The high resolution and sensitivity of electron microscopy is still a valuable tool, and for some diseases it is the gold standard in pathological diagnosis. Today the sample turnaround time for processing in the lab can be significantly reduced from days to hours by the microwave technology. In urgent clinical cases, microwave-assisted tissue processing, in combination with digital image acquisition, enables a "same-day" diagnosis. Ultrastructural telepathology allows instant and live second opinion retrieval from a remote expert worldwide.
Modern evidence-based medicine needs a cytology or tissue-based pathologic diagnosis describing the nature of a lesion and an interpretation of the data providing advice for an individual therapeutic strategy. This is achieved by pathologists mainly by light microscopy (LM) examination of H&E-stained sections (approx. 4 µm thick) of tissue embedded in paraffin wax. Special stains and techniques, like immunohistochemistry (IHC), flow cytometry, cytogenetics, and molecular techniques (gene rearrangement analysis, fluorescence in situ-hybridization (FISH) and polymerase chain reaction (PCR) analysis) provide additional information (genomics, proteomics, metabolomics) to refine the understanding of a disease and strengthen the diagnosis.
Why Diagnostic Transmission Electron Microscopy
If the analyzed features are smaller than the resolution limit of the classic light microscope (200 nm), the 1,000 times higher resolving power of transmission electron microscope (TEM) technology (0.2 nm) can visualize small intracellular and extracellular structures in great detail to facilitate a diagnosis that might be uncertain or impossible by Light Microscopy. Examples include cell organelle (nucleus/nucleoli, mitochondria, RER/SER, Golgi complex, lysosomes,melanosomes), various types of inclusions and secretory granules, cytoskeleton components (microtubules, filaments, centrioles), cilia, cell surface specializations (microvilli, cell protrusions, intercellular junctions), extracellular constituents (basal membrane, collagen, amyloid), and a variety of infectious agents (viruses, protozoa, fungi).
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In many diseases this components display peculiar abnormalities or lesions.
In conclusion, TEM examination of pathological samples is a method to extend morphologic analysis to the ultrastructural level, providing data not discernible by the other ancillary methods, e.g. on the basis of the antigens expressed by a neoplasm . The new stunning light diffraction limit breaking STED-microscopy (= Stimulated Emission Depletion; resolution range 16-80 nm) developed by Stephan Hell  is to date not available for routine diagnostic purposes . TEM was a very popular adjunct in diagnostic procedures in the 1970s and early 1990s, it importance declined in pathology with the emerging use of IHC and molecular methods as well as due to intrinsic EM limitations like insufficient sample processing automation and long turnaround time (TAT). Today a resurgence of TEM as an ancillary diagnostic modality is observed , however TEM-diagnostic expertise due to economic and staffing issues is generally available only in larger laboratories or centers with specific interest in ultrastructural pathology [4-6].
In TEM, instead of light, an electron beam passes through the examined section from a sophisticated electromagnetic lens system to give a high-resolution image of the specimen. The limited penetration depth of electrons and their interaction with the specimen (heat and ionization damage) require a special tissue preparation to produce ultrathin sections suitable for TEM examination.
In the literature there is a large number of protocols for embedding different specimens in a variety of dedicated resins according to the addressed issue . Briefly, after sample collection the standard approach is to immerse the specimen immediately in a buffered fixative (Karnovsky formulation = primary aldehyde-based fixative for protein preservation (fig. 1), subsequent osmium tetroxide postfixationfor lipid stabilization and contrast enhancement), dehydrate it in graded ethanols, and embedding in epoxy resin for polymerization (mostly by heat) into hard blocks (including ID-labels). This routine sample processing is nowadays performed in most diagnostic EM labs using computer controlled tissue processors saving reagents, time, and labour (batch processing overnight). The usual routine total sample turnaround time (TAT) is approximately 3 to 5 workdays. In case of urgent clinical cases, microwave-assisted tissue processing (AMW/Leica, KOS/Milestone) can reduce the TAT to less than 6 hours .
Once polymerized, tissue blocks are cut with an ultramicrotome equipped with a diamond knife to yield semithin sections for LM screening for specific features (approx. 0,8 µm, rapid staining on glass slides with toluidine blue and basic fuchsine) and to select areas for thin sectioning. The ultrathin resin sections (approx. 80 nm) are collected on copper grids and double-stained on drops of heavy metal salt solutions (uranyl acetate, lead citrate) for contrast enhancement of specimen structures (increased electron scattering). The air-dried sections are usually examined in a TEM with 80 to 100 kV acceleration voltage and equipped with a customized digital camera image acquisition system (recommended resolution 1kx1k or 2kx2k pixels) capable to store the images in a secure databank. Interactive remote TEM operation via Internet allows instant and live "second opinion" consultation of difficult cases worldwide ("ultrastructural telepathology") .
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