## 5 Discussion and Conclusions  
The aim of this practical guideline is to clarify the conceptual principles and define experimental operating conditions in order to facilitate implementation of MPM-FCS using TCSPC. Of specific interest has been to validate and explore the potential to perform MPM-FCS using settings compliable with biomedical studies in turbid media, to allow for combination of FCS and imaging in biomedical studies. In particular, the choices of objective lens, concentration range and laser power were investigated. Furthermore, a protocol for converting raw data photon counts to autocorrelated signal was presented and the effect of binning was explored. Table 1 describes a summary of key findings along with the parameters and conditions to be considered for MPM-FCS measurements. Two of the key parameters to be considered when performing FCS measurements using TCSPC are optimum time binning for autocorrelation and threshold range of excitation power, as also discussed by others [21]. In this study, autocorrelation curves extracted directly from the SPCM64 software using multi-tau binning and the linear-tau algorithm were compared. The analysis revealed that the data were distorted when multi-tau algorithm was used, as compared to the linear-tau binning. Instead, multi-tau was preferred for fast assessment of data during acquisition and Linear-tau algorithm was preferred for the analysis. The effect of time binning on FCS data was investigated. It was concluded from the results that it is important to find the optimum time binning for an experimental FCS which is essential to be validated before performing any FCS measurements. In this study, the optimal time binning was found to be 10\({}^{4}\)-10\({}^{5}\) ns in order to allow for measurement of diffusion times in the order of 0.01-10 ms. Furthermore, the FCS analyses were performed using different excitation powers. It is clear from the measurements that the range of applied excitation power varies for objective lenses with different NA. Thus, it is necessary to validate the optimal range of excitation power and time binning for a certain experimental setup to perform FCS measurements. Figure 9: (**a**) Plot of the diffusion time acquired using simple data fit (\(\tau\)-1/(I/I\({}_{0}\))\({}^{0.5}\) on autocorrelated data 10,000 ns) from experimental measurements of a Rhodamine B solution (10 nM), the 63\(\times\) (NA 1.2) objective lens as a function of different laser powers. Data points in blue. Red lines represent fitting to two different models (\(\tau\)-1/(I/I\({}_{0}\))\({}^{0.5}\) and \(\tau\)-(I/I\({}_{0}\))) demonstrating a 1/\(\tau\) dependency where r is related to the size of focal volume, which will increase as laser power increases as shown in (**b**). One of the important aspects of extending the applicability of FCS in biomedical research is the possibility of using the same objective lens for both imaging and FCS measurements. The results demonstrated the impact of numerical aperture on FCS measurements obtained from two different microscope objectives 63\(\times\) (NA1.2, working distance 0.28 mm) and 40\(\times\) (NA 0.8, working distance 1.8 mm) from Rhodamine B solution at 10 nM concentration. It is evident from the results that both objective lenses had the ability to enable FCS measurements; however, the amplitude of the autocorrelation curve, G(0), acquired using the 40\(\times\) (NA 0.8) objective lens was comparatively lower than 63\(\times\) (NA 1.2) objective lens. This is expected as, a higher NA generate a smaller excitation volume, thus less molecules results in a higher G(0) value [9; 16]. Furthermore, the concentration interval for which the measurements were possible was less for the 40\(\times\) objective. Thus, the applicability of utilizing the long working distance 40\(\times\) objective lens for FCS was found to be lower. An alternative approach for combining FCS and MPM would be to expand the feasibility of using high NA and short working distance objective lenses for imaging purposes.