A new HPF specimen carrier adapter for the use of high-pressure freezing with cryoscanning electron microscope: two applications: stearic acid organization in a hydroxypropyl methylcellulose matrix and mice myocardium.

Cryogenic transmission electron microscopy of high-pressure freezing (HPF) samples is a well-established technique for the analysis of liquid containing specimens. This technique enables observation without removing water or other volatile components. The HPF technique is less used in scanning electron microscopy (SEM) due to the lack of a suitable HPF specimen carrier adapter. The traditional SEM cryotransfer system (PP3000T Quorum Laughton, East Sussex, UK; Alto Gatan, Pleasanton, CA, USA) usually uses nitrogen slush. Unfortunately, and unlike HPF, nitrogen slush produces water crystal artefacts. So, we propose a new HPF specimen carrier adapter for sample transfer from HPF system to cryogenic-scanning electronic microscope (Cryo-SEM). The new transfer system is validated using technical two applications, a stearic acid in hydroxypropyl methylcellulose solution and mice myocardium. Preservation of samples is suitable in both cases. Cryo-SEM examination of HPF samples enables a good correlation between acid stearic liquid concentration and acid stearic occupation surface (only for homogeneous solution). For biological samples as myocardium, cytoplasmic structures of cardiomyocyte are easily recognized with adequate preservation of organelle contacts and inner cell organization. We expect this new HPF specimen carrier adapter would enable more SEM-studies using HPF.

Comparison of methods for the fabrication and the characterization of polymer self-assemblies: what are the important parameters?

The ability to self-assemble was evaluated for a large variety of amphiphilic block copolymers, including poly(ethyleneoxide-b-ε-caprolactone), poly(ethyleneoxide-b-d,l-lactide), poly(ethyleneoxide-b-styrene), poly(ethyleneoxide-b-butadiene) and poly(ethyleneoxide-b-methylmethacrylate). Different methods of formation are discussed, such as cosolvent addition, film hydration or electroformation. The influence of experimental parameters and macromolecular structures on the size and morphology of the final self-assembled structures is investigated and critically compared with the literature. The same process is carried out regarding the characterization of these structures. This analysis demonstrates the great care that should be taken when dealing with such polymeric assemblies. If the morphology of such assemblies can be predicted to some extent by macromolecular parameters like the hydrophilic/hydrophobic balance, those parameters cannot be considered as universal. In addition, external experimental parameters (methods of preparation, use of co-solvent, …) appeared as critical key parameters to obtain a good control over the final structure of such objects, which are very often not at thermodynamic equilibrium but kinetically frozen. A principal component analysis is also proposed, in order to examine the important parameters for forming the self-assemblies. Here again, the hydrophilic/hydrophobic fraction is identified as an important parameter.

Comparison of different cellular models measuring in vitro the whole human serum cholesterol efflux capacity.

BACKGROUND: Fu5AH rat hepatoma cells and cAMP (cyclic AMP)-pretreated J774 mouse macrophages are commonly used as models for SR-BI (scavenger receptor class B type I) and ABCA1 (ATP binding cassette transporter 1)-mediated free cholesterol efflux to whole serum, respectively. However, the responsiveness of Fu5AH, control or cAMP pretreated J774 cells to the various lipids and HDL (high-density lipoprotein)-parameters from both normo- and dyslipidaemic subjects has never been compared within the same study. MATERIALS AND METHODS: Fifty-eight men were classified into four groups: type IIa hypercholesterolaemic (n = 12), type IIb dyslipidaemic (n = 13), type IV hypertriglyceridaemic (n = 18) and normolipidaemic (n = 15) were recruited. A complete lipid profile including prebeta-HDL was performed. Cholesterol efflux from Fu5AH cells as well as from control or cAMP pretreated J774 cells were measured; the difference between these two latter values being taken as the ABCA1-mediated efflux. RESULTS: The Fu5AH and the control J774 cells delivered cholesterol to mature HDLs, especially to phospholipid (PL)-rich HDL. Using cAMP pretreated cells, the ABCA1-dependent efflux was highly sensitive to prebeta-HDL, which appeared to be a factor in determining the efflux. Consistent with the dependence of the SR-BI-mediated efflux on HDL-PL levels, which are not different between groups, all sera displayed similar efflux capacities from the Fu5AH cells. Conversely, in accordance with their high prebeta-HDL levels, the ABCA1-dependent efflux highlighted the efficiency of type IV sera. CONCLUSION: Two complementary cellular models providing SR-BI and ABCA1-dependent efflux should be used to measure the capacity of a biological fluid which contains a wide variety of components to promote cholesterol efflux.