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Since the recent boost in the usage of electron microscopy in life-science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused-ion beam scanning electron microscopy (FIB-SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold-labelled breast cancer SKBR3 cells was to visualise gold-labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 μm 2 cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back-scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB-SEM. Cross-sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms., (© 2018 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.)

In recent years, electron tomography has improved our three-dimensional (3D) insight in the structural architecture of cells and organelles. For studies that involve the 3D imaging of stained sections, manual annotation of tomographic data has been an important method to help understand the overall 3D morphology of cellular compartments. Here, we postulate that template matching can provide a tool for more objective annotation and contouring of cellular structures. Also, this technique can extract information hitherto unharvested in tomographic studies. To evaluate the performance of template matching on tomograms of stained sections, we generated several templates representing a piece of microtubule or patches of membranes of different staining-thicknesses. These templates were matched to tomograms of stained electron microscopy sections. Both microtubules and ER-Golgi membranes could be detected using this method. By matching cuboids of different thicknesses, we were able to distinguish between coated and non-coated endosomal membrane-domains. Finally, heterogeneity in staining-thickness of endosomes could be observed. Template matching can be a useful addition to existing annotation-methods, and provide additional insights in cellular architecture.

Transmission electron microscopy images acquired under tilted-beam conditions experience an image shift as a function of defocus settings - a fact that is exploited as a method for defocus determination in most of the automated tomography data collection systems. Although the method was shown to be highly accurate for a large variety of specimens, we point out that in its original design it can strictly only be applied to images of untilted samples. The application to tilted samples and thus in automated electron tomography is impaired mainly due to a defocus change across the images, resulting in reduced accuracy. In this communication we present a method that can be used to improve the accuracy of the basic autofocusing procedures currently used in systems for automated electron tomography.

Internal viewing of the cellular organization of hyphae by scanning electron microscopy is an alternative to observing sectioned fungal material with a transmission electron microscope. To study cytoplasmic organelles in the hyphal cells of fungi by SEM, colonies were chemically fixed with glutaraldehyde and osmium tetroxide and then immersed in dimethyl sulfoxide. Following this procedure, the colonies were frozen and fractured on a liquid nitrogen-precooled metal block. Next, the fractured samples were macerated in diluted osmium tetroxide to remove the cytoplasmic matrix and subsequently dehydrated by freeze substitution in methanol. After critical point drying, mounting, and sputter coating, fractured cells of several basidiomycetes were imaged with field-emission SEM. This procedure produced clear images of elongated and spherical mitochondria, the nucleus, intravacuolar structures, tubular- and plate-like endoplasmic reticulum, and different types of septal pore caps. This method is a powerful approach for studying the intracellular ultrastructure of fungi by SEM.

Dolipore septa and septal pore caps (SPCs) in filamentous basidiomycetes may play an important role in maintaining the integrity of hyphal cells. We have investigated the ultrastructure of the dolipore septum and the SPC in Rhizoctonia solani hyphal cells after high-pressure freezing, freeze substitution, and Spurr embedding. We visualized the SPC with associated cell ultrastructures in three dimensions by automated electron tomography of thick-sectioned cells, followed by 3D tomographic reconstructions. Using these methods we were able to document the passage of mitochondria through the SPC, small tubular membranous structures at the entrance of the septal pore channel, filamentous structures connecting the inner side of the SPC with pore-plugging material, thin filaments anchoring the pore-plugging material with the plasma membrane, small vesicles attached to the plugging material, and tubular endoplasmic reticulum continuous with the base of the SPC. We hypothesize that the SPC, the filamentous structures, the plugging material, and the endoplasmic reticulum act in a coordinated fashion to maintain cellular integrity, intercellular communication, and the transport of solutes and cell organelles in the filamentous fungus R. solani., (Copyright 2000 Academic Press.)

The septal pore cap (SPC) of Trichosporon sporotrichoides CBS 8245 is vesicular-tubular, connected with flat-tubular endoplasmic reticulum (ER), and stains densely with zinc/iodine/osmium tetroxide, as does the ER. The SPC of Schizophyllum commune CBS 340.81 is more complex, about 600 nm in diameter, with perforations of 80-120 nm diameter, and stains less densely with zinc/iodine/osmium tetroxide than the ER. In high-pressure frozen and freeze-substituted hyphae of T. sporotrichoides the ER is present parallel to the dolipore septa, and electron-dense material occurs opposite the septal pore channel; the SPC rarely showed smooth vesicular-tubular membranes, suggesting that this is an ephemeral function of the SPC. The SPC of S. commune has a smooth outer and inner membrane, which enclose a matrix with a palisade-like substructure. A thin layer of electron-dense material covers the inner surface of the SPC of S. commune, from which beaded filamentous structures connect the SPC and the pore-occluding material. These filamentous structures may maintain the intracellular position of the SPC and possibly play a role in plugging the septal pore channel. The septal pore swellings of T. sporotrichoides contain more 1,6-beta-glucan than the septum, and intracellular glucans are also present near the septal pore channel. This cytosolic 1,6-beta-glucan in T. sporotrichoides may serve as a matrix to keep the tubular membranous structures of the SPC together. In contrast, 1,6-beta-glucan is not observed in the SPC and in the pore-occluding material of S. commune, and hyphal septa of this species show less labelling of 1,6-beta-glucan than the septal swelling. The evolutionary transition from simple to more complex types of SPCs may have resulted in a requirement for different components to maintain the morphological integrity and cell biological function.

A combination of cryofixation without pre-treatment, freeze-substitution and low-temperature embedding was used to prepare specimens of Penicillium chrysogenum for electron microscopy. To produce specimens which are thin enough for appropriate cryofixation, the P.chrysogenum colonies were grown between dissected-dialysis tubing on an agar plate, which in addition allowed longitudinal sectioning. In contrast to classical chemical fixation, this preparation procedure resulted in excellent preservation of ultrastructure. Furthermore, the penicillin biosynthetic enzyme acyltransferase could be unequivocally located by immunogold labelling, indicating a preservation of antigenic properties of the specimen. Labelling density was not conspicuously affected when using different freeze-substitution media, but it was reduced after embedding in Epon 812.

We examined the suitability of freeze-substitution and Lowicryl HM20 embedding of aldehyde-fixed rat brain to localize several neural antigens at the ultrastructural level. The following rabbit polyclonal and mouse monoclonal antibodies were used: affinity-purified polyclonal immunoglobulins G raised to B-50/GAP43 (a membrane-anchored, growth-associated protein); affinity-purified polyclonal immunoglobulins G to human glial fibrillary acidic protein (GFAP; a subunit of glial filaments); a polyclonal antiserum raised to adrenocorticotropic hormone[25-39] (a neuropeptide present in dense-core granules); a polyclonal antiserum raised to myelin basic protein (a protein present in compact myelin of the central nervous system); and mouse monoclonal antibodies to synaptophysin (an integral membrane protein of small synaptic vesicles). Rat mesencephalon was fixed by perfusion with buffered 2% glutaraldehyde and 4% paraformaldehyde, cryoprotected, and frozen in liquid nitrogen. Freeze-substitution of tissue was performed with anhydrous methanol and 0.5% uranyl acetate at -90 degrees C. Semi-thin Lowicryl sections were used for light microscopic visualization of B-50 in the ventromedial mesencephalic central gray substance. The procedure preserves well the ultrastructure of this region and the immunoreactivity of the selected antigens. This study shows that dehydration by freeze-substitution, combined with Lowicryl HM20 embedding at sub-zero temperature, provides a successful method of preparation of fixed brain tissue for ultrastructural studies, allowing immunogold localization of several neural antigens by double labeling in the same section and in serial sections.

The localization of the enzymes involved in penicillin biosynthesis in Penicillium chrysogenum hyphae has been studied by immunological detection methods in combination with electron microscopy and cell fractionation. The results suggest a complicated pathway involving different intracellular locations. The enzyme delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase was found to be associated with membranes or small organelles. The next enzyme isopenicillin N-synthetase appeared to be a cytosolic enzyme. The enzyme which is involved in the last step of penicillin biosynthesis, acyltransferase, was located in organelles with a diameter of 200-800 nm. These organelles, most probably, are microbodies. A positive correlation was found between the capacity for penicillin production and the number of organelles per cell when comparing different P. chrysogenum strains.

An antiserum was raised against the phosphatidylcholine transfer protein from rat liver by immunization of rabbits. The antiserum was shown to be specific for this protein. A double-antibody radioimmunoassay for the phosphatidylcholine transfer protein was developed. In order to economize the use of second antibody (immunobeads), the specific anti-phosphatidylcholine transfer protein-IgG fraction isolated by affinity chromatography was used. Phosphatidylcholine transfer protein was labelled with 125I by the glucose oxidase-lactoperoxidase method and purified from the reaction mixture by affinity chromatography. Approx. 80% of the tracer was immunoprecipitable. The operating range of the assay was from 4 to 50 ng of transfer protein. This assay was used to determine the levels of phosphatidylcholine transfer protein in the 105000 x g supernatant fractions of rat liver and Morris hepatomas 7777, 7787 and 9633. The values obtained for the tumors were in good agreement with results previously obtained by immunotitration of the phosphatidylcholine transfer activity (Poorthuis, B.J.H.M., Van der Krift, T.P., Teerlink, T., Akeroyd, R., Hostetler, K.Y. and Wirtz, K.W.A., Biochim. Biophys. Acta 600 (1980) 376--386). For normal and host liver, the values determined by the radioimmunoassay were 2--4-fold higher.

The content of cholesterol and cholesterol ester as well as the levels of acyl coenzyme A:cholesterol acyltransferase activity were determined in the microsomes from Morris hepatomas 7777, 5123D, and 7787. The free cholesterol content, expressed per mg microsomal protein, was significantly increased only in the microsomes from Morris hepatoma 7777 [47.8 +/- 0.4 micrograms (S.D.); p less than 0.001] and hepatoma 7787 (37.6 +/- 6.2 micrograms; p less than 0.01) as compared to normal liver (28.8 +/- 2.4 micrograms). The cholesterol ester content in the microsomes of the three different tumors did not significantly differ from that of normal liver (2.1 +/- 1.2 micrograms cholesterol per mg microsomal protein). The microsomal acyl coenzyme A:cholesterol acyltransferase activity was decreased in Morris hepatoma 7777 (8.6 +/- 2.3 pmol/min/mg protein; p less than 0.01) and in hepatoma 5123D (7.5 +/- 1.7 pmol/min/mg protein; p less than 0.02), and was normal in the hepatoma 7787 (16.5 +/- 7.8 pmol/min/mg protein) as compared to rat liver (16.0 +/- 2.9 pmol/min/mg protein). In a previous study (B. J. H. M. Poorthuis and K. W. A. Wirtz, Biochim. Biophys. Acta, 710: 99-105, 1982), this acyltransferase activity was shown to be stimulated by preincubation of rat liver microsomes with cholesterol-containing vesicles and the nonspecific phospholipid transfer protein. In this paper, a similar 4-fold stimulation of activity was observed for the microsomes of the various hepatomas investigated. The possible role of the nonspecific phospholipid transfer protein in intracellular cholesterol esterification is discussed.

To study the possible effects of chemical fixation upon antigenicity and structural preservation, the subcellular localization of LamB-LacZ hybrid proteins in Escherichia coli K-12 strains pop3234 and pop3299 was investigated both by cryo-ultramicrotomy and freeze-substitution. Immuno-gold labelling of sections of freeze-substituted bacteria showed the same localization of the hybrid protein as found after cryo-ultramicrotomy. The efficiency of labelling of the accumulated form of the hybrid protein was lower after freeze-substitution whereas the efficiency of labelling of the membrane-bound form showed no difference. Different fixatives and Lowicryl resins had no clear effect on the label-efficiency but the complex substitution medium, containing osmium tetroxide, uranyl acetate and glutaraldehyde, in combination with the apolar Lowicryl HM20 gave the best sectioning properties and membrane contrast. For this specific problem, although the somewhat better preservation after freeze-substitution, cryo-ultramicrotomy is to be favored since it is much less time-consuming, there are no freezing problems, ultrastructural preservation is sufficient and the theoretical benefits of freeze-substitution are not expressed.

A radioimmunoassay for the phosphatidylcholine-transfer protein from rat liver was used to measure levels of PC-transfer protein in rat tissues. The assay as described before (Teerlink, T., Poorthuis, B.J.H.M., Van der Krift, T.P. and Wirtz, K.W.A., Biochim. Biophys. Acta 665 (1981) 74-80) was modified in order to measure PC-transfer protein in tissue homogenates and subcellular membrane fractions. To this end both a detergent (Triton X-100) and a proteolytic enzyme inhibitor (aprotinin) were added to the assay medium. The radioimmunoassay measured levels of PC-transfer protein in the range of 5-50 ng and was specific for PC-transfer protein from rat tissues. Subcellular distribution studies showed that in 10% (w/v) homogenates of liver approximately 60% of the PC-transfer protein was present in the 105000 X g supernatant fraction, the remainder being evenly distributed over the particulate fractions. PC-transfer protein associated with the particulate fractions was almost completely removed by a single washing step, suggesting a dynamic equilibrium between membrane-bound and soluble PC-transfer protein. Both 105000 X g supernatants and homogenates of various rat tissues were assayed. The highest levels of PC-transfer protein were measured in liver and intestinal mucosa. Lower values were found in kidney, spleen and lung, whereas heart and brain contained hardly any PC-transfer protein. PC-transfer protein levels in regenerating rat liver did not differ significantly from levels in normal liver. In fetal lung a change in PC-transfer protein content during development was observed, with a clear maximum 2 days before term, suggesting an involvement of PC-transfer protein in the secretion of lung surfactant.

Rat tissues contain a nonspecific transfer protein which in vitro mediates the transfer of diacylphospholipids as well as cholesterol between membranes. This protein appears identical to sterol carrier protein. A specific enzyme immunoassay for this protein was developed using antibodies raised in rabbits, against a homogeneous protein from rat liver. This assay was based on the very high affinity of the nonspecific lipid transfer protein for polyvinyl surfaces. A reproducible adsorption was achieved by presenting the protein to the surface in the presence of a large excess of bovine serum albumin. The adsorbed protein was detected with specific immunoglobulin (IgG) isolated by antigen-linked affinity chromatography and a goat anti-rabbit IgG-enzyme conjugate. Adsorption was proportional to the amount of protein present, giving rise to a linear standard curve. The enzyme immunoassay measured transfer protein levels in the range 0.2-2 ng. The highest concentrations of transfer protein were found in liver and intestinal mucosa. Levels in other tissues including brain, lung, kidney, spleen, heart, adrenals, ovary and testis were 5-10-fold lower than in liver. In the fast-growing Morris hepatoma 7777 the concentration of nonspecific lipid transfer protein was approximately one-tenth of that measured in the host liver, whereas a reduction of 65% was observed in the slow-growing Morris hepatomas 7787 and 9633. Subcellular distribution studies showed that approx. 70% of the transfer protein was present in the soluble supernatant fraction.

Phospholipid transfer activities for phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were measured in three hepatomas of increasing growth rate and degree of dedifferentiation, the hepatomas of 9633 and 7777, and compared to the activities found in normal and host liver. A 2-3-fold increase was found in the phosphatidylcholine and phosphatidylinositol transfer activities in the fast-growing 7777 hepatoma, while these activities were moderately or not increased in the 7787 and 9633 hepatomas. Phosphatidylethanolamine transfer was found to be extremely low in all three hepatomas. The possible significance of these findings with respect to the altered phospholipid content and composition of the hepatoma membranes is discussed. The contribution of the phosphatidylcholine specific exchange protein to the total phosphatidylcholine transfer activity was determined in normal and host liver and in the hepatomas 7777 and 9633 with the aid o f a phosphatidylcholine exchange protein specific antiserum. To this end a new procedure for the purification of the phosphatidylcholine exchange protein from rat liver was developed which leads to a final purification factor of 5300 and a high overall yield of 17%. In addition, this protein was chemically and immunologically characterized and its properties were compared to those of the bovine phosphatidylcholine exchange protein purified in our laboratory previously.

The nonspecific lipid transfer protein (i.e., sterol carrier protein 2) from human liver was purified to homogeneity using ammonium sulfate precipitation, CM-cellulose chromatography, molecular sieve chromatography and fast protein liquid chromatography. Its amino acid composition was determined and found to be very similar to that of the nonspecific lipid transfer protein from bovine and rat liver with, as main feature, the absence of arginine, histidine and tyrosine. By way of a specific enzyme immunoassay using affinity-purified antibodies, the levels of nonspecific lipid transfer protein were determined in human livers. Levels varied from approximately 150 ng nonspecific lipid transfer protein per mg 105,000 X g supernatant protein for juvenile and adult humans to 40 ng per mg supernatant protein for a young infant. Levels of nonspecific lipid transfer protein in livers of infants with cerebro-hepato-renal (Zellweger) syndrome were extremely low (i.e., 2 ng per mg supernatant protein). Immunoblotting revealed the presence of crossreactive proteins of molecular masses of 40,000 and 58,000. The 40 kDa and 58 kDa proteins occurred in control livers, whereas only the 40 kDa protein was present in Zellweger livers. As in rat the 58 kDa protein could be demonstrated in a peroxisomal preparation isolated from an adult liver. A possible link between the occurrence of nonspecific lipid transfer protein and the presence of peroxisomes is discussed.

An antibody against the non-specific lipid transfer protein from rat liver was purified by immunoabsorbent affinity chromatography. This antibody in conjunction with protein A-colloidal gold was used to localize the transfer protein in rat liver by electron microscopy. Labeling by this immunocytochemical technique was found to be mainly restricted to the peroxisomes; low labeling was observed in the cytoplasm. Subsequent analysis of isolated peroxisomes by immunoblotting indicated that the non-specific lipid transfer protein (mol. wt. 14800) was absent from this organelle and that a protein of molecular weight 58000 was responsible for the immunological response. Immunoblotting of the membrane-free cytosol showed the presence of both proteins. It remains to be established to what extent the non-specific lipid transfer protein in the cytosol and the high-molecular weight protein in the peroxisomes are related.