## 4 Discussion  
A method for early detection of [PERSON]'s disease using fluorescent probes in the CSF was previously presented by us [31] and validated in vitro and ex vivo. We have further tested and verified the method's accuracy and performance in animals, in this paper. Although the fluorescent probes were not injected directly to the blood and measured in the CSF as the method proposes, the experiments in this study were an additional important step towards testing the method in an AD animal model. Here we implanted tubes with fluorescent samples into two locations--subcutaneous and in the abdomen. The tubes with the clear fluorescent sample simulate the clear CSF layer, and the overlying tissue simulates the epidural fat layer. Since the abdomen tissue of the mouse consists mainly of fatty tissue, we can assume that the optical properties of the abdomen are similar to the epidural fat in the lumbar section. The experiments were performed also on a live tissue with blood flow and other vital signs, therefore making this a good model for the next in vivo step. The accuracy of the estimation of the ratio between two fluorescent probes was tested for a large range of ratios using these experiments, with an average estimation error of 19.9%. In our previous experiments [31], performed in vitro on polyester phantoms and ex vivo on chicken breast tissue, the average estimation error was lower. It was less than 5% for the in vitro and 11% for the ex vivo experiments. One can notice that there is an increase in the mean error with an increase in the complexity of the overlying medium. This result indicates that the optical properties of the reference phantom, which is used for creating the calibration curve, are not sufficiently close to the properties of a live tissue. The reference phantom is made of polyester resin with titanium dioxide, thus its scattering [33] is similar to the scattering of human tissue, which has a rather flat scattering spectra in the NIR range [34]. On the other hand, the absorption coefficient of human tissue varies in the NIR range with some peaks in the 700-800 nm range. These absorption peaks do not exist in the polyester absorption spectra, and therefore the polyester reference phantom differs in its absorption spectra from a living tissue, in mice or human. We can assume that chicken tissue has more features in its absorption spectra that are similar to human spectra, and obviously living mice are even more similar. The concentration ratio estimation relies on the fact that the optical properties variation in the 700-800 nm range is negligible, and the energy attenuation due to photon propagation is identical  
Figure 3: Time-resolved intensity curve. The triangle symbols represent the subcutaneous fluorescence curve and the circle symbols represent the abdomen fluorescence curve. in these wavelengths. Therefore, there may be inaccuracies when calculating the ratio based on a calibration curve, which was created using a medium with a different absorption spectrum than the measured tissue. Nevertheless, the errors are still rather small and reasonable. The system's accuracy may be improved using a different reference phantom, which better resembles the human tissue optical properties, for instance by adding an absorber, such as blood to the polyester. Such an absorber may assist in creating an absorption spectrum similar to the human tissue spectrum. The fluorescent probes that were implanted subcutaneously had slightly lower estimation errors compared to those implanted deeper in the abdomen. This was explained by the low fluorescence intensity in the abdomen tubes, caused by a thicker overlying tissue. The decrease in the fluorescence intensity caused a lower signal to noise ratio and bigger errors. In addition, the concentration ratio estimation is based on the assumption that the measured fluorescence intensity ratio is affected mainly by the concentrations of the fluorophores and that the energy attenuation due to photon propagation is identical in both wavelengths.