## 5 Discussion and Conclusions
Possibly image filtering and processing could potentially aid in improving image quality using nonoptimal imaging settings. The feasibly of this approach would be possible, particularly in increasing field of view; however, it is unlikely that after processing, techniques can compensate for restrictions in working distance, which why this issue is problematic. In addition to the limitation discussed above, the diffusion time acquired when using the two objective lenses were different. According to the theoretical relationship between the diffusion constant and diffusion time in the context of size of detection volume [11], the observed difference is larger than the expected shift. This discrepancy in the observed diffusion time acquired for the two different objective lenses cannot be explained by the size of focal volume. To date, a limited numbers of studies have investigated the relationship between focal volume optics and FCS data [39]. We further speculate that this effect is related to the results demonstrating an inverse relationship of laser power on the measured diffusion time. The study confirms an expected relationship between autocorrelation and laser power close to the equilibrium condition, i.e., at short lag times (\(\tau\to 0\)); however, at \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Parameters** & **Conditions** & \multicolumn{2}{c}{**Observations and Recommendations**} \\ \hline Objective lens & High NA objective lenses (NA \(>\) 1) are preferred for FCS; however, for imaging MPM the long working distances normally restricts NA. & \(\bullet\) & Higher NA leads to less distortions in FCS data and more reliable diffusion measurements. \\ \hline \multirow{4}{*}{Concentration range} & The concentration should preferably be in nM range for FCS, but for imaging the concentration range can vary by several orders of magnitudes. & \(\bullet\) & Lower NA (NA \(<\) 1) objective lenses can be used but careful validation is required. \\ \cline{2-3}  & In MPM-FCS the excitation power will determine the excitation and detection volume. & \(\bullet\) & Look for inverse relationship of laser power on the measured diffusion time using simple model fit. \\ \hline \multirow{4}{*}{Time binning} & TCSPC raw data has a sparse binary & \(\bullet\) & The optimal range of time binning should be validated for the specific experiments. \\  & data format, and time binning is & \(\bullet\) & Photon count per time bin should increase above binary value \\  & required in order to perform & \(\bullet\) & Photon distribution over time binning should follow Poison distribution. \\ \hline \multirow{4}{*}{Autocorrelation algorithm} & Multi-tau and extended data fitting models have been proposed in the literature. Here it is shown that linear-tau and simple model fitting is preferred for the data analysis. & \(\bullet\) & Multi-tau view can be used for fast data assessment during acquisition \\ \cline{1-1} \cline{2-3}  & & \(\bullet\) & For data analysis, linear tau and simple model are preferable to enable systematic validation of parameters above. \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Summary of results describing the parameters to be considered for FCS measurements. nonequilibrium condition, i.e., time intervals corresponding to the diffusion, the measured diffusion time was surprisingly found to be dependent on laser power. This finding can be conceptually understood in view of a dimensional analysis of [PERSON]'s diffusion law, as the diffusion time will vary when the surface area for which the diffusion constant is defined changes given that the diffusion constant remains constant. Therefore, the measured diffusion time should be inversely proportional to the surface area to size ratio, i.e., the square root of the radius of the detection volume, which in turn is proportional to the excitation power. This inverse relationship was confirmed in the data. To conclude, this guideline provides concepts and principles required for implementing FCS in combination with MPM.