used as a simple measurement system in laboratories, medical diagnosis, and environmental measurements.  
Conceptualization, J.Y.; methodology, J.Y.; software, J.Y.; validation, J.Y. and A.S.; formal analysis, J.Y.; investigation, J.Y. and A.S.; resources, A.S.; data curation, J.Y.; writing--original draft preparation, J.Y.; writing--review and editing, J.Y.; visualization, J.Y.; supervision, J.Y.; project administration, J.Y.; funding acquisition, J.Y. Both authors have read and agreed to the published version of the manuscript.  
This work was supported by the Adaptable and Seamless Technology Transfer Program through Target-driven R & D (A-STEP) from Japan Science and Technology Agency (JST) (JPMJTM19Y0).  
The authors thank [PERSON] (Hokkaido University, Japan) for his valuable discussions.  
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [PERSON] has Japanese Patent No. 6667868 issued to Hokkaido University.