Continuous fluorescence microphotolysis (CFM) is a powerful tool to analyze molecular mobilities and association reactions in single living cells but has mostly been restricted to diffraction limited focal volumes. Recently, we introduced the theoretical and experimental framework to combine CFM with super-resolution microscopy by utilizing a 4Pi point spread function in mobility analysis.Here we show that this methodology can be readily applied to living yeast cells under physiological buffer conditions using water-immersion lenses. Yeast cells are relatively small with a typical diameter of 5 μm and the intracellular individual compartmentalization with a large vacuole and the nucleus results in small volume elements with unhindered diffusion. Therefore diffusion measurements with an engineered point spread function should be beneficial to recover the diffusion coefficient as this approach is less susceptible to improper positioning of the laser.Using 4Pi CFM we were able to clearly recover the diffusion coefficient of GFP in the cytoplasm and the nucleus of living yeast cells. Additionally, the mobility of GFP-tagged proteins involved in nucleo-cytoplasmatic transport was analyzed. While the diffusion coefficient of a GFP-tagged cargo was determined to be in a range expected for a molecule of this respective size, we found evidence that the diffusion coefficient of a GFP-tagged transport receptor was reduced compared to the expected value for purely free diffusion. This might indicate that the molecule is subject to a certain degree of unspecific binding in the cytoplasm of yeast.