Rafts are tethered to the actin cytoskeleton which has a pivotal role in maintaining their structure and integrity. The size of membrane rafts, like that of fenestrations, is below the limits of resolution of light microscopy and their visualization has mostly been achieved with fluorescence microscopy. In this study, we used 3D-SIM to establish the threedimensional structure of membrane rafts and liver sieve ALK5 Inhibitor II in vivo plates in the cell membranes of LSECs, and the topographical relationship between them. Furthermore, by manipulating membrane rafts and actin, we show how rafts might influence fenestrations, and conclude that rafts are the final regulatory step in the formation of transcellular pores, fenestrations and liver sieve plates. Fenestrations and rafts are both cell membrane structures that are below the limit of resolution of light microscopy. The morphology of fenestrations has been studied primarily using electron microscopy while that of rafts has been studied using fluorescence microscopy. 3D-SIM is a super-resolution fluorescence microscopy technique that provides the opportunity to simultaneously study both membrane rafts and fenestrations and their distribution in isolated LSECs. 3D-SIM provides high resolution images of fenestrations and associated structures, such as the cellular cytoskeleton. Here, we also applied 3D-SIM to visualize membrane rafts. Using the fluorescent raft stain, Bodipy FL C5 ganglioside GM1, membrane rafts were found to be aggregated preferentially in the perinuclear region of LSECs, with a more diffuse distribution in the peripheral cytoplasmic extensions, and were generally thicker than the surrounding cell membrane. This pattern of distribution of rafts was confirmed using TIRFM with two raft stains, Bodipy FL C5 ganglioside GM1 and NBD-cholesterol. With 3D-SIM, a few clustered rafts sections were also apparent in the peripheral regions of the cells. These were about 1–2 mm in diameter and some had a raised perimeter, consistent with predictions based on line tension. As reported previously, 3D-SIM revealed that fenestrations are clustered in groups of 10–100 fenestrations called liver sieve plates that occupy 5–10% of the entire cell membrane. Moreover, the SIM images revealed that there is a distinct inverse distribution between liver sieve plates and membrane rafts. On the basis of this observation, we investigated whether manipulating membrane rafts had any effect on fenestrations. Most studies of membrane rafts have used various agents to isolate raft or non-raft membranes. At low concentrations, 7KC disrupts membrane rafts by disordering lipid membranes, while at much higher concentrations than we used 7KC can also induce apoptosis. Triton X-100 is a detergent that has been used to separate detergent-resistant membranes from cells. This has usually been undertaken using high concentrations above the Critical Micelle Concentration which are associated with cell lysis. In our experiments we used a much lower concentration in order to study cell membranes that have remained intact. Triton X-100 still penetrates cell membranes in the monomeric form and also increases the formation of rafts. Here we used these two agents to assess their effect on the morphology of the LSEC cell membrane. Treatment with 7KC was associated with increased number and diameter of fenestrations in vitro and increased diameter of fenestrations in vivo. Triton X-100 was associated with a reduced number of fenestrations. The fluorescent stains, LAURDAN and NBD-cholesterol confirmed an effect of low concentrations of 7KC and Triton X-100 on membrane rafts.