A Compartmentalized Neuronal Cell-Culture Platform Compatible With Cryo-Fixation by High-Pressure Freezing for Ultrastructural Imaging

The human brain contains a wide array of billions of neurons and interconnections, which are often simplified for analysis in vitro using compartmentalized microfluidic devices for neuronal cell culturing, to better understand neuronal development and disease. However, such devices are traditionally incompatible for high-pressure freezing and high-resolution nanoscale imaging and analysis of their sub-cellular processes by methods including electron microscopy. Here we develop a novel compartmentalized neuronal co-culture platform allowing reconstruction of neuronal networks with high variable spatial control, which is uniquely compatible for high-pressure freezing. This cryo-fixation method is well-established to enable high-fidelity preservation of the reconstructed neuronal networks and their sub-cellular processes in a near-native vitreous state without requiring chemical fixatives. To direct the outgrowth of neurites originating from two distinct groups of neurons growing in the two different compartments, polymer microstructures akin to microchannels are fabricated atop of sapphire disks. Two populations of neurons expressing either enhanced green fluorescent protein (EGFP) or mCherry were grown in either compartment, facilitating the analysis of the specific interactions between the two separate groups of cells. Neuronally differentiated PC12 cells, murine hippocampal and striatal neurons were successfully used in this context. The design of this device permits direct observation of entire neuritic processes within microchannels by optical microscopy with high spatial and temporal resolution, prior to processing for high-pressure freezing and electron microscopy. Following freeze substitution, we demonstrate that it is possible to process the neuronal networks for ultrastructural imaging by electron microscopy. Several key features of the embedded neuronal networks, including mitochondria, synaptic vesicles, axonal terminals, microtubules, with well-preserved ultrastructures were observed at high resolution using focused ion beam – scanning electron microscopy (FIB-SEM) and serial sectioning – transmission electron microscopy (TEM). These results demonstrate the compatibility of the platform with optical microscopy, high-pressure freezing and electron microscopy. The platform can be extended to neuronal models of brain disease or development in future studies, enabling the investigation of subcellular processes at the nanoscale within two distinct groups of neurons in a functional neuronal pathway, as well as pharmacological testing and drug screening.

Fine cryo-SEM observation of the microstructure of emulsions frozen via high-pressure freezing

An emulsion, a type of soft matter, is complexed with at least two materials in the liquid state (e.g. water and oil). Emulsions are classified into two types: water in oil (W/O) and oil in water (O/W), depending on the strength of the emulsifier. The properties and behavior of emulsions are directly correlated with the size, number, localization and structure of the dispersed phases in the continuous phase. Therefore, an understanding of the microstructure comprising liquid-state emulsions is essential for producing and evaluating these emulsions. Generally, it is impossible for conventional electron microscopy to examine liquid specimens, such as emulsion. Recent advances in cryo-scanning electron microscopy (cryo-SEM) could allow us to visualize the microstructure of the emulsions in a frozen state. Immersion freezing in slush nitrogen has often been used for preparing the frozen samples of soft matters. This preparation could generate ice crystals, and they would deform the microstructure of specimens. High-pressure freezing contributes to the inhibition of ice-crystal formation and is commonly used for preparing frozen biological samples with high moisture content. In this study, we compared the microstructures of immersion-frozen and high-pressure frozen emulsions (O/W and W/O types, respectively). The cryo-SEM observations suggested that high-pressure freezing is more suitable for preserving the microstructure of emulsions than immersion freezing.

Dry Ice Self-Pressurised Rapid Freezing (DryIce SPRF): all you need to cool for cryofixation of nematodes is dry ice

Summary Cryofixation immediately arrests all biochemical, physiological and dynamic processes underway in the sample in their present state, resulting in both excellent preservation of the specimen’s ultrastructure and its antigenicity. Cryofixation involves extremely rapid cooling of specimens, creating an amorphous, or ‘non-crystalline’, state of water containing no detectable ice crystals, a process dependent on pressure, medium composition and temperature. Self-Pressurised Rapid Freezing (SPRF) employs plunge freezing of specimens in a sealed copper tube into a cryogen such as nitrogen slush (−210°C), liquid nitrogen (−196°C), ethane (−183°C) or propane (−120°C). In this study we have explored the use of SPRF with cooled acetone on dry ice (−80°C) as the cryogen, a method named DryIce SPRF. Although with this relatively high temperature amorphous water cannot be formed, we have demonstrated that the ultrastructural and antigenicity results after DryIce SPRF on Caenorhabditis elegans are perfectly comparable with those achieved using High Pressure Freezing and SPRF. Thus, with sufficient pressure optimal results, with ice crystals below the resolution of transmission electron microscopy, can be achieved even at −78°C. Furthermore, a huge advantage of DryIce SPRF over other techniques is its use of affordable, easily available and safe products.

Cryo-electron tomography of native Drosophila tissues vitrified by plunge freezing

Cryo-focused ion beam (cryo-FIB) milling allows thinning vitrified cells for high resolution imaging by cryo-electron tomography (cryo-ET). However, it remains challenging to apply this workflow to tissues, as they usually require high-pressure freezing for vitrification. Here we show that dissected Drosophila tissues can be directly vitrified by plunge freezing upon a short incubation in 10% glycerol. This expedites subsequent cryo-FIB/ET, enabling systematic analyses of the molecular architecture of native tissues.

Cytonemes with complex geometries and composition extend into invaginations of target cells

Cytonemes are specialized filopodia that mediate paracrine signaling in Drosophila and other animals. Studies using fluorescence confocal microscopy (CM) established their general paths, cell targets, and essential roles in signaling. To investigate details unresolvable by CM, we used high-pressure freezing and EM to visualize cytoneme structures, paths, contents, and contacts. We observed cytonemes previously seen by CM in the Drosophila wing imaginal disc system, including disc, tracheal air sac primordium (ASP), and myoblast cytonemes, and identified cytonemes extending into invaginations of target cells, and cytonemes connecting ASP cells and connecting myoblasts. Diameters of cytoneme shafts vary between repeating wide (206 ± 51.8 nm) and thin (55.9 ± 16.2 nm) segments. Actin, ribosomes, and membranous compartments are present throughout; rough ER and mitochondria are in wider proximal sections. These results reveal novel structural features of filopodia and provide a basis for understanding cytoneme cell biology and function.

Frozen strawberry quality enhancement using high hydrostatic pressure and vacuum infusion with pectin methylesterase and calcium chloride solution

Fresh strawberry(Fragaria x ananassa) is a delicious fruit that is an excellent source of micronutrients and an array of beneficial phytochemicals. Emerging technologies like high pressure freezing and thawing have gained interest recently due to their ability to preserve the structure of the food during storage, albeit the possible shortcomings of degradation of texture and sensory quality. In this study, the effects of high hydrostatic pressure freezing and thawing on the physical quality attributes (texture, drip loss and Degree of Methylation(DM)) of strawberry were investigated. Strawberry samples were infused in a vacuum using pectin methyl esterase(PME) and CaCl2solutionat170 hPa pressure for 5 min at room temperature to improve textural integrity; infused with distilled water; and another group of samples further incubated at 40°C for 20 min. Fresh samples and non-pre treated samples were also studied. All the samples were subjected to high pressure freezing and thawing processes at 200 MPa and the effect of the processing conditions on texture were evaluated. The high pressure processed samples were compared with conventionally frozen and thawed samples.Pectin methyl esterase,CaCl2infusion and incubation gave a relative hardness of 1.5 pre treatment compared to water only infused at 0.5.TheCaCl2and PME-infused samples had a higher relative hardness after one day of storage at -18°C compared to non-pretreated and water-infused samples at 0.3 compared to 0.1,respectively. Pretreated, water-infused, and PME withCaCl2-infused samples showed no significant difference in hardness when measured immediately after conventional thawing and high pressure induced thawing HPIT but are the former are a third less hard compared to the latter upon storage after 3 hours and three months. Galacturonic acid per mg of Alcohol insoluble residues AIR sample for the fresh strawberrywas0.002±2.699E-05 and 0.002±1.070E-05molesfor the one-day vs.three months storage,respectively,for samples conventionally frozen and thawed in duplicate experiments.Therefore,a combination of high-pressure shift freezing (HPSF) with PME and CaCl2infusion improved the texture of the strawberry compared to those that were not pretreated,and gained weight by 15%. The PME and CaCl2-infused strawberry showed less degradation than the non-pretreated and water-infused during both long-and short-time frozen storage. Conventional freezing processes caused more degradation compared to HPSF. Degradation of 3 months conventional freezing was comparable to 1-day storage. For frozen storage, enzyme pretreatment combined with high pressure freezing can be used to enhance the quality of strawberry.