Direct attachment of cell suspensions to high-pressure freezing specimen planchettes

We describe a procedure for high‐pressure freezing (HPF) of cultured cells using the HPF aluminium planchettes as a substrate. Cells are either grown directly on planchettes covered with Matrigel or allowed to attach to poly‐l ‐lysine‐coated planchettes. This method allows for rapid transfer of the cells into the HPF and minimizes physical and physiological trauma to the cells. Furthermore, the yield of well‐frozen cells approaches 100% for every cell type we have tried so far. In this report, we show well‐preserved ultrastructure in mitotic and interphase HeLa cells, isolated gastric parietal cells and isolated gastric glands. Immunogold labelling of H⁺/K⁺‐ATPase is shown in parietal cells of isolated gastric glands embedded in LR White resin. The aluminium planchettes appear to have little effect on cell physiology, as demonstrated by the fact that parietal cells cultured for 24–28 h on the planchettes retain their responsiveness to stimulation with histamine.     

Controlled microaspiration for high-pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography

High‐pressure freezing is the preferred method to prepare thick biological specimens for ultrastructural studies. However, the advantages obtained by this method often prove unattainable for samples that are difficult to handle during the freezing and substitution protocols. Delicate and sparse samples are difficult to manipulate and maintain intact throughout the sequence of freezing, infiltration, embedding and final orientation for sectioning and subsequent transmission electron microscopy. An established approach to surmount these difficulties is the use of cellulose microdialysis tubing to transport the sample. With an inner diameter of 200 μm, the tubing protects small and fragile samples within the thickness constraints of high‐pressure freezing, and the tube ends can be sealed to avoid loss of sample. Importantly, the transparency of the tubing allows optical study of the specimen at different steps in the process. Here, we describe the use of a micromanipulator and microinjection apparatus to handle and position delicate specimens within the tubing. We report two biologically significant examples that benefit from this approach, 3D cultures of mammary epithelial cells and cochlear outer hair cells. We illustrate the potential for correlative light and electron microscopy as well as electron tomography.     

A new approach for cryofixation by high-pressure freezing

A newly designed high-pressure freezing machine for cryofixation was established and tested (Leica EMPACT), based on ideas originally proposed by Moor & Riehle in 1968. The new machine, essentially an improved version of our prototype, pressurizes the sample to 2000 bar in a small container (using methylcyclohexane as hydraulic fluid) and at the same time cools the outer surface of the container with a jet of liquid nitrogen. The advantage of this approach is that the machine uses little liquid nitrogen and can be built small and light. The machine is able to vitrify and freeze well a variety of specimens, for example, plant leaves, yeast cells, liver or nerve tissue (more samples are shown at: http://www.ana.unibe.ch/empact ). Cooling efficiency is the same as in the traditional machines that use liquid nitrogen to pressurize and simultaneously cool the sample.     

A review of high-pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues

In this paper, we review some published studies using correlative light and electron microscopy methods. We further refined our criteria to include only those studies using live cells for light microscope and where high-pressure freezing was the method of specimen preparation for electron microscopy. High-pressure freezing is especially important for some difficult-to-fix samples, and for optimal preservation of ultrastructure in samples larger than a few micrometres. How the light microscope observations are done is completely sample dependent, but the choice of high-pressure freezer depends on the speed required to capture (freeze) the biological event of interest. For events requiring high time resolution (in the 4–5 s range) the Leica EM PACT2 with rapid transfer system works well. For correlative work on structures of interest that are either non-motile or moving slowly (minutes rather than seconds), any make of high-pressure freezer will work. We also report on some efforts to improve the capabilities of the Leica EM PACT2 rapid transfer system.     

Ultrastructure of the frog retina after high-pressure freezing and freeze substitution

In many types of tissue, high-pressure freezing (HPF), followed by freeze substitution, can produce excellent ultrastructural preservation at depths over 10 times that obtained by other cryofixation techniques. However, in the case of neural tissue, the benefits of HPF have not been realized. In the present study, isolated frog ( Rana pipiens) retina was sliced at a thickness of 150 or 350 μm, rapidly frozen in a Balzers HPM 010 high-pressure freezer, and freeze substituted with 1% OsO₄ and 0.1% tannic acid in acetone. Specially designed HPF chambers and specific freezing media (35% high-MW dextran for 150-μm slices or 15% low-MW dextran for 350-μm slices) were required for adequate freezing.
The quality of preservation after HPF was excellent throughout the retina in both the 150- and 350-μm slices, compared with chemically fixed slices. Specifically, HPF resulted in better preserved cellular, mitochondrial and nuclear membranes in all retinal layers.
This is the first study to successfully cryofix all of the layers of the retina. The increased depths of adequate freezing achieved by HPF should facilitate various ultrastructural studies of retina, as well as of other CNS tissues, where preservation approaching that of the 'native' state is required.     

Aclar discs: a versatile substrate for routine high-pressure freezing of mammalian cell monolayers

High‐pressure freezing avoids the artefacts induced by conventional chemical fixation, and, in combination with freeze‐substitution and plastic embedding, is a reliable method for the ultrastructural analysis of mammalian cell monolayers. In order to high‐pressure freeze mammalian cell monolayers, cells have to be seeded on a suitable substrate. Unfortunately, electron microscopy analysis is often hampered by poor cell growth, changes in cell morphology induced by the cell substrate or cell loss during processing. We report a method to culture, high‐pressure freeze, freeze‐substitute and plastic embed mammalian cell monolayers. The method is based on the use of Aclar, a copolymer film with properties very similar to those of tissue culture plastic. We show that Aclar discs support the normal growth and morphology of a wide variety of mammalian cell types, and form an ideal starting point for high‐pressure freezing, freeze‐substitution and plastic embedding. We present a complete protocol, which, because of its simplicity and reproducibility, provides a method suitable for the routine analysis of mammalian cell monolayers by electron microscopy and tomography.     

The use of filter membranes for high-pressure freezing of cell monolayers

Rapid freezing of cells and tissues, followed by freeze‐substitution fixation and plastic embedding, has become a highly reliable method for preparing samples for imaging in the electron microscope. High‐pressure freezing is an efficient means of immobilizing suspensions of yeasts, thick pellets of mammalian cells, or small (< 0.5 mm) pieces of plant or animal tissue. Monolayers of cultured mammalian cells that are too thick for efficient immobilization by other modes of rapid freezing have also been successfully preserved by this method. Monolayer cultures are often important because they can be imaged by light microscopy (LM) both before and after their preparation for electron microscopy (EM). Additionally, some monolayer cultures serve as model systems for physiological processes, so it is important that cells under study can grow on a substrate that is both physiologically appropriate and convenient for EM processing. Here we describe a reliable method for preparing mammalian cell monolayers (PtK₁ and polarized MDCK) for EM. Our protocol results in good preservation of cellular ultrastructure, it is a useful companion to studies of cell physioloy and, with some limitation, is suitable for correlative LM and EM.     

Freeze-substitution: the addition of water to polar solvents enhances the retention of structure and acts at temperatures around –60°C

High‐pressure freezing followed by freeze substitution and plastic embedding is becoming a more widely used method for TEM sample preparation. Here, we have investigated the influence of solvents, fixative concentrations and water content in the substitution medium on the sample quality of high‐pressure frozen, freeze‐substituted and plastic embedded mammalian cell culture monolayers. We found that the visibility of structural details was optimal with acetone and that extraction increased with both increasing and decreasing solvent polarity. Interestingly, the addition of water to polar solvents increased the sample quality, while being destructive when added to apolar solvents. The positive effect of water addition is saturable in acetone and ethanol at 5%(v/v), but even addition of up to 20% water has no negative effect on the sample structure. Therefore, a medium based on acetone containing fixatives and 5% water is most optimal for the substitution of mammalian cell cultures. In addition, our results suggest that the presence of water is critical for the retention of structure at temperatures around –60°C.     

Comparison of slam-freezing and high-pressure freezing effects on the DNA cholesteric liquid crystalline structure

Using in parallel electron microscopy of ultrathin frozen-hydrated sections and freeze-fracture replicas, we compare the ultrastructural consequences of two freezing techniques: slam-freezing at liquid helium temperature and high-pressure freezing, on a model system, the DNA cholesteric liquid crystalline phase. Both freezing techniques are able to vitrify DNA liquid crystalline solutions containing up to 85% water, but induce structural rearrangements of the molecular organization. The cholesteric structure is preserved by the slam-freezing method despite the formation of periodic distortions induced by the mechanical compressive stress. In contrast, high-pressure freezing does not preserve the structure of the liquid crystal: the long-range cholesteric stratification disappears, and the local continuous twist between molecules is modified. These results show that vitrification, though necessary, may not be a sufficient token of preservation of the native state of hydrated materials. We discuss the possible origins of the molecular rearrangements that have time to occur in the specimens as a result of the low freezing rate permitted by the high-pressure freezing process.     

Moving EM: the Rapid Transfer System as a new tool for correlative light and electron microscopy and high throughput for high-pressure freezing

In this paper, the Rapid Transfer System (RTS), an attachment to the Leica EMPACT2 high‐pressure freezer, is described as a new tool for special applications within the cryofixation field. The RTS is an automated system that allows for fast processing of samples (<5 s) that make it possible for the first time to use high‐pressure freezing in combination with high time resolution correlative light and electron microscopy. In addition, with a working cycle of 30 s this rapid turn over time allows one to acquire more samples of biopsy material before it deteriorates than with other HPF machines with longer cycle times. With the use of the RTS it was possible to obtain three samples each of four different tissues in 6 min. Together with the finding that 90% of samples of cells grown on sapphire discs were well frozen, the RTS was both fast and reliable. Most important, together with other newly developed accessories, the RTS made it possible to capture specific events occurring live in the cell as observed by light microscopy, to cryofix that sample/event within 4 s, and then to analyze that event at high resolution in the electron microscope with excellent preservation of ultra‐structure. These developments should give us the tools to unravel intracellular processes that can be observed by live cell imaging but are too rare or fast to be picked up by routine EM methods or where the history of a structure is necessary to be able to discern its nature.     

A rapid microbiopsy system to improve the preservation of biological samples prior to high-pressure freezing

A microbiopsy system for fast excision and transfer of biological specimens from donor to high‐pressure freezer was developed. With a modified, commercially available, Promag 1.2 biopsy gun, tissue samples can be excised with a size small enough (0.6 mm × 1.2 mm × 0.3 mm) to be easily transferred into a newly designed specimen platelet. A self‐made transfer unit allows fast transfer of the specimen from the needle into the specimen platelet. The platelet is then fixed in a commercially available specimen holder of a high‐pressure freezing machine (EM PACT, Leica Microsystems, Vienna, Austria) and frozen therein. The time required by a well‐instructed (but not experienced) person to execute all steps is in the range of half a minute. This period is considered short enough to maintain the excised tissue pieces close to their native state. We show that a range of animal tissues (liver, brain, kidney and muscle) are well preserved. To prove the quality of freezing achieved with the system, we show vitrified ivy leaves high‐pressure frozen in the new specimen platelet.     

Microcarriers for high-pressure freezing and cryosectioning of adherent cells

A method is described employing microcarrier spheres of cross‐linked dextran for obtaining ultra‐ and semithin vitreous sections from high‐pressure frozen anchorage‐dependent (mammalian) cells. Avoiding trypsination or scraping cells off from the culture surface, the presented approach allows for cryoimmobilization, cryosectioning and cryoelectron microscopy/tomography of frozen‐hydrated cells in an unperturbed manner which is important to preserve the native state of, for instance, the cytoskeleton. Furthermore, our studies on the ‘life cycle’ of Herpes simplex virus in Vero cells demonstrate that cell monolayers on microcarrier beads are well suited for fluorescence microscopic characterization of the sample prior to high‐pressure freezing.     

Freeze substitution of high-pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water

Biological membranes are often poorly visible with the electron microscope after high‐pressure freezing and freeze‐substitution. The water content of the sample and of the substitution medium is one factor among others that strongly influences membrane visibility. In order to investigate this effect, high‐pressure frozen yeast cells, rat‐pancreas tissue and arthropod tissue were freeze‐substituted with and without adding water to the substitution medium. The visibility of the biological membranes was generally improved if the substitution medium contained 1–5% water. The effect was especially pronounced in yeast cells, where membrane visibility was poor after freeze‐substitution with water‐free medium but good after addition of 5% water to the substitution medium.     

Role of parietal and principal gastric mucosa cells in the phenomenon of concentration of aluminum and indium

The subcellular behavior of aluminum and indium, used in medical and industrial fields, was studied in the gastric mucosa and the liver after their intragastric administration to rats, using, two of the most sensitive methods of observation and microanalysis, the transmission electron microscopy, and the secondary ion mass spectrometry. The ultrastructural study showed the presence of electron dense deposits, in the lysosomes of parietal and principal gastric mucosa cells but no loaded lysosomes were observed in the different studied hepatic territories. The microanalytical study allowed the identification of the chemical species present in those deposits as aluminum or indium isotopes and the cartography of their distribution. No modification was observed in control rats tissues. In comparison to previous studies describing the mechanism of aluminum concentration in the gastric mucosa and showing that this element was concentrated in the lysosomes of fundic and antral human gastric mucosa, our study provided additional informations about the types of cells involved in the phenomenon of concentration of aluminum and indium, which are the parietal and the principal cells of the gastric mucosa. Our study demonstrated that these cells have the ability to concentrate selectively aluminum and indium in their lysosomes, as a defensive reaction against intoxication by foreign elements.     

A new method for cell culture on an electron-transparent melamine foil suitable for successive LM,TEM and SEM studies of whole cells

A new cell culture technique is described which is based on the observation that foils cast from the melamine resin hexamethylol-melamine-ether are suitable for the cultivation of beating heart muscle cells and fibroblasts of the rat. This foil can be flamed for sterilization, is about 80 nm in thickness, homogeneous and smooth, withstands dehydration and critical point-drying, can be removed from glass and permits the imaging of whole cells successively by light microscopy, transmission and scanning electron microscopy. The method is capable of narrowing the gap between light and electron microscopy, yielding excellent whole cell preparations in various kinds of microscopic studies to be performed on one and the same cell.     

A variable cell culture chamber for ‘open’ and ‘closed’ cultivation,perfusion and high microscopic resolution of living cells

A multipurpose chamber is described for growing and testing cultured cells. The chamber can be converted from a perfusion chamber to an ‘Open’ or ‘closed’ culture system. The chamber provides optimum conditions for microscopy using all common objectives and condensers for different microscopic methods.     

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.     

A new approach for the automatic evaluation of the solidification structure in steel using orientational entropy filtering

We present a new method for the automatic evaluation of the dendritic solidification structure in metallurgical micrographs of solidified steel. The evaluation of the microstructural parameters such as the primary dendrite arm spacing and the primary grain size are of high importance due to their direct relationship with the internal quality and mechanical properties of the cast product. Given the repeated geometric features in the micrographs and the regular pattern in colour intensity, we applied a filter mask to determine the local entropies within the masks in order to detect the centre coordinates of each individual dendrite. The orientation of the dendrites was determined by rotating the filter mask over each pixel to find the orientation which corresponds to the lowest entropy value. The segmentation of the microstructure was then performed via Delaunay tessellation and subsequent transformation of the triangular mesh into a rectangular grid, enabling the determination of the desired microstructural parameters.     

MRT Letter: 3D culture of isolated cells: A fast and efficient method for optimizing their histochemical and immunocytochemical analyses

The rapid development of three‐dimensional (3D) culture systems and engineered cell‐based tissue models gave rise to an increasing need of new techniques, allowing the microscopic observation of cell behavior/morphology in tissue‐like structures, as clearly signalled by several authors during the last decennium. With samples consisting of small aggregates of isolated cells grown in suspension, it is often difficult to produce an optimal embedded preparation that can be further successfully processed for classical histochemical investigations. In this work, we describe a new, easy to use, efficient method that enables to embed an enriched “preparation” of isolated cells/small 3D cell aggregates, without any cell stress or damage. As for after tissue‐embedding procedures, the cellular blocks can be further suitably processed for efficient histochemical as well as immunohistochemical analyses, rendering more informative‐and attractive‐studies onto 3D cell‐based culture of neo‐tissues. Microsc. Res. Tech. 78:249–254, 2015. © 2015 Wiley Periodicals, Inc.