Characterization of Complex Drug Formulations Using Cryogenic Scanning Electron Microscopy (Cryo-SEM).

The physicochemical properties of complex drug formulations, including liposomes, suspensions, and emulsions, are important for understanding drug release mechanisms, quality control, and regulatory assessment. It is ideal to characterize these complex drug formulations in their native hydrated state. This article describes the characterization of complex drug formulations in a frozen-hydrated state using cryogenic scanning electron microscopy (cryo-SEM). In comparison to other techniques, such as optical microscopy or room-temperature scanning electron microscopy, cryo-SEM combines the advantage of studying hydrated samples with high-resolution imaging capability. Detailed information regarding cryo-fixation, cryo-fracture, freeze-etching, sputter-coating, and cryo-SEM imaging is included in this article. A multivesicular liposomal complex drug formulation is used to illustrate the impact of different cryogenic sample preparation conditions. In addition to drug formulations, this approach can also be applied to biological samples (e.g., cells, bacteria) and soft-matter samples (e.g., hydrogels). © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Cryo-fixation to preserve the native structure of samples using planchettes Alternate Protocol: Cryo-fixation to preserve the native structure of biological samples on sapphire disks Basic Protocol 2: Sample preparation for cross-sectional cryo-SEM imaging Basic Protocol 3: Cryo-SEM imaging and microanalysis.

Direct comparison of optical and electron microscopy methods for structural characterization of extracellular vesicles.

As interest in the role of extracellular vesicles in cell-to-cell communication has increased, so has the use of microscopy and analytical techniques to assess their formation, release, and morphology. In this study, we evaluate scanning electron microscopy (SEM) and cryo-SEM for characterizing the formation and shedding of vesicles from human breast cell lines, parental and hyaluronan synthase 3-(HAS3)-overexpressing MCF10A cells, grown directly on transmission electron microscopy (TEM) grids. While cells imaged with conventional and cryo-SEM exhibit distinct morphologies due to the sample preparation process for each technique, tubular structures protruding from the cell surfaces were observed with both approaches. For HAS3-MCF10A cells, vesicles were present along the length of membrane protrusions. Once completely shed from the cells, extracellular vesicles were characterized using nanoparticle tracking analysis (NTA) and cryo-TEM. The size distributions obtained by each technique were different not only in the range of vesicles analyzed, but also in the relative proportion of smaller-to-larger vesicles. These differences are attributed to the presence of biological debris in the media, which is difficult to differentiate from vesicles in NTA. Furthermore, we demonstrate that cryo-TEM can be used to distinguish between vesicles based on their respective surface structures, thereby providing a path to differentiating vesicle subpopulations and identifying their size distributions. Our study emphasizes the necessity of pairing several techniques to characterize extracellular vesicles.

Cryo-Electron Microscopy Reveals That Sperm Modification Coincides with Female Fertility in the Mosquito Aedes aegypti.

Manipulating mosquito reproduction is a promising approach to reducing mosquito populations and the burden of diseases they carry. A thorough understanding of reproductive processes is necessary to develop such strategies, but little is known about how sperm are processed and prepared for fertilization within female mosquitoes. By employing cryo-electron microscopy for the first time to study sperm of the mosquito Aedes aegypti, we reveal that sperm shed their entire outer coat, the glycocalyx, within 24 hours of being stored in the female. Motility assays demonstrate that as their glycocalyx is shed in the female's sperm storage organs, sperm transition from a period of dormancy to rapid motility-a critical prerequisite for sperm to reach the egg. We also show that females gradually become fertile as sperm become motile, and that oviposition behavior increases sharply after females reach peak fertility. Together, these experiments demonstrate a striking coincidence of the timelines of several reproductive events in Ae. aegypti, suggesting a direct relationship between sperm modification and female reproductive capacity.

Physical Principles of Membrane Shape Regulation by the Glycocalyx.

Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in communication between cells and with the extracellular matrix.

Connectivity of centermost chromatophores in Rhodobacter sphaeroides bacteria.

The size of whole Rhodobacter sphaeroides prevents 3D visualization of centermost chromatophores in their native environment. This study combines cryo-focused ion beam milling with cryo-electron tomography to probe vesicle architecture both in situ and in 3D. Developing chromatophores are membrane-bound buds that remain in topological continuity with the cytoplasmic membrane and detach into vesicles when mature. Mature chromatophores closest to the cell wall are typically isolated vesicles, whereas centermost chromatophores are either linked to neighboring chromatophores or contain smaller, budding structures. Isolated chromatophores comprised a minority of centermost chromatophores. Connections between vesicles in growing bacteria are through ~10 nm-long, ~5 nm-wide linkers, and are thus physical rather than functional in terms of converting photons to ATP. In cells in the stationary phase, chromatophores fuse with neighboring vesicles, lose their spherical structure, and greatly increase in volume. The fusion and morphological changes seen in older bacteria are likely a consequence of the aging process, and are not representative of connectivity in healthy R. sphaeroides. Our results suggest that chromatophores can adopt either isolated or connected morphologies within a single bacterium. Revealing the organization of chromatophore vesicles throughout the cell is an important step in understanding the photosynthetic mechanisms in R. sphaeroides.

Manifestations of probe presence on probe dynamics in supercooled liquids.

Experimental studies that follow behavior of single probes embedded in heterogeneous systems are increasingly common. The presence of probes may perturb the system, and such perturbations may or may not affect interpretation of host behavior from the probe observables typically measured. In this study, the manifestations of potential probe-induced changes to host dynamics in supercooled liquids are investigated via molecular dynamics simulations. It is found that probe dynamics do not necessarily mirror host dynamics as they exist either in the probe-free or probe-bearing systems. In particular, for a binary supercooled liquid, we find that smooth probes larger than the host particles induce increased translational diffusion in the host system; however, the diffusion is anisotropic and enhances caging of the probe, suppressing probe translational diffusion. This in turn may lead experiments that follow probe diffusion to suggest Stokes-Einstein behavior of the system even while both the probe-free and probe-bearing systems exhibit deviations from that behavior.