We also inves gated phase separa on as a func on of sampling depth. For this, we recorded images at different depths over a total depth range of 22 μm within a 20% drug loaded film (replicate experiment) at the 30 min mepoint (Figure ). The images reveal significantly different distribu ons as a func on of sampling depth. At the top and bo om imaging depths, in the vicinity of the glass cover slips, near con nuous drug rich domains are observed, sugges ng that the drug has preferen ally phase separated toward the glass during water penetra on, while the polymer has concentrated towards the middle of the film. Four planes at different depths are also shown in Figure (top). While phase separa on is evident at each of these layers, the layers near the top and bo om (closest to the glass cover slips) show more bicon nuous drug and polymer domains across large sec ons of the image matrix area, while in the middle of the layer, the drug rich phase is more predominantly represented by discrete droplets in a con nuous polymer-rich phase with the con nuous drug layer being restricted to the matrix-buffer interface. These substan ally different observa ons highlight the importance of sampling setup, and in this case in par cular, being aware of the poten al of glass-matrix interfaces introducing artefacts into the observed phase behavior. In our studies, we have by default imaged the film close to coverslip closest to the microscope objec ve to maximize spa al resolu on and signal strength and therefore image quality (the detrimental effect of sampling depth on image quality is evident from the four planes shown in Figure ). However, these effects should be kept in mind, when interpre ng the images with different drug loadings.