Membranes are involved in almost every biological process and yet their organization and regulation are still not fully understood. Current membrane model, proposed by Simons and Van Meer in 1988, suggests that lipids form different microdomains within the bilayer and provide platforms for lipid-protein, protein-protein interactions and cell signaling processes. Since these membrane microdomains have small sizes---from 10 and up to 700 nm, consequently advanced microscopy techniques are necessary to investigate their properties and behaviors. In this dissertation, we used a combination of different spectroscopic and microscopic methods namely Generalized Polarization (GP), Fluorescence Correlation Spectroscopy (FCS), Fluorescence Lifetime Imaging Microscopy (FLIM), Forster Resonance Energy Transfer (FRET), and probabilistic GP-Lifetime (GPtau) to study microdomains fluidity effects on proteins dynamics and functions. Using Giant Unilamellar Vesicles (GUVs) made of native apical membranes, isolated from two regions of rat small intestine, we were able to resolve a mystery behind a function of an integrated membrane protein- sodium dependent phosphate co-transporter type 2b (NaPi2b), behavior. It was known for several years that this protein has more abundant expression in the jejunum, compare to the duodenum, but activity within these two regions was similar. Therefore, performing single point FCS measurements on both labeled NaPi2b and membrane microdomains in same location, we were able to show that jejunum has a unique NaPi2b protein cluster (pentamer), which resides in bigger microdomains compare to other clusters of NaPi2b. Measuring local fluidity of these microdomains, in contrast to mean fluidity of a whole membrane, we showed that they are more solid, compare to other microdomains, where NaPi2b reside, suggesting that these pentamers are inactive. Applying combination of FLIM-FRET techniques and fluidity measurements we were able to show that soluble Klotho binds specific lipid within rafts; disrupting them and shifting basolateral membrane in live cells towards more fluid state. Even though we could measure all of these parameters, visualization of the microdomains within the membranes was still challenging, given the resolution of the microscopy techniques. To improve membrane microdomains visualization, we developed a probabilistic GPtau method as a novel approach to enhance a contrast in intensity GP images.