principles and techniques of electron microscopy biological applications pdf

Principles And Techniques Of Electron Microscopy Biological Applications Pdf

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Transmission Electron Microscopy of Biological Samples

During the last 70 years, transmission electron microscopy TEM has developed our knowledge about ultrastructure of the cells and tissues.

Another aim is the determination of molecular structure, interactions and processes including structure-function relationships at cellular level using a variety of TEM techniques with resolution in atomic to nanometre range.

Even with the best transmission electron microscope, it is impossible to obtain real results without optimal sample preparation, respecting both the structure and the antigenicity preservation. Preparation techniques for high-resolution study of both macromolecular complex and organelles within cellular complex are based on fast cryoimmobilisation process, where the sample is in the most native, hydrated state.

Next, thin samples are directly visualised under cryo-transmission electron microscopy cryo-TEM , while thicker samples require a thinning step via cryo-electron microscopy of vitreous sections CEMOVIS or cryo-focused ion beam cryo-FIB before visualisation.

Alternatively, vitrified samples are freeze substituted and embedded in chosen resin for room temperature ultramicrotomy. This preparation technique is suitable for morphological study, 3D analysis of cellular interior and immunoelectron microscopy. A different route for immunolocalisation study is cryosectioning according to the Tokuyasu technique that is a choice for rare or methacrylate-sensitive antigens.

Most recently, new hybrid techniques have been developed for difficult-to-fix organisms and antigens or labile and anoxia-sensitive tissues. Another preparation technique is, the oldest but still important, conventional chemical fixation dedicated in a wide range of research interest, involving morphological and immunolocalisation study.

In this chapter, we present different sample preparation approaches for transmission electron microscopy of biological samples, including its methodological basis and applications. The first transmission electron microscope was constructed in the early s by Ernst Ruska and Max Knoll [ 1 ]. Roughly a decade later, the first electron microscope picture of eukaryotic cells was taken by Keith Porter [ 2 ]. Since then TEM made it possible to study cells and tissue structure and function at nanoscale.

Although fluorescence techniques allow for imaging dynamic process in living cells and modern fluorescence microscopes overcoming the diffraction limits that makes it possible to zoom in on cellular structure with resolution under nm [ 3 ], TEM remains the main technique which makes it possible to study biological systems owing to its near-atomic-level resolution [ 4 ].

Moreover, TEM gives opportunities to visualise an interesting target with surrounding structure, when unlabelled surroundings still remain hidden at fluorescence sample [ 5 ].

Additionally, TEM comprises different branches: electron crystallography and single-particle analysis are dedicated to study proteins and macromolecular complexes, cryo- electron tomography and CEMOVIS for cellular organelles and molecular architectures and conventional TEM for gross morphology. Such a wide range of electron microscope techniques gives opportunity to find the relation between different macromolecules, their supramolecular complexes and organelles assembled into an intricate network of cellular compartments.

Knowledge of the cellular ultrastructure can contribute to an understanding of how cells and tissues function in both normal physiological and pathological state. Since the invention of the first TEM, the aim has been to image liquid samples at higher resolution but as easily as with light microscopy.

At the beginning, it was impossible to accomplish due to low technological knowledge and lack of appropriate tools. Thus, scientists have introduced sample preparation techniques for observing soft and frail living matter in the inhospitable environment of an electron microscope.

The TEM column is under ultrahigh vacuum, where electrons as a coherent beam are directed on the sample. Moreover, biological matter compose mainly of light elements e.

Consequently, native biological materials are of extremely low contrast. On the other hand, the two aforementioned factors oriented sample preparation strategies. Larger samples need to be sectioned for analysis, but cells and tissues are too soft to be sectioned thinly enough without earlier sample preparation. Many laboratories have been ingenious in designing and implementing different preparation techniques over the years, and as a result, scientists have found at least a partial remedy to these problems.

Therefore, a biological sample can be prepared either by removing or by freezing water. The oldest method is conventional sample preparation which uses chemical fixation, sample dehydration at room temperature and embedding with chosen resin.

In the s, Tokuyasu introduced an alternative to conventional sample preparation dedicated for immunocytochemistry. An alternative method to chemical fixation is cryofixation via vitrification process. Taking into consideration the size of the sample, electron microscopists have a wide range of freezing techniques at their disposal.

Small or thin sample after plunge freezing can be directly observed at low temperature under cryo-transmission electron microscope cryo-TEM. Finally, thin frozen-hydrated samples are directly observed under cryo-TEM. Another option is freeze substitution FS which bridges the gap between vitreous states and room temperature ultramicrotomy. Lastly, different combinations of mentioned techniques offer a new research possibility, especially for difficult-to-fix organisms or antigens.

In a particular situation, chemical pre-fixation step is a prerequisite for successful sample vitrification, although it seems to be contradicted. Biological systems are very complex; thus, it is impossible to understand structure-function relationship outside the surrounding context. These days, dynamic developing of correlative light and electron microscopy CLEM approach can be observed. This approach relies on two steps. Firstly, the object of interest is located and imaged with fluorescence microscopy FM , and then the sample is imaged in TEM.

This technique is highly demanding according to cell biologists because high-resolution data can be fitted in the cellular context. However, new possibilities introduce new challenges in the preparative stage, and protocols for TEM and FM often are incompatible. Therefore, it is worth to mention that technological progress stimulates new sample preparation design, but often existing preparation schemes initiate new ideas. In this chapter, we present different specialised preparation techniques dedicated to cells and tissues; but at the beginning, we would like to impress the importance of water in life on readers, because for a long time, its role in living organisms was neglected.

Another point is that for a long time, water was treated as a foe by electron microscopists. Nevertheless, readers should bear in mind that selection of an appropriate technique strongly depends on the material and aims of the study. Thus, a general rule of thumb is that the higher the resolution is important, the closer to the native state sample preparation is desired.

Moreover, the higher the resolution, the thinner sample should be, but at the same time, less information is achievable. During morphological study, more important is the sample size; hence, the preparation technique based on resin-embedded sample is an adequate choice. However, for immunolabelling research, compromise between antigens and ultrastructure preservation is the major challenge. Although the main aim of this chapter is to present different preparation techniques of biological specimens for TEM, we would like to also point out that the preparation step is important for correlative approach.

We strongly encourage further reading of proposed positions where the reader can find practical insights of the presented subject, e. Many important hints in sample preparation for TEM are also connected with CLEM field [ 15 , 16 ] and immunoelectron microscopy [ 17 ]. In our opinion, a complete library should also include the Handbook of Cryo-Preparation Methods for Electron Microscopy because this position is strongly oriented to the practical side of sample preparation art [ 18 ].

It is also important to know what was done so far and thus where we should go. Among many old books, but with still-current knowledge, Cryotechniques in Biological Electron Microscopy [ 19 ] captured our attention. The last but not the least position is the Principles and Techniques of Electron Microscopy: Biological Applications [ 20 ]. Organisms consist in major part of liquid water which is the medium in which life takes place.

Hence, life on our planet and its probability elsewhere in the universe cannot have evolved or continue without water. In view of the abundant presence of water in living organisms, this substance cannot be perceived as an inert diluent. Water performs many functions: it transports, reacts, lubricates and structures and is used in signalling.

Water is also a metabolite and a temperature buffer. The physical properties of water, which result from its structure, play a key role in the orchestration of the cell machinery. Biological molecules and water should be thought as equal partners where one is required and structured by the other.

From a chemical point of view, a water molecule contains one oxygen atom covalently bounded with two hydrogen atoms. Due to positively charged hydrogen atoms and a negatively charged oxygen atom, where negative charge comes from two lone electron pairs, water is a dipole. Water as a dipole has the most important property: water molecules are able to form multiple hydrogen bonds between each other.

A hydrogen bond occurs when a partially positively charged hydrogen atom lies between partially negatively charged oxygen of H 2 O molecules. A hydrogen bond is naturally formed from a complex combination of different interactions: an electrostatic, a polarisation and a covalent attraction, and a dispersive attractive interactions, an electron repulsion and a nuclear quantum effects.

In theory, one water molecule can interact with four other water molecules, thereby forming a tetrahedron configuration. In practice, hydrogen bonds are very dynamic and heterogeneous structures, both on energetic and structural levels, and a single water molecule can form two or four hydrogen bonds.

As a result, in liquid water, hydrogen bonds behave in cooperative and anticooperative manner [ 21 ]. At the higher level of organisation, water molecules in liquid state tend to create tetrahedral pentameric clusters, which are linked to other water molecules and clusters to form a complex network or liquid phases [ 22 ].

Such a network of hydrogen bonds is dynamic and ordered in a nanometre range structure. The dipolar nature of water enables to arrange molecules of water into an ordered, very constrained manner on the surface of biological molecules. Depending on the chemical nature of surface domains, hydrophobic or hydrophilic, water order is different. Water molecules are strongly attracted by ionised and hydrophilic domains than by apolar domains, where H 2 O molecules arrange themselves into clathrate-like structures [ 23 ].

They form a hydration shell, called also interfacial water, built from several water layers. Hydration shells are critical for solubility of molecules and prevent them from aggregating. When two particles meet, they do not stick together, but separate [ 24 ]. Moreover, protein folding is mediated and guided by aqueous solvation, and protein structure is stabilised by water clusters and their hydrogen bonding capabilities.

Water also gives proteins flexibility during conformational changes, and its molecules mediated protein-ligand interaction. Another interesting example of water role in the cellular world is nucleic acid-water interactions. Firstly, water molecules stabilise structure of double helix.

Secondly, water hydrates both the major and the minor grooves of DNA. The specific arrangement of interfacial water governs protein binding to the DNA. The enumerated examples are further discussed in detail in [ 25 ]. Water inside the cell, which is not bound in hydration shell, is unaffected by the biomolecules.

Additionally, cellular unbounded water behaves differently from water outside, e. The cytoplasm has a sol-gel nature. The local parts of the cytoplasm may manifest itself as a more highly viscous and stiff environment, likened to a gel-state, or as a low-viscosity sol-state solution [ 21 ]. In the former case, water molecules form more strongly hydrogen-bounded water clusters. This reduces local fluctuations in the nearby macromolecules and slows down metabolite and ion migration [ 23 ].

An additional function of strong hydrogen-bonded network existence is transmission of information about solutes and surfaces at distances of several nanometres. The state of the water is thus essential for the biological activity of the cell, and the state of metabolites controls water structure. Thus, water is defined as an engine of life [ 26 ]. From the physical point of view, water is usually perceived as an ordinary substance because people interact with it all the time in their everyday lives.

Learn & Share: Correlative Light Electron Microscopy (CLEM)

The advantage of LM is that it can provide wide field images of whole, often living, cells, but its resolution is limited. The advantage of EM is that it can provide much higher resolution images, up to molecular dimensions, but only over specific regions of a cell at a time and not in living cells. CLEM combines the advantages of both techniques, allowing scientists to spot cellular structures and processes of interest in whole cell images with LM and then zoom in for a closer look with EM. This dual examination provides valuable complementary and often unique information. Your bookmarks are missing?

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Electron microscopy EM is a technique for obtaining high resolution images of biological and non-biological specimens. It is used in biomedical research to investigate the detailed structure of tissues, cells, organelles and macromolecular complexes. The high resolution of EM images results from the use of electrons which have very short wavelengths as the source of illuminating radiation. Electron microscopy is used in conjunction with a variety of ancillary techniques e. EM images provide key information on the structural basis of cell function and of cell disease.

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Principles and techniques of electron microscopy biological applications vol 9

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Principles and techniques of electron microscopy vol 2 biological applications. Hayat, M. Biological Applications. Xv p. Xv, Principles and techniques of electron microscopy.

Synopsis: Principles and Techniques of Electron Microscopy is the standard work for biological electron microscopists wishing to learn how to prepare their specimens for electron microscopic investigation. This fully revised and expanded fourth edition includes three new chapters covering such topics as plant tissues, immunocytochemistry, and applications of microwave irradiation to microscopy. It provides practical instructions on how to process biological specimens, as well as a detailed discussion on the principles underlying the various processes. Hayat presents methods in a self-explanatory form and includes alternative procedures and points of disagreement to help the reader interpret data accurately. What sets this book apart from its competition is that it not only describes techniques but also explains their fundamental principles; that is, those chemical reactions underlying the use of various reagents for preserving and staining cellular components.. Hayat Synopsis: Principles and Techniques of Electron Microscopy is the standard work for biological electron microscopists wishing to learn how to prepare their specimens for electron microscopic investigation.


Hayat MA. Principles and techniques of electron microscopy: biological applications. 4th edn. PDF; Split View. Views. Article contents.


Introduction

Nigel Chaffey, Hayat MA. Principles and techniques of electron microscopy: biological applications. Cambridge: Cambridge University Press. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide.

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Он выбежал из помещения Третьего узла и направился к люку. Чатрукьяна во что бы то ни стало следовало остановить. ГЛАВА 51 Джабба был похож на гигантского головастика.

 Мы тонем! - крикнул кто-то из техников. ВР начала неистово мигать, когда ядро захлестнул черный поток. Под потолком завыли сирены.

 Может быть, для того, чтобы вы не заподозрили, что это приманка. Может быть, Танкадо защитил его ровно настолько, чтобы вы на него наткнулись и сочли, что вам очень повезло. Это придает правдоподобность его электронной переписке.

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During the last 70 years, transmission electron microscopy TEM has developed our knowledge about ultrastructure of the cells and tissues.

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