[25,26,27,28]. In order to analyse subcellular structures by ET in the state ‘close to native’, samples should be fixed by high pressure freezing, which allows immobilization within milliseconds, fol- lowed by freeze substitution [29]. Because FVs are relatively large compartments, their 3D reconstruction requires serial sectioning and joining of tomograms. Here we demonstrate that high pressure freezing and ET of serial sections, supported by freeze-fracture and immunocyto- chemistry, give a new insight into the structure and organization of FVs in umbrella cells. We also compared the arrangement of FVs during distension-contraction cycle of the urinary bladder, and analysed intermediate cells with respect to the occurrence of FVs. Results High pressure freezing gives a new insight into the ultrastructure of urothelial cells Rapid immobilization of structures by cryo-fixation is currently the method of choice for EM studies of biological tissues. In this work, we compared the ultrastructure of urothelial cells obtained by high pressure freezing (HPF) followed by freeze substitution (FS) with that obtained by chemical fixation followed by ethanol dehydration. In both cases, the specimens were embedded in Epon. The general appearance of the urothelium was comparable; a clear distinction between the structure of basal, intermediate and umbrella cells was seen. On the other hand, samples prepared by the two fixation methods differed significantly in details (Figs. 1A, B). In samples prepared by HPF-FS, numerous vesicles were flat, with thickened and non-thickened membrane domains clearly discriminated in cross-sections (Figs. 1B, 2A, B). In chemically fixed cells, vesicles were often dilated (Fig. 1A). The ultrastructure of other membrane compartments, such as Golgi apparatus, endoplasmic reticulum, mitochondria, were also superior after HPF-FS compared to chemical fixation. Next, we compared HPF-FS and chemical fixation, followed by Lowicryl embedding, for their use in immunocytochemistry. Urothelial cells immunolabelled with anti-AUM antibody in both cases occupied the same location and yielded comparable densities of gold particles (Figs. 1C, D). The difference between HPF-FS and chemically fixed, Lowicryl embedded samples was not in the density of the immunolabelling, but in the preservation of the ultrastructure. In the HPF-FS samples, anti-AUM labelled vesicles were flattened, while in the chemically fixed samples, vesicles were more dilated (Figs. 1C, D). Mature fusiform vesicles of umbrella cells are flattened discs Umbrella cells contained numerous mature FVs (mFVs), recog- nized as elongated structures measuring up to 1.2 mm in their transverse diameter (Fig. 2A). Their lumen appeared as a very narrow slit (ranging 5–10 nm in the central region of the vesicle), slightly dilated only at the rims of vesicles (Fig. 2A). The two opposing membranes were clearly seen as 12 nm thick AUM, identical to those seen on the apical cell surface (Figs. 2A, B). Immunolabelling showed that flattened parts of mFVs were densely labelled with anti-AUM antibodies (Figs. 1C, D). Furthermore, freeze-fracturing showed that 16-nm uroplakin particles formed the central region of mFVs (Fig. 2C). On the other hand, the rounded, omega shaped rim of mFVs, contained non-thickened membrane, which was UP-negative and smooth on freeze-fracture replicas (Figs. 2A, C, 5A). On HPF-FS urothelial samples, we performed ET to study the 3D structure of mFVs. Since FVs measured up to 1.2 mm in diameter, we had to join 3 to 5 serial tomograms, each 300 nm thick, in order to reconstruct the whole volume of an mFV (Table S1). In the model Electron Tomography of Fusiform Vesicles of an individual mFV, it was seen that it had the shape of a flattened disc, which is circular when viewed from the side (Figs. 2D–G, Movies S1, S2). The rim of the vesicle was slightly dilated. The organization of mature fusiform vesicles changes between central and subapical cytoplasm of umbrella cells Mature FVs in umbrella cells had the morphology of flattened disks. However, there were two distinct regions of the cytoplasm regarding the organization of mFVs: central and subapical (Fig. 3A). The border between central and subapical cytoplasm corresponded to the position of the cytokeratin 20 network [30,31], which was seen as a narrow line by anti-CK20 immunofluorescence (Fig. S1). In the central cytoplasm of umbrella cells, mFVs were usually organized into stacks, where mFVs were closely packed in parallel arrangement to each other (Fig. 3B). Stacks were more obvious in the HPF-FS than in chemically fixed umbrella cells (Figs. 1B, D versus Figs. 1A, C). Stacks contained 4 to 15 FVs of similar lengths. mFVs in stacks were densely labelled with anti-AUM antibody (Fig. 1D). Stacks of mFVs were not surrounded by other membrane compartments, which suggested that they represented a relatively static, mature situation. Three-dimensional recon- structions showed that mFVs in stacks have the same flattened-disk shape as individually positioned mFVs (Fig. 3C, Table S1and Movies S3, S4). In the subapical cytoplasm of umbrella cells, mFVs were sparser than in the central cytoplasm, and were usually seen as individual entities (Fig. 3A). Immunolabelling of ultrathin cryo-sections with anti-Rab27b antibody showed that some mFVs from both central and subapical region were labelled (Fig. 3D), while some mFVs remained non- labelled. In addition, weak anti-Rab27b labelling was observed also on some immature FVs. Orientation of mature fusiform vesicles changes during distension-contraction cycle of the urinary bladder We compared the organization of mFVs in the umbrella cells of distended and contracted bladders. In contrast to other studies, which employed different solutions and manipulations (e.g. post-mortem stretching, in vitro chambers), to acquire distended or contracted bladders [7,32,33], we collected bladders just before and immediately after micturition in order to obtain physiologically distended and contracted bladder, respectively. During bladder filling, the shape of umbrella cells changed from cubical to flattened, the distinction between cells subapical and central cytoplasm became less obvious, but the morphology of mFVs did not change (Figs. 4A–G). The reorientation of mFVs was observed in the subapical cytoplasm during distension and contraction of the urinary bladders. In the contracted bladders, 62% of mFVs were oriented perpendicular and 38% parallel to the apical cell surface (Fig. 4A). Contacts of hinge regions of mFVs with the apical plasma membrane were observed (Figs. 4B–F). In the distended bladder, 35% of mFVs were oriented perpendicular and 65% parallel to the apical cell surface (Fig. 4G). The difference was statistically significant (p = 0, 0375). Mature fusiform vesicles are limited to umbrella cells Umbrella cells contained numerous vesicles, which mainly represented individually positioned or stacked mFVs described above. Occasionally, stacks were not uniform; they had mFVs only at one side of the stack, while on the other side of the stack vesicles were shorter and more convex. These types of the stack were PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e32935