In this study we present an innovative method for the preparation of fully hydrated samples of microbial biofilms of cultures Staphylococcus epidermidis, Candida parapsilosis and Candida albicans. Cryo-scanning electron microscopy (cryo-SEM) and high-pressure freezing (HPF) rank among cutting edge techniques in the electron microscopy of hydrated samples such as biofilms. However, the combination of these techniques is not always easily applicable. Therefore, we present a method of combining high-pressure freezing using EM PACT2 (Leica Microsystems), which fixes hydrated samples on small sapphire discs, with a high resolution SEM equipped with the widely used cryo-preparation system ALTO 2500 (Gatan). Using a holder developed in house, a freeze-fracturing technique was applied to image and investigate microbial cultures cultivated on the sapphire discs. In our experiments, we focused on the ultrastructure of the extracellular matrix produced during cultivation and the relationships among microbial cells in the biofilm. The main goal of our investigations was the detailed visualization of areas of the biofilm where the microbial cells adhere to the substrate/surface. We show the feasibility of this technique, which is clearly demonstrated in experiments with various freeze-etching times.
- Klíčová slova
- Biofilm, Candida, Freeze-fracturing, High-pressure freezing (HPF), Staphylococcus, cryo-SEM,
- MeSH
- biofilmy MeSH
- Candida albicans ultrastruktura MeSH
- Candida parapsilosis ultrastruktura MeSH
- elektronová kryomikroskopie metody MeSH
- extracelulární matrix ultrastruktura MeSH
- mikroskopie elektronová rastrovací metody MeSH
- mrazové lámání metody MeSH
- Staphylococcus epidermidis ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The purpose of this work is to investigate the entrapment of protein molecules in cubosomic nanocarriers that are sterically stabilized by an amphiphilic poly(ethylene glycol) (PEG) derivative. Toward that aim, the mechanism of fragmentation of a self-assembled, PEGylated cubic lipid phase into nanoparticles (NPs) is investigated in excess aqueous medium. The molar ratio between the cubic-phase-forming lipid monoolein (MO) and its PEGylated derivative (MO-PEG(2000)) is selected as to favor the formation of inverted-type liquid-crystalline (LC) structures (permitting one to reveal the stages of the fragmentation and bicontinuous membrane NP assembly process) rather than a phase transformation to lamellar or micellar phases. The PEGylated amphiphile considerably affects the interfacial curvature of the cubic lipid membrane and, under agitation, contributes to the fragmentation of the bicontinuous cubic lattice into NPs. Freeze-fracture electron microscopy (FF-EM), quasi-elastic light scattering (QELS), and confocal laser scanning fluorescence microscopy (CLSFM) are applied for determination of the NPs' sizes, inner organization, and stability with regard to a thermal stimulus. Entrapped protein molecules can essentially stabilize the cubosomic particles (proteocubosomes), which display well-defined inner organization of nanochannels in their freeze-fracture planes. The protein α-chymotrypsinogen A is studied in proteocubosome dispersions by means of far-UV synchrotron radiation circular dichroism (SRCD) spectroscopy. It is suggested that the protein molecules are entrapped in the interior of the PEGylated cubosomes via a "nanopockets" mechanism. The LC PEGylated proteocubosomes offer new possibilities for investigation of protein loading in sterically stabilized ("Stealth") nanostructured lipid carriers, which differ from Poloxamer-stabilized isasomes.
- MeSH
- chymotrypsinogen chemie MeSH
- cirkulární dichroismus metody MeSH
- elektronová mikroskopie metody MeSH
- glyceridy chemie MeSH
- kapalné krystaly chemie MeSH
- konfokální mikroskopie MeSH
- mrazové lámání metody MeSH
- nanočástice chemie ultrastruktura MeSH
- poloxamer chemie MeSH
- polyethylenglykoly chemie MeSH
- synchrotrony MeSH
- voda chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chymotrypsinogen MeSH
- glyceridy MeSH
- monoolein MeSH Prohlížeč
- poloxamer MeSH
- polyethylenglykoly MeSH
- voda MeSH
