Rate of kinetically stable proteins compared to urea titrations at room temperature, e.g. for ligand-bound maltose binding protein. 2. High temperature has little effect on the intrinsic proteolysis rate; high urea concentrations however inhibit the enzyme. 3. Temperature gradients reveal quickly self-aggregating unfolded species while urea may dissolve aggregates. Taken together, both approaches have complementary benefits: FASTpp gives insight into thermal stability, Pulse Proteolysis into equilibrium unfolding. FASTpp, however, requires less experimental time. Considering the broad range of folds that can be analysed by FASTpp and the specificity, robustness and speed of the method, we anticipate a broad range of future applications. Minimal sample preparation requirements and use of standard molecular biological techniques allow applications in protein INCB28060 abmole engineering, cell biology and biomedical research. Liver sinusoidal endothelial cells act as a filter between the lumen of the hepatic sinusoid and the surrounding hepatocytes. A major role of the LSEC is to minimize any barrier for the bi-directional transfer of small or soluble substrates between blood and the extracellular space of Disse, while excluding larger circulating particles such as blood cells, platelets and chylomicrons. This physiological role is achieved by the presence of numerous transcellular pores in LSECs called fenestrations. Fenestrations are approximately 50–150 nm in diameter and most are aggregated into groups of 10–100, so-called liver sieve plates. The diameter and number of fenestrations are altered by various liver diseases, diabetes mellitus and old age and are influenced by cytokines and hormones. Alteration in the size and number of fenestrations influences the hepatic trafficking of lipoproteins, clearance of pharmaceutical agents, liver regeneration and interactions between lymphocytes and hepatocytes. No markers have been reported that specifically label fenestrations and the mechanisms for the regulation of their formation and size remain unclear. The most consistent findings of biological relevance are that fenestrations are increased by actin-disrupting agents and by the angiogenic cytokine, vascular endothelial growth factor. The mechanisms that regulate fenestrations need to be clarified in order to develop strategies to improve lipoprotein metabolism in old age and liver disease, and to enhance liver regeneration. Fenestrations are smaller than the limit of resolution of light microscopy and most studies have relied upon electron microscopy with inherent problems related to fixation of tissue. Recently three dimensional structured illumination fluorescence light microscopy was applied to LSECs and their fenestrations. 3DSIM is an ultra-high resolution light microscopy technique that uses interference patterns to convert structures below the resolution limit of light microscopy into observable ones by generating difference/beat frequencies called Moire´ fringes. The morphology of the fenestrations and sieve plates was very effectively resolved by 3D-SIM, providing for the first time a detailed three-dimensional map of their structure. Using the plasma cell membrane stain Cell-Mask Orange, discrete membrane structures were identified between the sieve plates. On the basis of their size and appearance we postulated that these structures are membrane rafts and potentially involved in the regulation of sieve plates. Membrane rafts are lipid-ordered domains in cell membranes that vary in size from 10–200 nm, and may aggregate to form micrometer-sized structures.