## 5 Discussion and Conclusions
It includes crucial validation experiments that should be performed on each experimental system before undertaking FCS-MPM and that should be adapted for each specific study. Furthermore, it highlights some important limitations of the methodology, particularly when aiming at combining FCS with an experimental set up allowing for imaging MPM. Based on the novel insights on the relationship between laser power and measured diffusion times, the hope is to stimulate for the future development of protocols compensating for this effect potentially being able to work around these issues and ultimately allow MPM-FCS to become the highly useful tool in biomedical research as it is promised to be. The following are available online at [[https://www.mdpi.com/2076-341](https://www.mdpi.com/2076-341)]([https://www.mdpi.com/2076-341](https://www.mdpi.com/2076-341)) 7/11/5/2122/s1, Figure S1: The effect of time binning on generating FCS curve obtained from Rhodamine B solution at a concentration of 10 nM. The data was obtained from red channel (580/150 nm) at 800 nm excitation wavelength, 25 mW excitation intensity using 40\(\times\) (NA 0.8) objective lens, Figure S2: The effect of time binning on photon count rate obtained from Rhodamine B solution at concentration 10 nM. The data was obtained from red channel (580/150 nm) at 800 nm excitation wavelength, 25 mW excitation intensity using 40\(\times\) (NA 0.8) objective lens, Figure S3: Plot of the (a) autocorrelation amplitude function G(0) acquired using simple data fit (G\({}_{0}\)-(I/I\({}_{0}\))\({}^{3/2}\)) and (b) number of particles N using data fit (N-(I/I\({}_{0}\))\({}^{3/2}\)) on autocorrelated data (time binning 10,000 ns) from experimental measurements of a Rhodamine B solution (10 nM), the 40\(\times\) (NA 0.8) objective lens as a function of different laser powers, (c) average photon count as a function of excitation power. Data points in blue and red line is fitting. Figure S4: (a) Plot of the diffusion time acquired using simple data fit (\(\tau\)-1/(I\({}_{0}\))\({}^{0.5}\) on autocorrelated data 10,000 ns) from experimental measurements of a Rhodamine B solution (10 nM), the 40\(\times\) (NA 0.8) objective lens as a function of different laser powers. Data points in blue. Red lines represents fitting to two different models (\(\tau\)-1/(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 (b). Project design and planning, J.J. and M.B.E. ; data acquisition, J.J.; data analysis, J.J. and M.B.E. ; data and results discussion, J.J., M.B.E. and J.E. ; measurements and data analysis, J.J.; supervision, M.B.E. and J.E. ; writing--original draft preparing, J.J. and M.B.E. ; writing--editing and reviewing, J.J., M.B.E. and J.E. ; fund acquisition and project administration, M.B.E. All authors have read and agreed to the published version of the manuscript. Financial support was provided by Swedish Research Council (VR 2015-05002) and the [PERSON] foundation. The authors would like to thank [PERSON], Center for Cellular Imaging Core Facility, University of Gothenburg, for valuable input on the project. The authors declare no conflict of interest.