We report that the dye nile red, 9-diethylamino-5H-benzo[a]phenoxazine-5-one, is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry. The specificity of the dye for lipid droplets was assessed on cultured aortic smooth muscle cells and on cultured peritoneal macrophages that were incubated with acetylated low density lipoprotein to induce cytoplasmic lipid overloading. Better selectivity for cytoplasmic lipid droplets was obtained when the cells were viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, > 528 nm) rather than red fluorescence (excitation, 515-560 nm; emission, > 590 nm). Nile red-stained, lipid dropletfilled macrophages exhibited greater fluorescence intensity than did nile red-stained control macrophages, and the two cell populations could be differentiated and analyzed by flow cytofluorometry. Such analyses could be performed with either yellow-gold or red fluorescence, but when few lipid droplets per cell were present, the yellow-gold fluorescence was more discriminating.Nile red exhibits properties of a near-ideal lysochrome. It is strongly fluorescent, but only in the presence of a hydrophobic environment. The dye is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution. Nile red can be applied to cells in an aqueous medium, and it does not dissolve the lipids it is supposed to reveal.

A rapid and simple method for the isolation of membranes from subcellular organelles is described. The procedure consists of diluting the organelles in ice-cold 100 mM Na 2COs followed by centrifugation to pellet the membranes. Closed vesicles are converted to open membrane sheets, and content proteins and peripheral membrane proteins are released in soluble form . Here we document the method by applying it to various subfractions of a rat liver microsomal fraction, prepared by continuous density gradient centrifugation according to Beaufay et al . (1974, J. Cell Biol . 61 :213-231) . The results confirm and extend those of previous investigators on the distribution of enzymes and proteins among the membranes of the smooth and rough endoplasmic reticulum . In the accompanying paper (1982, J. Cell Biol . 93 :103-110) the procedure is applied to peroxisomes and mitochondria .

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Improved, largely automated methods are described for the purification and analysis o peroxisomes, lysosomes, and mitochondria from the livers of rats injected with Triton WR-1339. With these new methods, it has become possible to obtain, in less than 6 hr and with reliable reproducibility, mitochondria practically free of contaminants, as well as the rarer cytoplasmic particles in amounts (about 100 mg of protein) and in a state of purity (95%) that make them suitable for detailed biochemical studies. The results obtained so far on these preparations have made more conclusive and precise previous estimates of the biochemical and morphological properties of the three groups of cytoplasmic particles. In addition, peroxisomes were found to contain essentially all the L-a-hydroxy acid oxidase of the liver, as well as a small, but significant fraction of its NADP-linked isocitrate dehydrogenase activity. Another small fraction of the latter enzyme is present in the mitochondria, the remainder being associated with the cell sap. The mitochondrial localization of the metabolically active cytoplasmic DNA could be verified. The relative content of the fractions in mitochondria, whole peroxisomes, peroxisome cores, lysosomes, and endoplasmic reticulum was estimated independently by direct measurements on electron micrographs, and by linear programming (based on the assumption that the particles are biochemically homogeneous) of the results of enzyme assays. The two types of estimates agreed very well, except for one fraction in which low cytochrome oxidase activity was associated with mitochondrial damage.

A review of the development and implementation of a 4-year medical student integrated ultrasound curriculum is presented. Multiple teaching and assessment modalities are discussed as well as results from testing and student surveys. Lessons learned while establishing the curriculum are summarized. It is concluded that ultrasound is a well received, valuable teaching tool across all 4 years of medical school, and students learn ultrasound well, and they feel their ultrasound experience enhances their medical education.

Nile red is a phenoxazone dye that fluoresces intensely, and in varying color, in organic solvents and hydrophobic lipids. However, the fluorescence is fully quenched in water. The dye acts, therefore, as a fluorescent hydrophobic probe. We utilized this novel property of nile red to develop a sensitive fluorescent histochemical stain for tissue lipids. Nile red was prepared by boiling Nile blue A under reflux for 2 hr in 0.5% H2SO4, and extracting the product into xylene. For staining, the purified dye is dissolved in 75% glycerol (1-5 micrograms/ml) and applied to frozen tissue sections. Tissue lipids then fluoresce yellow-gold to red, depending on their relative hydrophobicity. Using sections of liver and aorta from a cholesterol-fed rabbit, we assessed the value of Nile red as a stain for neutral lipids by comparing the staining pattern obtained with that produced by oil red O, a commonly used dye for tissue cholesteryl esters and triacylglycerols. In the cholesterol fatty liver, Nile red staining was comparable to that of oil red O. In contrast, Nile red staining of rabbit aortic atheroma revealed ubiquitous lipid deposits not observed with oil red O staining. These latter results suggest that Nile red can detect neutral lipid deposits, presumably unesterified cholesterol, not usually seen with oil red O or other traditional fat stains.

Interest in ultrasound education in medical schools has increased dramatically in recent years as reflected in a marked increase in publications on the topic and growing attendance at international meetings on ultrasound education. In 2006, the University of South Carolina School of Medicine introduced an integrated ultrasound curriculum (iUSC) across all years of medical school. That curriculum has evolved significantly over the 9 years. A review of the curriculum is presented, including curricular content, methods of delivery of the content, student assessment, and program assessment. Lessons learned in implementing and expanding an integrated ultrasound curriculum are also presented as are thoughts on future directions of undergraduate ultrasound education. Ultrasound has proven to be a valuable active learning tool that can serve as a platform for integrating the medical student curriculum across many disciplines and clinical settings. It is also well-suited for a competency-based model of medical education. Students learn ultrasound well and have embraced it as an important component of their education and future practice of medicine. An international consensus conference on ultrasound education is recommended to help define the essential elements of ultrasound education globally to ensure ultrasound is taught and ultimately practiced to its full potential. Ultrasound has the potential to fundamentally change how we teach and practice medicine to the benefit of learners and patients across the globe.Electronic supplementary materialThe online version of this article (doi:10.1186/s13089-015-0035-3) contains supplementary material, which is available to authorized users.

Membranes were isolated from highly purified peroxisomes, mitochondria, and rough and smooth microsomes of rat liver by the one-step Na2CO3 procedure described in the accompanying paper (1982, J. Cell Biol . 93 :97-102) . The polypeptide compositions of these membranes were determined by SDS PAGE and found to be greatly dissimilar . The peroxisomal membrane contains 12% of the peroxisomal protein and consists of three major polypeptides (21,700, 67,700 and 69,700 daltons) as well as some minor polypeptides . The major peroxisomal membrane proteins as well as most of the minor ones are absent from the endoplasmic reticulum (ER) . Conversely, most ER proteins are absent from peroxisomes.By electron microscopy, purified peroxisomal membranes are -6 .8 nm thick and have a typical trilaminar appearance . The phospholipid/protein ratio of peroxisomal membranes is -200 nmol/mg; the principal phospholipids are phosphatidyl choline and phosphatidyl ethanolamine, as in ER and mitochondrial membranes. In contrast to the mitochondria, peroxisomal membranes contain no cardiolipin . All the membranes investigated contain a polypeptide band with a molecular mass of 15,000 daltons. Whether this represents an exceptional common membrane protein or a coincidence is unknown. The implications of these results for the biogenesis of peroxisomes are discussed.Knowledge of the peroxisomal membrane's properties is essential to an understanding both of the organelle's functions and of its biogenesis . The membrane separates the peroxisomal contents from the cytosol and defines the peroxisomal interior as a distinct intracellular space. The permeability properties of the membrane determine to what extent the peroxisome functions as a separate biochemical compartment. Knowledge of how the membrane is formed is essential to an understanding of the biogenesis of the organelle as a whole. If the membrane is derived from some other intracellular membrane, for example the endoplasmic reticulum (ER) (as is widely assumed), then one might expect to see some similarity in composition between them . If, on the other hand, the peroxisomes exist as a separate intracellular compartment, as has recently been suggested (1), then the peroxisomal membrane needs to have no structural similarity to the ER . THE JOURNAL OF CELL BIOLOGY " VOLUME 93 APRIL 1982 103-110 ©The Rockefeller University Press -0021-9525/82/04/0103/08 $1 .00We have applied the newly-developed sodium carbonate procedure described in the accompanying paper (2) to isolate peroxisomal, mitochondrial, and ER membranes.' We have partially characterized these three membranes, and found that their polypeptide compositions are almost entirely different, but their phospholipid compositions are similar. Some of these results have appeared in abstract form (3, 4) .' Rat liver microsomes were subfractionated by isopycnic centrifugation in linear sucrose gradients according to Beaufay et al. (7). The fractions selected as the "rough microsomal fraction" have been shown by these authors to c...