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.     

A new approach for high-pressure freezing of primary culture cells: the fine structure and stimulation-associated transformation of cultured rabbit gastric parietal cells

A newly designated procedure for high‐pressure freezing of primary culture cells provided excellent ultrastructure of rabbit gastric parietal cells. The isolated parietal cells were cultivated on Matrigel‐coated aluminium plates for conventional subsequential cryoimmobilization by high‐pressure freezing. The ultrastructure of different organelles (Golgi apparatus, mitochondria, multivesicular bodies, etc.) was well preserved compared to conventional chemical fixation. In detail, actin filaments were clearly shown within the microvilli and the subapical cytoplasm. Another striking finding on the cytoskeleton system is the abundance of microtubules among the tubulovesicles. Interestingly, some microtubules appeared to be associating with tubulovesicles. A large number of electron‐dense coated pits and vesicles were observed around the apical membrane vacuoles in cimetidine‐treated resting parietal cells, consistent with an active membrane uptake in the resting state. Immunogold labelling of H⁺/K⁺‐ATPase was seen on the tubulovesicular membranes. When stimulated with histamine, the cultured parietal cells undergo morphological transformation, resulting in great expansion of apical membrane vacuoles. Immunogold labelling of H⁺/K⁺‐ATPase was present not only on the microvilli of expanded apical plasma membrane vacuoles but also in the electron‐dense coated pits. The present findings provide a clue to vesicular membrane trafficking in cultured gastric parietal cells, and assure the utility of the new procedure for high‐pressure freezing of primary culture cells.     

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.     

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.     

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.     

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.     

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.     

An improved procedure for low-temperature embedding of high-pressure frozen and freeze-substituted plant tissues resulting in excellent structural preservation and contrast

Here we describe refinements in the processing of high-pressure frozen samples of delicate plant tissues for immuno-electron microscopy. These involve: shortened freeze-substitution schedules, lower temperatures during processing and polymerisation, the avoidance of temperature fluctuations and the optimisation of heat transfer from the specimens using small disposable aluminium containers. The application of these modifications leads to very good structural preservation and selective membrane contrast. As a result, the versatility of the method is increased since not only immuno-electron microscopical studies can be performed but often the quality is also quite suitable for structural investigations.     

Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography

Cryoimmobilization is regarded as the most reliable method to preserve cellular ultrastructure for electron microscopic analysis, because it is both fast (milliseconds) and avoids the use of harmful chemicals on living cells. For immunolabelling studies samples have to be dehydrated by freeze‐substitution and embedded in a resin. Strangely, although most of the lipids are maintained, intracellular membranes such as endoplasmic reticulum, Golgi and mitochondrial membranes are often poorly contrasted and hardly visible. By contrast, Tokuyasu cryosectioning, based on chemical fixation with aldehydes is the best established and generally most efficient method for localization of proteins by immunogold labelling. Despite the invasive character of the aldehyde fixation, the Tokuyasu method yields a reasonably good ultrastructural preservation in combination with excellent membrane contrast. In some cases, however, dramatic differences in cellular ultrastructure, especially of membranous structures, could be revealed by comparison of the chemical with the cryofixation method. To make use of the advantages of the two different approaches a more general and quantitative knowledge of the influence of aldehyde fixation on ultrastructure is needed. Therefore, we have measured the size and shape of endosomes and lysosomes in high‐pressure frozen and aldehyde‐fixed cells and found that aldehyde fixation causes a significant deformation and reduction of endosomal volume without affecting the membrane length. There was no considerable influence on the lysosomes. Ultrastructural changes caused by aldehyde fixation are most dramatic for endosomes with tubular extensions, as could be visualized with electron tomography. The implications for the interpretation of immunogold localization studies on chemically fixed cells are discussed.     

Candida albicans biofilms: comparative analysis of room‐temperature and cryofixation for scanning electron microscopy

Biofilms are frequently related to invasive fungal infections and are reported to be more resistant to antifungal drugs than planktonic cells. The structural complexity of the biofilm as well as the presence of a polymeric extracellular matrix (ECM) is thought to be associated with this resistant behavior. Scanning electron microscopy (SEM) after room temperature glutaraldehyde‐based fixation, have been used to study fungal biofilm structure and drug susceptibility but they usually fail to preserve the ECM and, therefore, are not an optimised methodology to understand the complexity of the fungal biofilm. Thus, in this work, we propose a comparative analysis of room‐temperature and cryofixation/freeze substitution of Candida albicans biofilms for SEM observation. Our experiments showed that room‐temperature fixative protocols using glutaraldehyde and osmium tetroxide prior to alcohol dehydration led to a complete extraction of the polymeric ECM of biofilms. ECM from fixative and alcohol solutions were recovered after all processing steps and these structures were characterised by biochemistry assays, transmission electron microscopy and mass spectrometry. Cryofixation techniques followed by freeze‐substitution lead to a great preservation of both ECM structure and C. albicans biofilm cells, allowing the visualisation of a more reliable biofilm structure. These findings reinforce that cryofixation should be the indicated method for SEM sample preparation to study fungal biofilms as it allows the visualisation of the EMC and the exploration of the biofilm structure to its fullest, as its structural/functional role in interaction with host cells, other pathogens and for drug resistance assays.     

Optimal preservation of the shark retina for ultrastructural analysis: An assessment of chemical, microwave, and high-pressure freezing fixation techniques

Recent advances in microwave chemical fixation (MCF) and/or high pressure freezing (HPF) combined with transmission electron microscopy have resulted in superior ultrastructural detail in a variety of tissue types. To date, selachian tissue has been fixed and processed using only standard chemical fixation (CF) methods, and the resulting ultrastructure has been less than ideal. In this study, we compared the ultrastructure of the fragile retinal tissue from the brown banded bamboo shark, Chiloscyllium punctatum, obtained using CF, MCF, and HPF methods. For all fixation protocols, ultrastructural preservation was improved by keeping the tissue in oxygenated Ringer solution until the time of fixation. Both MCF and HPF produced superior retinal ultrastructure compared to conventional CF. Although HPF occasionally resulted in very high quality ultrastructure, microwave fixation was almost comparable, quicker and far more consistent. Microsc. Res. Tech. 75:1218–1228, 2012. © 2012 Wiley Periodicals, Inc.     

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.     

Postembedding ultrastructural immunocytochemistry of neural antigens on tissues previously processed for routine light and electron microscopy: Preservation of antigenicity up to 22 years after initial fixation

A postembedding immunocytochemical technique is described that allows ultrastructural localization of glial fibrillary acidic protein and neuron-specific enolase on tissues originally processed only for routine light and electron microscopy. Use of the oxidizing agent sodium metaperiodate prior to incubation with the primary antiserum sufficiently removes osmium tetroxide (OsO₄) from potential antigen—antibody combining sites to allow specific localization of these neural antigens by colloidal gold immunolabelling. Both human and monkey neural tissues, prepared for routine ultrastructural examination with aldehyde fixatives and OsO₄ postfixation, show excellent ultrastructural morphology and antigen localization. In addition, formalin-fixed, paraffin-embedded pathological human brain tissues, obtained at autopsy up to 22 years previously, show good ultrastructural immunolocalization of glial fibrillary acidic protein when re-embedded for electron microscopy. Thus, ultrastructural immunolocalization of certain neural antigens is easily achieved in tissues originally processed for routine light and electron microscopy. This allows re-examination of archival tissues using current immunocy-tochemical advances, including that of selected pathological tissues previously prepared solely for light microscopy.     

Transmission electron microscopy staining methods for the cortex of human hair: a modified osmium method and comparison with other stains

For wool, superior staining of a wide range of ultrastructural components is achieved by en bloc treatment of fibres with a chemical reductant followed by osmium tetroxide. For human scalp hair, although staining quality is similar, the penetration of reagents is poor, resulting in large parts of the fibre cortex remaining unstained. Here we describe a modification to the reduction-osmication method in which reagents penetrate through a cut fibre end, allowing visualization of a wide range of features across the cortex. We compare the staining quality, artefacts and range of structure rendered visible using transmission electron microscopy for en bloc reduction-osmication to other staining alternatives including en bloc silver nitrate and section stains based on uranyl acetate and lead citrate, phosphotungstic acid, potassium permanganate, ammoniacal silver nitrate and some combinations of these stains. The effects of hair-care treatments are briefly examined.     

Atomic force microscope (AFM) combined with the ultramicrotome: a novel device for the serial section tomography and AFM/TEM complementary structural analysis of biological and polymer samples

A new device (NTEGRA Tomo) that is based on the integration of the scanning probe microscope (SPM) (NT‐MDT NTEGRA SPM) and the Ultramicrotome (Leica UC6NT) is presented. This integration enables the direct monitoring of a block face surface immediately following each sectioning cycle of ultramicrotome sectioning procedure. Consequently, this device can be applied for a serial section tomography of the wide range of biological and polymer materials. The automation of the sectioning/scanning cycle allows one to acquire up to 10 consecutive sectioned layer images per hour. It also permits to build a 3‐D nanotomography image reconstructed from several tens of layer images within one measurement session. The thickness of the layers can be varied from 20 to 2000 nm, and can be controlled directly by its interference colour in water. Additionally, the NTEGRA Tomo with its nanometer resolution is a valid instrument narrowing and highlighting an area of special interest within volume of the sample. For embedded biological objects the ultimate resolution of SPM mostly depends on the quality of macromolecular preservation of the biomaterial during sample preparation procedure. For most polymer materials it is comparable to transmission electron microscopy (TEM). The NTEGRA Tomo can routinely collect complementary AFM and TEM images. The block face of biological or polymer sample is investigated by AFM, whereas the last ultrathin section is analyzed with TEM after a staining procedure. Using the combination of both of these ultrastructural methods for the analysis of the same particular organelle or polymer constituent leads to a breakthrough in AFM/TEM image interpretation. Finally, new complementary aspects of the object's ultrastructure can be revealed.     

Oolong tea and LR‐White resin: a new method of plant sample preparation for transmission electron microscopy

Simplifying sample processing, shortening the sample preparation time, and adjusting procedures to suitable for new health and safety regulations, these issues are the current challenges which electron microscopic examinations need to face. In order to resolve these problems, new plant tissue sample processing protocols for transmission electron microscopy should be developed. In the present study, we chose the LR‐White resin‐assisted processing protocol for the ultrastructural observation of different types of plant tissues. Moreover, we explored Oolong tea extract (OTE) as a substitute for UA in staining ultrathin sections of plant samples. The results revealed that there was no significant difference between the OTE double staining method and the traditional double staining method. Furthermore, in some organelles, such as mitochondria in root cells of tomatoes and chloroplast in leaf cells of watermelons, the OTE double staining method achieved little better results than the traditional double staining method. Therefore, OTE demonstrated good potentials in replacing UA as a counterstain on ultrathin sections. In addition, sample preparation time was significantly shortened and simplified using LR‐White resin. This novel protocol reduced the time for preparing plant samples, and hazardous reagents in traditional method (acetone and UA) were also replaced by less toxic ones (ethanol and OTE).