Transmission electron microscopy has been responsible for most of what is presently known about the ultra-structural organization of tissues; cells, and organelles, thanks to the greater resolution achieved than that of the light microscope.
Due to advancements in the field for histological use of TEM, almost any biological specimen can be visualized and studied at a high resolution (atomic level). However, although the TEM is capable of resolving images on an atomic scale, artefacts introduced by biological sample preparation (aldehyde fixation; dehydration and staining), and section thickness, reduce this resolution to nanometres.
The development of Cryo-fixation, by high pressure freezing (HPF), avoids most artefact deposition by vitrification of samples thereby immobilizing the complex macromolecular structures within, and maintaining their native state.
In able to exploit these new techniques, further development of the conventional TEM were realised. With the addition of sophisticated instrumentation, Cryo-electron tomography (Cryo- TEM) was born. This made it possible to generate high resolution 3D tomograms (3D images of cellular structures in biological samples). The only processing after tomogram generation is contrast inversion for better visibility of details.
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Cryo-electron microscopy of vitreous sections (CEMOVIS) (Al-Amoudi et al. 2004) is the one and only method, currently available, which allows for the visualization of the true 'in-situ' structure of the specimen being analysed. Since solvents; fixatives; resins and stains are not capable of penetrating and thus dehydrating the sample, the only concern lies with sample damage via the electron beam.
HPF, as a primary step, in the fixation procedure, appears to be the most promising approach available at present. Post sample acquisition and exclusion, preservation an insight into the specimen as a 'living state' is thus made possible. Immunolabelling, by the use of specific antibodies, has become a popular technique and boosted by recent advances in this area. (see appendices). Although currently at the fore front of TEM use in histology, improvements must be made before it is of routine use.
Glossary of Terms
Melanoma - A dark-pigmented benign or malignant tumour that arises from a melanocyte and occurs most commonly in the skin. Malignant melanoma metastasizes quickly and is associated with sun exposure.
Neoplasm - Neoplasia (new growth in Greek) is abnormal and purposeless proliferation of cells in a tissue or organ. A neoplastic growth is called a neoplasm. Most neoplasms proliferate to form distinct masses, or tumours, but there are also many examples of neoplastic processes which are not grossly apparent, a commonly diagnosed example being cervical intraepithelial neoplasia, a pre-cancerous lesion of the uterine cervix. It is important to note that the term "neoplasm" is not synonymous with cancer, since neoplasms can be either benign or malignant. Leiomyoma (fibroids of the uterus) and melanocytic nevi (moles) are the most common types of neoplasms - both are benign. On the other hand, cancer is a typical example of malignant neoplasia or tumour. Hence, it is important to be able to differentiate between neoplasia, tumour and cancer.
Weibel-Palade body - ultrastructural markers of endothelial cells in primates and horses. The importance of Weibel-Palade bodies is highlighted by some human disease mutations. Mutations within vWF are the usual cause of the most common inherited bleeding disorder, von Willebrand disease. VWD has an estimated prevalence in some human populations of up to 1%, and is most often characterised by prolonged and variable mucocutaneous bleeding. Type III von Willebrand Disease is a severe bleeding disorder, not unlike severe haemophilia type A or B. VWF acts in primary haemostasis to recruit platelets at a site of injury, and is also important in secondary haemostasis, acting as a chaperone for coagulation factor VIII (FVIII).
Rhabdomyosarcoma - a highly malignant neoplasm derived from striated muscle.
Melanosomes - are bound by a lipid membrane and are, in general, rounded, sausage-like, or cigar-like in shape. The shape is constant for a given species and cell type. They have a characteristic ultrastructure on electron microscopy, which varies according to the maturity of the melanosome, and, for research purposes, a numeric staging system is sometimes used.
Ultra structure-Â is the detailed structure of a biological specimen, such as a cell, tissue, or organ which can be observed only by electron microscopy.
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Alport syndromeÂ -is aÂ genetic disorderÂ characterized by glomerulonephritis, end stage kidney disease, and hearing loss. Alport syndrome can also affect the eyes. The presence ofÂ bloodÂ in theÂ urineÂ (haematuria) is almost always found in this condition.
Nephropathy-Â refers to damage to or disease of theÂ kidney.
Fabry's disease- This isÂ an X-linked lysosomal storage disease of glycosphingolipid catabolism resulting from deficiency of Î±-galactosidase A and leading to accumulation of ceramide trihexoside in the cardiovascular and renal systems'.
Lymphoma-Â any neoplastic disorder of lymphoid tissue. Often used to denoteÂ malignancy, classifications of which are based on predominant cell type and degree of differentiation; various categories may be subdivided into nodular and diffuse types depending on the predominant pattern of cell arrangement
Lupus nephritis-Â glomerulonephritis associated with systemic lupus erythematous.
Percutaneous renal biopsy- is the removal of kidney tissue for microscopic examination. It is conducted to establish the diagnosis of a renal disorder and to aid in determining the stage of the disease, the appropriate therapy, and the prognosis.
Adenocarcinoma- Carcinoma derived from glandular tissue or in which the tumour cells form recognizable glandular structures.
MesotheliomaÂ -A primary benign or malignant tumour composed of cells resembling the mesothelium.
Carcinoembryonic antigen (CEA) - This has been thought to be a diagnostic and prognostic indicator of colorectal cancer.
CA 19-9 (Cancer Antigen 19-9)-This screening may be ordered along with other tests, such as carcinoembryonic antigen (CEA), bilirubin, and/or a liver panel, when a person has symptoms that may indicate pancreatic cancer. Its main use is as aÂ tumour marker.
S-100 Protein - A tumour marker (not widely used) in the diagnosis of Metastatic Melanoma.
HMB-45 - is considered by some as a specific tool to detect early metastatic melanoma. HMB-45Â is aÂ monoclonal antibodyÂ that reacts against an antigen present inÂ melanocyticÂ tumoursÂ such asÂ melanomas. It is used inÂ anatomic pathologyÂ as a marker for such tumours.
HMB stands forÂ HumanÂ MelanomaÂ Black.
Trump's FixÂ - Tissue fixed in Trump's fixative (a glutaraldehyde based fixative) and processed for electron microscopy.
ParaffinÂ - Tissue fixed in 10% neutral buffered formalin then retrieved from paraffin blocks and reprocessed for electron microscopy.
Etiology - the set of factors that contributes to the occurrence of a disease
Histology is defined as the microscopic study of cellular and tissue anatomy (both plant and animal). Histopathology, is the microscopic study/analysis of diseased tissue, and is thus, an important resource in anatomical pathology, since accurate diagnosis of cancers and other diseases usually require histopathological examination of these specimens.
These are performed by the examination of thin sections of tissues via light and/or electron microscopy - (LM/EM respectively).
The ability to visualize/differentially identify microscopic structures can often be enhanced by the use of both these methods in conjunction with specific histological stains.
In the past, LM has allowed for exceptional contributions to our understanding of the macromolecular structure of cells. This was aided by the development of special stains and advances in optics and digital imaging. However, the spatial resolution of LM is limited, thus, an alternative means of imaging, yielding better resolution thereby allowing another level of observation was necessary. EM Provides this, giving a greater amount of data and discovery possibilities than LM could ever hope to achieve.
Electron microscopy (EM) involves the use of an electron microscope for the examination of tissue. It allows for greater magnification, and visualization of organelles within cells, than LM. The transmission electron microscope (TEM) was once the main diagnostic tool, routinely used, to screen human tissues, at ultrastructural level. Its main use, in the early 1980's, was in the identification of tumours, and also in renal disease.
EM is not without its limitations. These include the need for specimens to be able to withstand a high vacuum and damage that may be inflicted upon them by the electron beam.
More recently, due to technological advances, TEM is often only used in conjunction with other methods, such as; LM and immunofluorescence (IF) which has enabled EM to be utilized in such a way as to decrease these factors.
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However, immunolabelling techniques are now more widely used rather than TEM for tumour diagnosis, but TEM still remains in use in many histopathology laboratories (Ireland and globally), and plays a role in the diagnosis of renal disease among a range of other disorders.
TEM is specifically used for diagnostic purposes when it enables:
useful and/or complementary information in respect to a differential diagnosis
it enables a more confirmatory diagnosis that would ultimately result in better treatment
Is more cost and time effective than its counterparts / alternative techniques.
The role of TEM remains "...an important and sometimes essential tool in diagnostic pathology." "...The techniques of EM and immunohistochemistry should be viewed not as competing with each other, but rather as complementary methods for achieving the same goal." (Rosai & Erlandson 1995).
Both techniques should be used to supplement the other in the recognition of diagnostic structures and features of differentiation in neoplasms such as; cilliary abnormalities, melanosomes in amelonotic malignant melanoma or sacromeres in poorly differentiated rhabdomyosarcoma.It is apparent, upon comparing both techniques, that the detailed fine structure of cellular and extracellular components can only be resolved by the high resolution of the TEM, whereas it can only be assumed that these potentially diagnostic structures in neoplasms are present using immunohistochemical stains.
There are a number of drawbacks in the use of TEM.Many specimens require extensive preparations to yield a sample thin enough to be electron transparent. This makes the whole process time consuming with a low turnaround time not suitable for use in a busy hospital laboratory. There is also the potential for the specimen to become damaged by the electron beam, and this is of particular concern for biological specimens.Therfore, only the most appropriate and cost- effective diagnostic procedure should be used in the evaluation of cases that cannot be resolved by routine methods in place. The choice of the initial diagnostic method should ideally be based upon the specific problem and the resources available to the laboratory. (Dr. Andra R. Frost 1994)
1. Transmission Electron Microscopy
The transmission electron microscope (TEM) has long been of use as a diagnostic tool in hospital pathology laboratories due to its ability to allow the user to view the ultrastructure (magnification of up to 1 Ã- 106).
As knowledge, at the nano scale of biological functions continues to grow, so does the understanding of pathological disorders/diseases.TEM has therefore proved to be a valuable resource in the definitive diagnosis of many diseases which are characterized by morphological changes in cells and tissues,that are too small to be resolved by LM. Examples of such cases include; variations in membrane thickness in renal diseases, mitochondrial and viral infections and certain skin cancers. in other instances, the use of TEM is useful as a confirmatory tool in diagnosis. (Hoenger & McIntosh 2010)
1.1 TEM and how it works
The TEM is based upon the use of a beam of electrons to magnify an object, such as tissues and cells. Normal optical (LM) microscope use only visible light and thus can only achieve magnification of between 40 - 2000 times. A TEM can resolve the ultrastructural features of a specimen;
Magnification capabilities of 50 - 1 million
The specimen appears flat
FEI's Morganiâ„¢ and Tecnaiâ„¢Spirit are two types of TEM which are widely used in pathology and medical diagnostics. The Morganiâ„¢ is a TEM that has the main functions, including digital imaging capabilities, for easy use in pathology laboratories. The Tecnnaiâ„¢ Spirit gives better scope for automated microscope functionality. It can also allow for the incorporation of tomography (ET) as well as Cryo - EM. These features of EM will be discussed in later chapters. (Nebraska-Lincoln 2010)
2. The role of TEM in Histology -
Past and present
The use of diagnostic TEM in histology was once invaluable for the diagnosis of certain diseases, by permitting the study of tissue and cell ultra-structure to answer diagnostic anomalies. By this approach, a specialized means for the improvement in clinical diagnosis and research was exploited.
Through examination of specially prepared tissue sections (refer to appendix), changes could be observed by TEM, which would not have been detectable by LM, leading to increased definitive diagnosis. For example, in certain renal diseases, the appropriate diagnosis could only be made by TEM, which in turn determined the prognosis and type of treatment/therapy administered. Likewise, certain neoplasms were only accurately identified via the use of TEM (enabled ultrastructural study of the specimen), again with obvious implications in prognosis and treatment administration.
In the past, the use of TEM added new possibilities to the scope of diagnostic pathology; however, due to advancements in techniques, such as, immunohistichemistry, the continuing importance of TEM in a diagnostic role for pathology is questionable.
Many experts now agree that TEM can still be vital for the diagnosis of certain neoplasms; nephropathies and neuropathies, and that both modern immunohistochemical techniques and TEM should be used to complement each other to provide a means of powerful diagnostics in histologically based disease diagnosis.
Rationalists would argue that the benefits of TEM are overshadowed by the costs incurred. However, from a broader perspective, centralization of a dedicated EM facility to service hospital pathology laboratories and smaller facilities to focus on cancer, renal and other subspecialties, in which TEM could play an essential role in diagnosis and evaluation, may optimize the cost effectiveness of TEM. GUH currently forward Renal biopsies for TEM to the US. Therefore, a central EM lab, based in Ireland, would be more appropriate and prove more cost effective.
Consequently, the question remains as to whether any pathology Dept. could afford not to have an EM facility at their disposal since the relative costs of TEM are small in respect of the benefits bestowed upon patient care
3. Indications for the use of TEM
The implications in the use of TEM for pathologic diagnosis fall into six major factions:
Renal Disease: TEM plays a very important role in the diagnosis of many renal disorders and in many circumstances; a diagnosis cannot be obtained without its use. TEM allows for the refinement of the diagnosis -i.e. sub classification of Lupus nephritis or may provide confirmatory evidence of the disease.TEM is mainly useful in the analysis of percutaneous renal biopsies. The Examination of a single glomerulus by TEM may be enough to permit a definitive diagnosis. (Ackermans Surgical Pathology 1989) (ESMA 1991).
Neoplasms: The role of TEM in the diagnosis of neoplasms has been well documented in many texts. The indications for the use of TEM in the diagnosis of tumours includes;
Poorly Differentiated Carcinomas where TEM is valuable in the identification of squamous, glandular and neuroendocrine differences. These are easily missed by LM.
Poorly differentiated endocrine V's non endocrine neoplasms with differential diagnosis of adenocarcinoma versus malignant mesothelioma.
Poorly differentiated spindle cell neoplasms
Possible cutaneous T-cell lymphomas. (Diagnostic EM of Tumours 1985)
Tumours with unexpected or confusing immunophenotypes. Refer appendices
Non-immunoreactive tumours - Tumours are encountered from time to time which are non-immunoreactive for a very large panel of antibodies,
The use of TEM has been shown to be valuable for the pathologic study of skin and lymph node biopsies, along with buffy coats from the peripheral blood of patients suspected of having cutaneous T-cell lymphomas, since mycosis and Sezary cells may be unnoticeable by LM, but are easily seen by TEM. (Dianostic Electron Microscopy 1979) (Cancer Treatment Reports 1979)
The contribution of TEM in the diagnosis of tumours in a general hospital was investigated in a three year study (Fisher et al. 1985) , in which 235 cases were examined. Of these, 28% proved to be problematic in diagnosis via LM and 64% of these were resolved via the use of EM by ultrastructural examination.
Metabolic Diseases: Applications for past TEM use in the diagnosis of metabolic diseases included;
Skeletal muscle biopsies with possible mitochondrial myopathy. Here LM changes were not detectable. (Diagnostic Ultrastructure of Non- Neoplastic Diseases 1992)
Peripheral nerve biopsies (Diagnostic Ultrastructure of Non- Neoplastic Diseases 1992) (1990)
Liver biopsies in patients with suspected alcohol related liver disease. This type of liver disease gives characteristic ultrastructural findings of mitochondrial hyperplasia; gigantism and fibrillar Mallory bodies which may be missed by LM. (Dianostic Electron Microscopy 1979)
Hypersensitivity disorders of the skin. The use of TEM has been proven to reveal dermal vascular injury which can be very difficult to detect by LM and/or immunohistochemically. (1990) (Human Pathology 1979/1993).
Infectious Disease: The use of TEM has been shown to be of use, past and present, whenever the etiology of a suspected infectious disease is unknown and more specifically:
When an infectious disease is suspected, but no infectious agent is detectable in tissue sections via LM. Infections of the intestine due to microsporidia and possibly very small Gram negative bacteria, which are barely/not visible by LM, are good examples. These are easily visible in tissue sections, by TEM. (1995) (Ann Int Med. 1996)When there may be discrepancies among pathologists using LM to establish the etiology of an infectious disease (1995)
When contradictory evidence, in relation to the cause of an infectious disease arises.ie clinical; LM; serologic...
Bone Marrow Biopsies: The bone marrow (BM) biopsy is a standard diagnostic procedure, which allows for the histological evaluation of marrow specimens, especially when aspirate material is difficult to obtain. The ultrastructural study of human BM biopsies was often fraught with issues surrounding the decalcification stage. To overcome this, cryofracture enabled the dissection and fixation of BM biopsies without decalcification. This also reduced the specimen deformation and enabled small segments of the specimen to be embedded and cut into thin sections for viewing by TEM. By use of this technique, TEM can be extended for use in the histopathological study of human BM biopsy specimens. (Kuto et al. 1981)
A disease in which little is known about the nature and etiology, TEM has been shown to be beneficial. (1996)
TEM use in microprobe analysis continues to be of relevance -primarily in the diagnosis of asbestosis. (Microprobe analysis 1989)
Advancements in TEM
TEM has allowed for the visualization of many previously unseen cellular structures and substances. Numerous ground-breaking techniques have emerged to facilitate this. Examples include the transformation of biological samples by chemical fixation; dehydration; resin embedding and special staining. However, these techniques tend to be quite labour intensive. The decreased popularity of basic TEM use in histological diagnosis has led to the advancement in its operational capacity.
1. Combined Light and Electron Microscope (LEM 2000)
A newer model of TEM, which combined the virtues of the LM and TEM, (LEM 2000), was developed to provide the opportunity to visualize the same specimen in both modes, by a combination of selective staining and high resolution microscopy. The LEM has a built in microprocessor which can record and relocate areas of interest and photographs are automatically numbered, with the coordinates of the fields also marked on them.
The main advantage of the LEM is down to its ability to facilitate large grid sizes. (7mm versus 3mm -TEM/LM respectively). This enabled resolution of issues regarding sample orientation and relocation of fields of diagnostic relevance. This all led to a reduction in sampling errors (Diagnostic EM of Tumours 1985).Ultramicrotomes are not essential- thus allowing for cheaper and simpler specimen preparation. The LEM does not need to be operated in a darkened environment. (Jones et al. 1982).
2. Phase Contrast TEM
This is based upon the use of thin-film phase plates to facilitate an increased visibility of transparent specimens. The main disadvantage of phase plate use in TEM is the electron loss as a result of plate scattering. Refer to appendices.
Since specimen preparation is crucial in the application of TEM to biological samples, many properties contribute to their preparation and are often viewed as being significantly complicated. A standard method, Cryo technique, has now been established to resolve these issues. However, this method does have its own disadvantages. These include:
Increased work up time (labour intensive).
3. Cryo- Electron Microscopy
To solve the issue of significant tissue damage, vitrification via cryofixation was introduced. Thereafter, cryomicroscopy was developed to avoid electron beam damage to specimens prepared by cryofixation. This enabled the recovery of vital structural information.
This technique allowed for excellent preservation of cellular structures by rapid freezing of specimens and the extraction of thin slices to enable detailed views of the cellular structure. In order to obtain the 3D information, Different orientations of the sample were realized by alternating the position of the specimen, via sample rotation. These cryo-technologies have since been combined with tomography to give rise to electron cryotomography.
4. Cryo-Electron Tomography (ECT)
Cryo-electron tomographyÂ (Cryo-ETÂ orÂ electron cryotomography) is a type ofÂ cryomicroscopyÂ that enables the 3D structural studies of biological samples, such as cells and organelles, in a close-to-native state (Baumeister, 2005; LucË‡icÂ´ et al., 2005).ECT is becoming an increasingly valuable tool for biochemists, microbiologists, and cell biologists to determine the ultrastructure of macromolecular complexes, supramolecular assemblies, or even whole cells, and should therefore precipitate a wonderful revival in transmission electron microscopy. (Murphy & Grant 2007). However, the phase-contrast imaging method used in conventional cryo-electron tomography fails to faithfully represent the full range of structural features in such specimens. Only certain features are recorded with adequate contrast, and overall contrast is low. The recently developed Zernike phase contrast (ZPC-EM) method has the potential to solve this problem. This new method has uniform transfer characteristics for a wide range of spatial frequencies, leading to improved overall signal-to-noise ratio and increasing the prospects of higher resolution and quantitative representation of specimen densities in the reconstructed tomograms.
3D spatial complexity of cellular organization as visualized by electron microscope tomography.
The frozen-hydrated sample is typically a few hundred nanometres thick and is maintain at close to liquid-nitrogen temperature (195.8Â°C) during observation. Vitreous freezing retains the specimen in its native, fully hydrated state and helps to reduce the adverse effects of radiation damage, which is the principle limiting factor in all high-resolution Cryo-electron microscopy studies (LucË‡icÂ´ et al., 2005; Frank, 2006).Images are recorded in the transmission electron microscope (TEM) as the sample is tilted through a wide angular range.
All observations are performed on a TEM with the microscope optimized for phase-plate applications by the installation of a specially designed wide-gap objective lens pole piece and a heated phase-plate holder.
A high tilt tomography, liquid-nitrogen-cooled Cryo-transfer specimen holder is also used (Model 914, Gatan Pleasanton, CA, USA). Data can then be recorded via a charge-coupled device (CCD) camera, which permits TV monitoring of the TEM specimen image. (Radostin et al. 2010)
Clarification of the 3D structural organization of molecules, cells and tissues is essential to the development of a complete understanding of basic cell and molecular biology, thus allowing for greater understanding to improve therapeutic interventions for the prevention and/or treatment of many diseases such as cancer. High resolution 3D imaging studies of cells have already provided new insights into the complexity of 3D cellular organization. These have affected how scientists, analyse specimens at the subcellular level.
(Dubrokci, Marsh & Kalinowski 2009)
Use of phase plates in electron microscopy has until recently been impractical because of various limitations of the microscope systems, as well as difficulty in manufacturing the phase plates.
Due to advances in technologies,most of these problems can now be overcome,and excellent results can routinely be achieved using TEM
Although several device types are currently under development (Majorovits et al., 2007; Cambie et al., 2007; Schröder et al., 2007; Shiue et al., 2009), the only one which has been used extensively in applications is the Zernike phase plate made of a thin carbon film. In a recent study, Zernike phase contrast TEM (ZPC-TEM) demonstrated improved performance for 3D structural investigations of proteins based on the ''single particle" reconstruction method (Danev and Nagayama, 2008). Another recent study (Barton et al., 2008) demonstrated improved visibility of details in reconstructed tomograms of thin sections of resin embedded biological specimens by utilizing a Hilbert 100 type carbon film phase plate.