Sample Preparation Handbook for Transmission Electron Microscopy: Techniques
Secondly, this filtering removes electrons that are scattered to high angles, which may be due to unwanted processes such as spherical or chromatic aberration, or due to diffraction from interaction within the sample. Apertures are either a fixed aperture within the column, such as at the condenser lens, or are a movable aperture, which can be inserted or withdrawn from the beam path, or moved in the plane perpendicular to the beam path.
Aperture assemblies are mechanical devices which allow for the selection of different aperture sizes, which may be used by the operator to trade off intensity and the filtering effect of the aperture. Aperture assemblies are often equipped with micrometers to move the aperture, required during optical calibration. Imaging methods in TEM use the information contained in the electron waves exiting from the sample to form an image.
The projector lenses allow for the correct positioning of this electron wave distribution onto the viewing system. Different imaging methods therefore attempt to modify the electron waves exiting the sample in a way that provides information about the sample, or the beam itself. From the previous equation, it can be deduced that the observed image depends not only on the amplitude of beam, but also on the phase of the electrons, although phase effects may often be ignored at lower magnifications. Higher resolution imaging requires thinner samples and higher energies of incident electrons, which means that the sample can no longer be considered to be absorbing electrons i.
Instead, the sample can be modeled as an object that does not change the amplitude of the incoming electron wave function, but instead modifies the phase of the incoming wave; in this model, the sample is known as a pure phase object. For sufficiently thin specimens, phase effects dominate the image, complicating analysis of the observed intensities. Figure on the right shows the two basic operation modes of TEM — imaging and diffraction modes. In both cases specimen is illuminated with the parallel beam, formed by electron beam shaping with the system of Condenser lenses and Condenser aperture.
After interaction with the sample, on the exit surface of the specimen two types of electrons exist — unscattered which will correspond to the bright central beam on the diffraction pattern and scattered electrons which change their trajectories due to interaction with the material. In Imaging mode, Objective aperture is inserted in a back focal plane BFP of Objective lens where diffraction spots are formed.
If using the Objective aperture, the central beam is selected the rest signal is blocked and the bright field image BF image is obtained. If we allow the signal from the diffracted beam, the dark field image DF image is received. Then selected signal is magnified and projected on a screen or on a camera with the help of Intermediate and Projector lenses. Image of the sample is received. In Diffraction mode, Selected area aperture is used to determine the specimen area from which the signal will be displayed.
By changing the strength of Intermediate lens, the Diffraction pattern is projected on a screen. Diffraction is a very powerful tool for doing a cell reconstruction and crystal orientation determination. The contrast between two adjacent areas in a TEM image can be defined as the difference in the electron densities in image plane.
Due to the scattering of the incident beam by the sample, the amplitude and phase of the electron wave change, which results in amplitude contrast and phase contrast , correspondingly. Most of images have both contrast components. Amplitude—contrast is obtained due to removal of some electrons before the image plane. During their interaction with the specimen some of electrons will be lost due to absorption, or due to scattering at very high angles beyond the physical limitation of microscope or are blocked by the objective aperture.
While the first two losses are due to the specimen and microscope construction, the objective aperture can be used by operator to enhance the contrast. Figure on the right shows a TEM image a and the corresponding diffraction pattern b of Pt polycrystalline film taken without an objective aperture. In order to enhance the contrast in the TEM image the number of scattered beams as visible in the diffraction pattern should be reduced.
This can be done by selecting a certain area in the diffraction plane like only the central beam or a diffracted beam, or combinations of beams with objective aperture to form BF c in case the central beam is included or DF d-e images in case the central beam is blocked. DF images d-e are obtained using the diffracted beams indicated in diffraction pattern with circles b. Grains from which electrons are scattered into these diffraction spots appear brighter. More details about diffraction contrast formation are given further.
There are two types of amplitude contrast — mass—thickness and diffraction contrast. When the beam illuminates two neighbouring areas with low mass or thickness and high mass or thickness , the heavier region scatters electrons at bigger angles. As a result, heavier regions appear darker in BF images have low intensity. Mass—thickness contrast is most important for non—crystalline, amorphous materials.
Diffraction contrast occurs due to a specific crystallographic orientation of a grain. In such a case the crystal is in a so-called Bragg condition, whereby atomic planes are oriented in a way that there is a high probability of scattering. Thus diffraction contrast provides information on the orientation of the crystals in a polycrystalline sample.
Note that in case diffraction contrast exists, the contrast cannot be interpreted as due to mass or thickness variations. Samples can exhibit diffraction contrast, whereby the electron beam undergoes Bragg scattering , which in the case of a crystalline sample, disperses electrons into discrete locations in the back focal plane. By the placement of apertures in the back focal plane, i. If the reflections that are selected do not include the unscattered beam which will appear up at the focal point of the lens , then the image will appear dark wherever no sample scattering to the selected peak is present, as such a region without a specimen will appear dark.
This is known as a dark-field image. Modern TEMs are often equipped with specimen holders that allow the user to tilt the specimen to a range of angles in order to obtain specific diffraction conditions, and apertures placed above the specimen allow the user to select electrons that would otherwise be diffracted in a particular direction from entering the specimen.
Applications for this method include the identification of lattice defects in crystals. By carefully selecting the orientation of the sample, it is possible not just to determine the position of defects but also to determine the type of defect present. If the sample is oriented so that one particular plane is only slightly tilted away from the strongest diffracting angle known as the Bragg Angle , any distortion of the crystal plane that locally tilts the plane to the Bragg angle will produce particularly strong contrast variations.
However, defects that produce only displacement of atoms that do not tilt the crystal to the Bragg angle i. Crystal structure can also be investigated by high-resolution transmission electron microscopy HRTEM , also known as phase contrast. When using a field emission source and a specimen of uniform thickness, the images are formed due to differences in phase of electron waves, which is caused by specimen interaction. As such, the image is not only dependent on the number of electrons hitting the screen, making direct interpretation of phase contrast images more complex.
However this effect can be used to an advantage, as it can be manipulated to provide more information about the sample, such as in complex phase retrieval techniques. As previously stated, by adjusting the magnetic lenses such that the back focal plane of the lens rather than the imaging plane is placed on the imaging apparatus a diffraction pattern can be generated. For thin crystalline samples, this produces an image that consists of a pattern of dots in the case of a single crystal, or a series of rings in the case of a polycrystalline or amorphous solid material.
For the single crystal case the diffraction pattern is dependent upon the orientation of the specimen and the structure of the sample illuminated by the electron beam. This image provides the investigator with information about the space group symmetries in the crystal and the crystal's orientation to the beam path. This is typically done without using any information but the position at which the diffraction spots appear and the observed image symmetries.
Diffraction patterns can have a large dynamic range, and for crystalline samples, may have intensities greater than those recordable by CCD. As such, TEMs may still be equipped with film cartridges for the purpose of obtaining these images, as the film is a single use detector. Analysis of diffraction patterns beyond point-position can be complex, as the image is sensitive to a number of factors such as specimen thickness and orientation, objective lens defocus, spherical and chromatic aberration.
Although quantitative interpretation of the contrast shown in lattice images is possible, it is inherently complicated and can require extensive computer simulation and analysis, such as electron multislice analysis. More complex behaviour in the diffraction plane is also possible, with phenomena such as Kikuchi lines arising from multiple diffraction within the crystalline lattice. In convergent beam electron diffraction CBED where a non-parallel, i. Using the advanced technique of electron energy loss spectroscopy EELS , for TEMs appropriately equipped, electrons can be separated into a spectrum based upon their velocity which is closely related to their kinetic energy, and thus energy loss from the beam energy , using magnetic sector based devices known as EEL spectrometers.
These devices allow for the selection of particular energy values, which can be associated with the way the electron has interacted with the sample. For example, different elements in a sample result in different electron energies in the beam after the sample. This normally results in chromatic aberration — however this effect can, for example, be used to generate an image which provides information on elemental composition, based upon the atomic transition during electron-electron interaction.
EELS spectrometers can often be operated in both spectroscopic and imaging modes, allowing for isolation or rejection of elastically scattered beams. As for many images inelastic scattering will include information that may not be of interest to the investigator thus reducing observable signals of interest, EELS imaging can be used to enhance contrast in observed images, including both bright field and diffraction, by rejecting unwanted components.
As TEM specimen holders typically allow for the rotation of a sample by a desired angle, multiple views of the same specimen can be obtained by rotating the angle of the sample along an axis perpendicular to the beam. This methodology was proposed in the s by Walter Hoppe. Under purely absorption contrast conditions, this set of images can be used to construct a three-dimensional representation of the sample. The reconstruction is accomplished by a two-step process, first images are aligned to account for errors in the positioning of a sample; such errors can occur due to vibration or mechanical drift.
This three-dimensional image is of particular interest when morphological information is required, further study can be undertaken using computer algorithms, such as isosurfaces and data slicing to analyse the data. Using such arrangements, quantitative electron tomography without the missing wedge is possible.
All the above-mentioned methods involve recording tilt series of a given specimen field. This inevitably results in the summation of a high dose of reactive electrons through the sample and the accompanying destruction of fine detail during recording. The technique of low-dose minimal-dose imaging is therefore regularly applied to mitigate this effect.
Low-dose imaging is performed by deflecting illumination and imaging regions simultaneously away from the optical axis to image an adjacent region to the area to be recorded the high-dose region. This area is maintained centered during tilting and refocused before recording. During recording the deflections are removed so that the area of interest is exposed to the electron beam only for the duration required for imaging. An improvement of this technique for objects resting on a sloping substrate film is to have two symmetrical off-axis regions for focusing followed by setting focus to the average of the two high-dose focus values before recording the low-dose area of interest.
Non-tomographic variants on this method, referred to as single particle analysis , use images of multiple hopefully identical objects at different orientations to produce the image data required for three-dimensional reconstruction. If the objects do not have significant preferred orientations, this method does not suffer from the missing data wedge or cone which accompany tomographic methods nor does it incur excessive radiation dosage, however it assumes that the different objects imaged can be treated as if the 3D data generated from them arose from a single stable object.
Sample preparation in TEM can be a complex procedure. Unlike neutron or X-Ray radiation the electrons in the beam interact readily with the sample, an effect that increases roughly with atomic number squared Z 2. Preparation of TEM specimens is specific to the material under analysis and the type of information to be obtained from the specimen.
Materials that have dimensions small enough to be electron transparent, such as powdered substances, small organisms, viruses, or nanotubes, can be quickly prepared by the deposition of a dilute sample containing the specimen onto films on support grids. Biological specimens may be embedded in resin to withstand the high vacuum in the sample chamber and to enable cutting tissue into electron transparent thin sections.
The biological sample can be stained using either a negative staining material such as uranyl acetate for bacteria and viruses, or, in the case of embedded sections, the specimen may be stained with heavy metals, including osmium tetroxide. Alternately samples may be held at liquid nitrogen temperatures after embedding in vitreous ice. Constraints on the thickness of the material may be limited by the scattering cross-section of the atoms from which the material is comprised.
The resin block is fractured as it passes over a glass or diamond knife edge.
Inorganic samples, such as aluminium, may also be embedded in resins and ultrathin sectioned in this way, using either coated glass, sapphire or larger angle diamond knives. TEM samples of biological tissues need high atomic number stains to enhance contrast. The stain absorbs the beam electrons or scatters part of the electron beam which otherwise is projected onto the imaging system. Compounds of heavy metals such as osmium , lead , uranium or gold in immunogold labelling may be used prior to TEM observation to selectively deposit electron dense atoms in or on the sample in desired cellular or protein region.
This process requires an understanding of how heavy metals bind to specific biological tissues and cellular structures. Mechanical polishing is also used to prepare samples for imaging on the TEM. Polishing needs to be done to a high quality, to ensure constant sample thickness across the region of interest. A diamond, or cubic boron nitride polishing compound may be used in the final stages of polishing to remove any scratches that may cause contrast fluctuations due to varying sample thickness.
Even after careful mechanical milling, additional fine methods such as ion etching may be required to perform final stage thinning. Certain samples may be prepared by chemical etching, particularly metallic specimens. These samples are thinned using a chemical etchant, such as an acid, to prepare the sample for TEM observation. Devices to control the thinning process may allow the operator to control either the voltage or current passing through the specimen, and may include systems to detect when the sample has been thinned to a sufficient level of optical transparency.
Ion etching is a sputtering process that can remove very fine quantities of material. This is used to perform a finishing polish of specimens polished by other means.
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Ion etching uses an inert gas passed through an electric field to generate a plasma stream that is directed to the sample surface. Acceleration energies for gases such as argon are typically a few kilovolts. The sample may be rotated to promote even polishing of the sample surface. The sputtering rate of such methods is on the order of tens of micrometers per hour, limiting the method to only extremely fine polishing. Ion etching by argon gas has been recently shown to be able to file down MTJ stack structures to a specific layer which has then been atomically resolved.
The TEM images taken in plan view rather than cross-section reveal that the MgO layer within MTJs contains a large number of grain boundaries that may be diminishing the properties of devices. More recently focused ion beam methods have been used to prepare samples. Because FIB can be used to micro-machine samples very precisely, it is possible to mill very thin membranes from a specific area of interest in a sample, such as a semiconductor or metal.
Unlike inert gas ion sputtering, FIB makes use of significantly more energetic gallium ions and may alter the composition or structure of the material through gallium implantation. Samples may also be replicated using cellulose acetate film , the film subsequently coated with a heavy metal such as platinum, the original film dissolved away, and the replica imaged on the TEM. Variations of the replica technique are used for both materials and biological samples. In materials science a common use is for examining the fresh fracture surface of metal alloys.
The capabilities of the TEM can be further extended by additional stages and detectors, sometimes incorporated on the same microscope. Particular attention is given to the type of material, conditioning, compatible analysis of a given preparation, and risks. This practical and authoritative reference companion deserves a place on the bench in every TEM lab. Combines all of the latest techniques for the preparation of mineral to biological samples.
To accomplish this step successfully, different approaches have been developed. One of the solutions is micromanipulator to hold and control the vitreous cryosections by eyelash when they come off from the knife edge. During the entire process, the ribbon is under constant tension as it grows longer, thereby keeping the ribbon as straight as possible.
When the appropriate length is obtained, the ribbon is attached to the grid surface by lowering the micromanipulator and holding it in optimal position while a second eyelash is used to affix the other end of the ribbon to the grid surface from the knife edge. It is possible to affix a few ribbons to a single grid. However, the discussed solution is time consuming and prone to ice contamination [ ]. The ribbons on the grid can be flattened by pressing with tools. The aim of this step is to reduce the probability of losing the sections during storage and transfer the grid, as well as improve the stability of section under electron beam [ ].
Another solution is electrostatic charging for attaching the sectioned ribbon to the grid [ ]. In comparison with micromanipulator solution, this method increases the successful attachment of frozen-hydrated sections to the carbon film, albeit both methods cannot guarantee uniform attachment of cryosections to the carbon film.
This results in higher sensitivity of the section to the beam exposure and section movement during image acquisition, especially during electron tomography [ ]. Recently, a new tool based on an aforementioned solution was presented [ ]. One of the micromanipulator is used to manipulate the section ribbon by electrically conductive fibre; the second one positions the grid beneath the newly formed ribbon, and with the help of an ioniser, the ribbon is attached to the grid. This tool greatly facilitates manipulations, but sectioning artefacts remain. This technique is widely used in material science; however, Marko et al.
FIB milling of vitreous samples is conducted using a dual-beam microscope. The dual-beam microscope is a combination of FIB system and scanning electron microscope.
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During FIB milling of vitreous sample, a finely focused beam of ions, usually gallium, is used to ablate the surface of the specimen through sputtering process. The whole procedure is under visual control of the SEM to ensure optimal procedure of sample preparation [ ]. However, direct interaction between the ion beam and vitreous material must be taken into consideration due to possible sample damage. The application of a gallium ion beam with current of 10 pA and 30 kV acceleration does not cause sample devitrification [ ]. Moreover, interaction of FIB with vitreous sample results in implantation of an ion layer, as thick as 5—20 nm into the FIB milling surface [ ].
Indeed, the thickness of implanted gallium layer is almost negligible in a vitreous specimen with a thickness of — nm. Furthermore, the ion layer is much thinner in comparison with crevasses found in vitreous sections. Cryo-FIB micromachining is a relatively new technique and remains in its early stages. Nevertheless, few sample preparation strategies have been introduced in the last 10 years.
To date, bacteria and small eukaryotic cells, like Mycobacterium smegmatis, Saccharomyces cerevisiae and Dictyostelium discoideum [ , ], Escherichia coli , HeLa cells [ , ], BHK cells [ ] and Aspergillus niger [ ], are deposited for culturing on the TEM grids and vitrified by plunge freezing technique. Next, vitrified material is transferred into the dual-beam microscope for a thinning process with a precision in the 10— nm range. At this stage, different FIB milling strategies for vitrified cellular samples are possible.
Eukaryotic and other cells similar in shape and size are milled in a thin self-supported membrane. During this process, a specific region of interest is localised and then rectangular sector below and above the selected volume is sputtered away, leaving behind a thin membrane, commonly referred to as a lamella, supported by the surrounding unmilled cells and ice [ ]. Another option to obtain lamellas is a traditional FIB lift-out method, although it was deemed impossible because of the difficulty in obtaining platinum deposition at cryogenic temperatures [ , ].
Shortly, after vitrification, the feature of interest is defined through SEM; next the sample is cryo-coated with platinum Pt and two trenches are milled on each side of the lamella to be extracted. In the next steps, the sides and bottom of the lamella are sputtered away, and by using cold nanomanipulator, lamella is then lifted out from the sample and finally attached to the TEM grid by cryo-Pt deposition. During the last step, the attached lamella is thinned enough to be transparent to electron beam. The lamella-based sample preparation has an advantage over the wedge-shaped strategy, because the simple ablation geometry would not permit to easily find and target structures of interests embedded deeply in cellular volume [ , ].
Thick samples and suspension of cells, e. After that, material hidden inside both the tube and the carrier is exposed during pretrimming step inside the cryo-ultramicrotome. Subsequently, vitreous sample is transferred to the dual-beam microscope and milled according to H-bar strategy, ultimately resulting in lamellas with the required thickness and surface area. Finally, prepared sample is transferred to the cryo-TEM in order to perform visualisation under low-dose mode.
Critical point is when each of the described steps must be carried under devitrification temperature and minimising frost or warming during transfer steps implicates customised transfer device introduction. For samples vitrified by plunge freezing, different cryo-FIB transfer stations and cryo-FIB shuttles were introduced [—], while high-pressure frozen samples require sophisticated and complex transfer systems like cryo-nano-bench system [ ] or an intermediate specimen holder [ , ].
The cryo-FIB technique provides controlled access to specific supramolecular structures buried inside the cell. However, this preparation technique is low throughput due to several factors. As it was mentioned, an incidence of the ion beam should be as low as possible because a smaller milling angle produces a larger viewing area for analysis and minimises the deposition effect of milled material.
The latter factor is not interrelated with milling currents or other parameters during sample thinning as such [ , ]. Inhomogeneous and varied composition of the vitreous samples is the main cause for curtaining effect which finally results in strong inhomogeneous lamella thickness [ , ]. These effect is reduced by deposition of organometallic platinum with a gas injection system without electron or ion beam radiation, prior to lamella preparation [ ].
Another issue is the amount of information obtained from prepared samples during cryo-electron tomography. During cryo-FIB milling process, part of the vitreous specimen is physically destroyed along all axes [ ]. In contrary, after vitreous sectioning, a series of cryosections is obtained, and information along these axes is partly remained, especially along z -axis. A further problem is the time needed to prepare sample prior to visualisation.
Due to the large size of eukaryotic cells, longer milling time is required in comparison with the prokaryotic samples [—]. Other challenges related to the increased size of eukaryotic cells are identifying and targeting specific sites for processing. Small organisms, such as E. Inversely, eukaryotic cells are surrounded by thick ice, thus identifying the area of interest is not simple.
A method to overcome this limitation is either milling of many adjacent places to find features of interest or application of correlative light and electron microscopy techniques. Cells are cultured on EM finder grids and optical images are recorded before vitrification. Appropriate regions are selected for subsequent cryo-FIB milling based on light microscopy photos [ , ]. Alternatively, the frozen-hydrated sample is imaged under cryo-fluorescence microscopy before sample preparation by cryo-FIB for further analysis.
The second approach allows direct correlation of the prepared vitreous sample between the two imaging modalities [ , , ]. Moreover, localisation of smaller molecules or structures that exist in low copy number is simplified because targeting is based on clonable labels, such as green fluorescent protein [ ]. The new idea presented recently is to localise structure of interest by fluorescent labels using cryo-light microscopy and then use it for coordinate transformation-based approach in the FIB-SEM system for milling [ ]. At present, cryo-FIB milling of vitreous samples remains cumbersome and far from routine [ ].
Much effort is required to improve the efficiency and repeatability of cryo-FIB milling process, such as reproducibility of lamella quality, i. Therefore, the main issue in the presented articles is improving sample preparation protocols. Nevertheless, some interesting results have been achieved. For example, ten nuclear pore complexes in D. Subtomogram averaging process yields the structure of nuclear pore with resolution of 7. To achieve 6 nm resolution, 4, protomers are required from isolated nuclei using the same type of analysis [ ].
Another result comes from bacterial cells, where membrane invaginations into both the cytoplasmic and periplasmic spaces of E. This technique will greatly facilitate high-resolution imaging of dynamic process, such as HIV particles travelling into the deep side of the host cell at different stages of infection, especially when viral capsid interacts with nuclear pore complex components [ ].
Sample Preparation Handbook for Transmission Electron Microscopy : Jeanne Ayache :
By using FIB milling process, ryanodine receptors in toadfish swimbladder muscle were determined. Obtained results agreed remarkably well with those described previously, albeit further study will be required to understand structural features of ryanodine receptor connected to the T-tubule [ ].
Cryo-FIB is also a promising thinning tool for describing new bacterial cytoplasmic structures termed as a stack. Stacks were defined as piles of oval disc subunits which are surrounded by a membrane-like structure. These structures are localised in the cell cytoplasm and are presented separately or grouped together in variable number within each cell. Due to compression created during CEMOVIS and visualisation limitations arising from plunged frozen samples, cryo-FIB technique could provide new insight into macromolecular assembly of membrane-enclosed discs [ ]. The sample in native state is very different from what has been seen before with conventional microscopy.
Vitrified material is as close as possible to the native state because during sample preparation, neither chemical fixation, staining nor dehydration is used. Therefore, the final images represent the real distribution of the immobilised biological material within vitrified water.
With frozen-hydrated samples, the contrast is proportional to the density and distribution of molecular inhabitants within the thickness of the sections. Moreover, structures in vitreous material are equally visible over the entire thickness, thus the native-state inherent low contrast due to low signal-to-noise ratio. In contrary to stained material, imaging of native biological material relies on phase contrast, which strongly depends on focus [ 71 ]. An additional issue is plethora of overlapping information for the reason that the typical fine details are much smaller than the section thickness.
The solution overcoming this limitation is both preparation of thin sections or lamellas and electron tomography to obtain a three-dimensional model of the material distribution in the vitreous material. Frozen-hydrated specimens behave differently under electron beam in comparison with plastic sections. A characteristic phenomenon that may be developed is bubbling. Bubbling is a result of gas accumulation produced by electron beam decomposition of biological matter. What is the most interesting is that different substances have different electron radioresistance. Another problem is beam-induced deformations which are seen twofold.
Vitreous material can be considered as high-viscosity liquid and can be rearranged by the electrons. On the one hand, sharp irregularities, such as crevasses and knife marks, under electron beam are removed from the vitreous section due to increasing the flow of the section. On the other hand, some biological structures, i. The main disadvantage of frozen-hydrated specimens is its uselessness to perform post-immunolocalisation of studied target.
Antibodies require proper conditions for working, that is, ambient conditions and water solution. Accordingly, some researchers have developed a specific label for the identification of molecules for cryo-electron microscopy. However, ligand labelling for cryo-EM is still an emerging field; hence, another preparation technique is dedicated for immunogold labelling and structural studies, namely, freeze substitution. Indeed, vitreous and freeze-substituted materials are very complementary [ ]. The latter preparation solution should be considered as the method of choice when high-resolution study is not the major aim.
Resin-embedded material is easier to obtain and is less sensitive to electron beam in comparison with vitreous material, and for this reason, analysis of larger sample area is possible. Additionally, plastic sections can be thicker than vitreous ones, and thus, the former enable studying a larger volume.
The other advantage of resin sections is the possibility of immunogold labelling. Freeze substitution FS is a hybrid method that bridges the gap between vitrified material and room temperature ultramicrotomy of resin-embedded material. Biological material after vitrification process is gradually dehydrated in the presence of chemical fixatives at low temperature. Later, the whole process is gradually warmed, and finally, the sample is embedded in resin.
This technique was first introduced in , as a preparative technique for light microscopy samples [ ]. The potential of FS at electron microscopy filed was explored by Fernandez-Moran [ ] and was further developed by others [—]. Freeze substitution process consists of dehydration and chemical fixation step followed by either low- or room-temperature embedding in chosen resin. The key point is that sample must be kept below devitrification temperature. What happens during water substitution at low temperatures is not fully understood.
Nevertheless, vitrified water turns into cubic ice, and then transition takes place into hexagonal ice. Cubic ice is a metastable state, thermodynamically more stable form of water. The most important event during these transitions is that water molecules probably rearrange by rotation with only small transitional displacement which leads to embedding the biological structure by ice without any segregation. Then the result is that the structural preservation is excellent down to molecular dimension.
In reality, FS process deals with cubic and sometimes with hexagonal ice but never with vitreous water [ 60 ]. This would imply that cubic ice has no significant influence on the observed morphology at the level of resolution of biological samples during FS process under controlled conditions.
Besides these theoretical bases, other aspects must be considered. From the biophysical side, low temperature influences on ultrastructure preservation through hydration shell preservation and infiltration of chemical fixatives. FS process preserves the hydration shell at least partially, although less hydrophilic organic solvents are used, e. It is well known that organic solvents cause protein aggregation, and chemical fixatives react relatively slowly; therefore, it cannot preserve all the cellular components simultaneously.
The consequences of chemical fixation are seen as osmotic changes and redistribution or extraction different molecules, i. The reason for the superior structural preservation is infiltration of stabilising or fixative compounds together with the dehydrating agents. Many different substitution media compositions were developed. The main fixatives are GA, osmium tetroxide and UA used in different combinations and at different percentages in acetone, methanol or ethanol [67,—]. Acetone is the most commonly used dehydrating agent because it substitutes at a slower rate than methanol, thus resulting in better structural preservation [ , ].
The most-used fixatives are OsO 4 with or without UA in acetone for morphological study and low concentrated GA in acetone for immunolabelling detection [—]. The interesting observation concerning reactivity of used fixatives was made. Uranyl acetate binds to proteins and phospholipids at an even lower temperature. For some immunolabelling study, pure solvent preserves well both the antigenicity and the ultrastructure of the cells [ , ]. In order to improve the membrane contrast and preservation of cultured cells, a few different substitution media were introduced.
A disadvantage of a medium containing water is antigen loss by extraction. Similar results were observed for pure solvents; thus, low concentration of glutaraldehyde can be generally used [ ], but the exact substitution protocol requires an individual approach. Alternatively, membrane contrast can be enhanced by using tannic acid-mediated osmium impregnation method [ ], tannic acid in acetone during FS [ 95 , ] or different combinations of glutaraldehyde, UA and OsO 4 [ , ].
Epoxide compounds react with proteins and lipids and provide interesting results which may become an important tool in getting information about influence of different reagents and protocols on ultrastructure preservation [ ]. Current FS procedures are measured in wide time range from less than 24 h for cell culture [ , , , ] to longer period of time such as four days for plant material [ , ]. However, other tissues are usually substituted during 2—4 days, e. On the one hand, such a wide range of different fixative cocktails gives opportunity to study different structures of interests in both structural and immunolocalisation research.
On the other hand, the variety of possibilities become a challenging task for optimisation of FS process because each sample is unique [ 67 ]. The last step during sample preparation for the room-temperature ultramicrotomy is sample embedding. Epoxide-based polymers are dedicated for morphological analysis including electron tomography by virtue of a larger stability in the electron beam and ease of sectioning. In contrast to epoxide resins, methacrylates do not bind covalently to cellular structures; hence, the antigens of interests remain unaltered and section surface has higher roughness, thereby higher access to antigens.
Another advantage of methacrylic resins is low-temperature embedding and polymerisation by UV light Figures 2 and 3 ; thus, harmful heat effects on epitopes is avoided [ , ]. However, this division is not a rule, because Epon sections were used to identify the subcellular localisation of proteins [ ], lipids [ ] or carbohydrates [ ]. Super-Quick FS takes only about 6 h from freezing process to resin blocks preparation ready to section.
For this protocol, organisms considered as difficult to fix were chosen. As a result, presented ultrastructure preservation was comparable to standard FS protocols, and high-temperature polymerisation does not affect antigen preservation. High-resolution study requires superior sample preparation via vitrification process. Sample such as protein, protein complexes, viruses, bacteria or organelles in vitro and in situ within whole organisms or single cell are prepared by plunge freezing.
Moreover, in modern structural biology, the main goal is in situ structure determination within unperturbed cells because purified objects are disintegrated during sample purification. The visualisation of frozen-hydrated biological samples is performed by single-particle analysis or cryo-electron tomography. The former technique enables to achieve near-atomic resolution and is applied to purified viruses, macromolecular complexes and single proteins [ 80 , , ].
On the other hand, cryo-ET bridges the gap between cellular ultrastructure and the structural analysis of macromolecular complexes within the cell with resolution in the sub-nanometre [ ] to 10 nm range [—]. Electron tomography of plastic sections is another technique dedicated for cellular structural biology, where the more important aim is to reveal functional-morphological relationships than macromolecular details.
Sample is prepared by cryoimmobilisation followed by freeze-substitution process. Next, polymerised specimen is sectioned in — nm range and analysed [ ]. Combination of subsequent section tomograms extended the depth of analysed volume to several micrometres. These advantages led to large-scale imaging where both detail and overview are necessary.
Besides three-dimensional analysis, both frozen-hydrated and plastic sections are also analysed at lower resolution at 2D morphological level. CEMOVIS has already provided unusual views of different structures with a molecular resolution in native cellular context including microtubules, mitochondria, Golgi apparatus [ ] or desmosomes [ ].
The reader is referred to review articles that cover mentioned topics with further references [79,—] due to limited space. Cryo-EM of vitreous sections gives opportunity to study different tissues, including skin biopsies. The unravelling of molecular organisation of the skin lipids will significantly improve molecular understanding of the tissue.
Until recently, six theoretical models for the molecular arrangement of the extracellular lipid matrix have been proposed. Nevertheless, combination of TOVIS, molecular modelling and EM simulations has revealed a new model of lipid organisation, which rationalises the skin nature and functions [ ].
These results will influence dermatology field and thereby further translates in technological developments of new, transdermal drug delivery systems, the development of noninvasive diagnostic sensors and dealing with toxicity from topical exposure to chemicals. In combination with fluorescence microscopy, it was showed that the induction of nucleolar stress in cancer cells resulted in both an increase in water content and a decrease in the element content in all cell compartments.
The presented study opens new way to understand cell functions, and future research could extend our knowledge about cell activities, depending on actual concentration of ions and the hydration status. The classical examples of superiority of the cryopreparation techniques, based on HPF followed by FS process, over conventional TEM were showed on the method-dependent bacterial mesosomes [ 55 ] and articular cartilage [ 85 ], although in the former, further investigation led to revisiting the mesosome as a site of hydrogen accumulation [ ] using quick-freezing preparation of TEM.
Among the advantages of plastic sections, one is the section thickness; thus, comparatively large cellular volume can be analysed that was exemplified by microtubule cytoskeleton architecture in yeast [ ], organellar relationship in the Golgi region of the pancreatic cells [ ] or architecture of the caveolar system [ ]. Besides, the reader can find many articles where either different techniques were compared [55,91,96,—] or examples were collected in reviews [ , ].
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Cells were chemically fixed, dehydrated and embedded in LR White resin . Visfatin is an enzyme which overexpression is correlated with poor prognosis in cancer patients. In this study, we tried to explore the association between visfatin distribution in subcellular compartments and increased apoptosis in cells treated with cytochalasin B. Figure and legend adapted and changed from  under terms of the CC BY 3.
In comparison with cells prepared by conventional method Figure 2 , vitrified and freeze-substituted material show outstanding ultrastructure preservation. Even without OsO4 fixation, membranes are clearly visible, mainly rough endoplasmic reticulum and nuclear membranes. Nevertheless, further protocol optimisation is required. Immunoelectron microscopy bridges the information gap between molecular biology and ultrastructural studies providing information regarding the function of the internal structures of the cell.
The main requirements to obtain valuable results are suitable fixation protocol and functional antibody conjugated with an appropriate nanoparticle to be detected. For many years different protocols were developed for structure localisation study [ ] based mainly on Tokuyasu technique, HPF followed by FS and low-temperature embedding [ ] or conventional chemical fixation [ , ] together with progressive-lowering temperature technique.
Nevertheless, this field is still amenable to new solutions, especially for difficult-to-fix samples and difficult-to-fix antigens. The hybrid techniques combine advantages of different cryopreparation techniques in order to eliminate the particular step limitations of each one, at least in part. Cryosectioning according to Tokuyasu is one of the most reliable and efficient immunolocalisation techniques for different types of sample.
An inherent limitation of Tokuyasu cryosectioning is mild chemical fixation at the beginning of the sample preparation. Thus, small molecules, including molecules of interests, may be dislocated or extracted during chemical fixation. Besides, it should be mentioned that process of chemical fixation is selective and results in pH-related and osmotic changes in the different organelles [ 58 ]. Another restriction is intractability of samples, which contain a hydrophobic cuticle or a rigid wall, such as C.
Cryoimmobilisation should be used to overcome difficulties arising from chemical fixation during sample fixation. In spite of the all above-mentioned issues, one more should be considered — the nature of the antigen. Some antigens are sensitive on chemical fixation at room temperature or resin components and solvents and thus cannot be immunolabelled either in thawed cryosections or after cryoimmobilisation, freeze substitution and resin embedding.
Therefore, the main aim was to introduce hybrid methods that combine the high-efficiency Tokuyasu cryosectioning labelling technique with an initial cryoimmobilisation step. Different approaches have been introduced with different results. The first attempts were taken by the group of Slot and Geuze [ 53 , 56 ] by combination of a frozen-hydrated cryosections with subsequent material fixation during thawing and after transfer to a grid.
This method turned out to be unsuitable to routine use because, besides technical requirements and lack of reproducibility, only a small area of obtained sections has got desired morphological quality [ 56 ]. Another strategy to improve the antigenicity and ultrastructure preservation is rehydration method RHM based on cryofixation, freeze substitution and rehydration process before entering Tokuyasu cryosectioning and immunolabelling [ 56 ].
After freeze substitution step, a rehydration process is carried out on ice. Rehydration step is necessary to enter the Tokuyasu procedure, i. During dehydration step, additional chemical fixation is performed because fixation during FS step turns out to be insufficient. This approach resulted in excellent preservation of HepG2 cells, primary chondrocytes, cartilage and exocrine pancreases and immunolabelling efficiency comparable to Tokuyasu method.
In the case of tested samples, authors suggested using the standard Tokuyasu technique because it is much easier, faster and allows the preparation of larger samples. The real power of the RHM methods was showed on Arabidopsis tissues, anthers containing pollen grains, D. For these organisms, the RHM method was slightly modified [ 58 , 59 ] on dehydration step which was started at subzero temperatures. Obtained results were similar to the Van Donselaar et al. Moreover, fixation-sensitive antigens were not inactivated, despite of using high concentration of fixatives. An additional benefit is usefulness of the hybrid techniques for fluorescence microscopy and CLEM due to the optimised ultrastructure preservation.
Green fluorescent protein signal could be observed even after OsO 4 treatment [ 58 ]. High lateral and axial fluorescence resolution can be obtained using thin cryosections; thereby, blurred signals are eliminated. Fluorescence-tagged antibody is much more sensitive than gold markers; thus, if fluorescence signal is not detected, then immunogold labelling is not worth to perform [ 59 ].
Hybrid techniques provide an alternative for worthlessness of vitrified cryosections in immunogold labelling. Next, the sections on the grid are fixed and brought to room temperature by means of FS and immunolabelling. Different combinations of fixatives were tested, including osmium tetroxide, glutaraldehyde, formaldehyde, acrolein and UA.
The variety of mentioned fixatives and short time needed for sample preparation give huge possibilities in protocol optimisation for different antibodies and samples. Although vitreous section is open structure and thus fixatives can penetrate it in high extent, the lack of embedding medium does not cause the material extraction.
The high accessibility for the fixatives results in outstanding preservation of vesicles, particularly in the Golgi area and organelles. Further superiority over other techniques was proved through immunolabelling of both resin and aldehyde-sensitive antigens.
As another option for making the impossible possible is vitrification of Tokuyasu-style immunolabelled sections, in brief VOS vitrification of sections technique. This approach was first time presented nearly a quarter of century ago by Sabanay et al. In contrary to the described hybrid approach, in VOS technique, the first step is based on Tokuyasu sample preparation.
The common steps are mild chemical fixation and cryoprotection in sucrose followed by immunogold labelling. After that, sample is re-vitrified in liquid ethane instead of treatment with methylcellulose and air-drying steps, as in the Tokuyasu technique [ ]. Therefore, VOS technique provides meaningful 3D information on nm thick sections.