###### Abstract  
Multiphoton laser scanning microscopy (MPM) has opened up an optical window into biological tissues; however, imaging is primarily qualitative. Cell morphology and tissue architectures can be clearly visualized but quantitative analysis of actual concentration and fluorophore distribution is indecisive. Fluorescence correlation spectroscopy (FCS) is a highly sensitive photophysical methodology employed to study molecular parameters such as diffusion characteristics on the single molecule level. In combination with laser scanning microscopy, and MPM in particular, FCS has been referred to as a standard and highly useful tool in biomedical research to study diffusion and molecular interaction with subcellular precision. Despite several proof-of-concept reports on the topic, the implementation of MPM-FCS is far from straightforward. This practical guideline aims to clarify the conceptual principles and define experimental operating conditions when implementing MPM-FCS. Validation experiments in Rhodamine solutions were performed on an experimental MPM-FCS platform investigating the effects of objective lens, fluorophore concentration and laser power. An approach based on analysis of time-correlated single photon counting data is presented. It is shown that the requirement of high numerical aperture (NA) objective lenses is a primary limitation that restricts field of view, working distance and concentration range. Within these restrictions the data follows the predicted theory of Poisson distribution. The observed dependence on laser power is understood in the context of perturbation on the effective focal volume. In addition, a novel interpretation of the effect on measured diffusion time is presented. Overall, the challenges and limitations observed in this study reduce the versatility of MPM-FCS targeting biomedical research in complex and deep tissue--being the general strength of MPM in general. However, based on the systematic investigations and fundamental insights this report can serve as a practical guide and inspire future research, potentially overcoming the technical limitations and ultimately allowing MPM-FCS to become a highly useful tool in biomedical research. laser scanning multiphoton microscope (MPM); fluorescence correlation spectroscopy (FCS); molecular diffusion +
Footnote †: journal: Applied Sciences  
can be avoided in MPM. Other advantages in applying MPM are increased imaging depth and reduced photo toxicity. Since the probability of nonlinear 2 PE depends on the intensity squared, tightly focused ultrashort pulsed lasers (i.e., operating in the femto- or picosecond regime) are required to yield efficient excitation probability enabled imaging. By using objective lenses with high numerical aperture (NA), the excitation volume is restricted to the order of less than one femtoliter inside sample. MPM has demonstrated to be particularly promising for 3D imaging of thick biological specimens such as human skin in vivo and ex vivo [2; 5; 6]; however, the technology is primarily qualitative and quantitative data are lacking. Therefore, MPM in combination with fluorescence correlation spectroscopy (FCS) being a quantitative technology has been proposed [7; 8; 9; 10]. The conceptual basis and experimental realization of FCS were in fact introduced already in the early 1970s [11; 12; 13]. The method is based on measuring the rates of decay of spontaneous concentration fluctuations of fluorophores present in a confined detection volume. In combination with mathematical autocorrelation of the recorded signal fluctuations, the acquired data potentially yields information related to both diffusion statistics and chemical kinetics. The early FCS experiments were based on measuring the fluorescence fluctuations over time in a defined open volume of solution.