There are various preparatory techniques for light microscopy permitting access to the inner structure of plant body and its development. Minute objects might be processed as whole-mount preparations, while voluminous ones should be separated into smaller pieces. Here we summarize some of the "classical" techniques to cut more voluminous objects into slices and access their inner structure either for simple anatomical analysis or for further processing (e.g., histochemistry, immunohistochemistry, in situ hybridization, enzyme histochemistry).

• MeSH
• Staining and Labeling methods   MeSH
• Tissue Fixation methods   MeSH
• Histocytochemistry methods   MeSH
• Histocytological Preparation Techniques methods   MeSH
• Microscopy methods   MeSH
• Microtomy methods   MeSH
• Plants chemistry   ultrastructure   MeSH
• Paraffin Embedding methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

We present a new method of multiple immunolabeling that is suitable for a broad spectrum of biomedical applications. The general concept is to label both sides of the ultrathin section with the thickness of 70-80 nm with different antibodies conjugated to gold nanoparticles and to distinguish the labeled side by advanced imaging methods with high resolution scanning electron microscopy, such as by correlating images acquired at different energies of primary electrons using different signals. From the Clinical Editor: The use of transmission electron microscopy has become an indispensible tool in the detection of cellular proteins. In this short but interesting article, the authors described their new method of labeling and the identification of four different proteins simultaneously, which represents another advance in imaging technique.

• MeSH
• Acrylic Resins chemistry   MeSH
• Staining and Labeling methods   MeSH
• Immunohistochemistry MeSH
• Metal Nanoparticles chemistry   ultrastructure   MeSH
• Microtomy methods   MeSH
• Reproducibility of Results MeSH
• Sensitivity and Specificity MeSH
• Microscopy, Electrochemical, Scanning methods   MeSH
• Image Enhancement methods   MeSH
• Gold chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Low voltage electron microscopes working in transmission mode, like LVEM5 (Delong Instruments, Czech Republic) working at accelerating voltage 5 kV or scanning electron microscope working in transmission mode with accelerating voltage below 1 kV, require ultrathin sections with the thickness below 20 nm. Decreasing of the primary electron energy leads to enhancement of image contrast, which is especially useful in the case of biological samples composed of elements with low atomic numbers. As a result treatments with heavy metals, like post-fixation with osmium tetroxide or ultrathin section staining, can by omitted. The disadvantage is reduced penetration ability of incident electrons influencing the usable thickness of the specimen resulting in the need of ultrathin sections of under 20 nm thickness. In this study we want to answer basic questions concerning the cutting of extremely ultrathin sections: Is it possible routinely and reproducibly to cut extremely thin sections of biological specimens embedded in commonly used resins with contemporary ultramicrotome techniques and under what conditions? Microsc. Res. Tech. 79:512-517, 2016. © 2016 Wiley Periodicals, Inc.

• MeSH
• Equipment Design MeSH
• Microscopy, Electron instrumentation   methods   MeSH
• Epoxy Resins chemistry   MeSH
• Microtomy methods   MeSH
• Myocardium ultrastructure   MeSH
• Mice MeSH
• Polymers chemistry   MeSH
• Heart diagnostic imaging   MeSH
• Plastic Embedding methods   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Výsledky studia mikroskopické struktury živočišných a lidských tkání získané významným českým badatelem 19. století Janem Evangelistou Purkyněm jsou již dostatečně popsány v různých starších a novějších publikacích. Obsahem stávajícího přehledu je popis mikroskopické techniky a přístrojů, které Purkyně a jeho asistenti na základě dochovaných pramenů používali k výzkumu histologie tkání a během výuky a na jejichž vývoji se přímo podíleli, jako jsou např. mikroskopy, mikrotomické tlačidlo a mikrotom. Z krátkého přehledu vývoje konstrukce krá- jecích zařízení vyplývá, že první mikrotom, ze kterého vycházejí moderní sáňkové mikrotomy, vznikl pod vedením Purkyně ve Fyziologickém ústavu ve Vratislavi. Purkyně a jeho asistenti tak nejen dosáhli prioritních výsledků v oblasti studia struktury živočišných a lidských tkání, ale rovněž nezanedbatelně přispěli k vývoji nástrojů a zařízení pro jejich studium, což je mnohdy v dnešní době opomíjeno.

The results obtained during the studies of the microscopic structure of animal and human tissues by the famous 19th century Czech scientist Jan Evangelista Purkyně are already sufficiently described in a variety of older and newer publications. The contents of the present paper are an overview of the microscopes and other tools and instruments that Purkyně and his assistants and pupils used for research of tissue histology and during teaching, and in whose development there were directly involved. A brief overview of the development of the cutting engines suggests that the first microtome, from which all modern sliding microtomes are derived, originated under the supervision of Purkyně at the Institute of Physiology in Wroclaw. Purkyně and his assistants thus not only obtained priority results in the field of the structure of animal and human tissues, but also substantially contributed to the development of instruments and equipment for their study, which is often forgotten today

• Keywords
• mikrotomické tlačidlo,
• MeSH
• History, 19th Century MeSH
• Histological Techniques history   methods   instrumentation   MeSH
• Microscopy * history   methods   instrumentation   MeSH
• Microtomy * history   methods   instrumentation   MeSH
• Check Tag
• History, 19th Century MeSH
• Publication type
• Historical Article MeSH
• Review MeSH

In this chapter, the basic principles and protocols of the electron microscopical detections of specific DNA and RNA sequences are described. We focused primarily on a comparison of various methods of electron microscopy in situ hybridization (EM-ISH) with respect to their sensitivity and the structural preservation of the sample with the aim of helping the readers select the appropriate hybridization protocol. As the post-embedding EM-ISH most frequently represents the optimal choice, the protocol for the post-embedding EM-ISH approach is described in detail. Concurrently, the alternative methods based on the enzymatic synthesis of the labeled nucleic acids chains that can be used for the detection of DNA or RNA molecules in situ are mentioned. In this respect, the technique enabling the enzymatic detection of the polyadenylated RNA sequences is described in detail.

• MeSH
• DNA chemistry   MeSH
• Microscopy, Electron methods   MeSH
• In Situ Hybridization methods   MeSH
• Microtomy methods   MeSH
• RNA chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Cíl: Snaha nastínit výhody histologického zpracování materiálu z radikální prostatektomie (RP) pomocí celoplošných řezů (whole-mount section - WMS) a její srovnání s jinými metodami, běžně užívanými pro vyhodnocování tohoto materiálu v patologické praxi. Materiál a metoda: Byly vyhledány všechny případy karcinomu prostaty řešené RP a histologicky zpracované metodou WMS za období 2009/VIII až 2013/ III. Případy byly zrevidovány. Sledovány byly následující parametry: histologicky typ, Gleasonovo skóre, přítomnost nádorové hmoty v apexu, multicentricita, postižení semenných váčků, dosah do resekční linie a přesah přes pseudopouzdro prostaty. Výsledky: Celkem bylo vyhodnoceno 191 preparátů z RP zpracovaných pomocí WMS. Tumory byly tvořeny z 97,38% acinárním prostatickým adenokarcinomem. Nejčastější Gleasonovo skóre bylo 6 (3 + 3) v 42,9 %. Ve 20,4 % šlo o leze multicentrické. Invaze apexu byla nalezena u 127 případů. Dosah přes fibrózní pseudopouzdro prostaty byl pozorován u 20,9 %. Metastatické postižení uzlin popsáno u 2,1 %. Dosah do resekční linie byl popsán u 36,1 %. Invaze semenných váčků byla přítomna v 12,04 %. Invaze nervově cévního svazku byla popsána v 3,7 %, invaze příčně pruhované svaloviny sfinkteru pak u 3,1 %. Závěr: WMS umožňuje přesné určení stage a Gleasonova skóre. Je rovněž možné naprosto přesně stanovit eventuální dosahování nádoru do resekční linie. Použití metody WMS v rutinní histopatologické diagnostice lze jednoznačně doporučit, jelikož představuje jasný benefit pro pacienta.

Aim: This work aims to present the advantages of histological processing using whole-mount sections (WMS) and to compare it with other methods commonly used for evaluation of tissue removed during the course of a radical prostatectomy (RP). Material and methods: All cases of carcinoma of the prostate, treated by radical prostatectomy, and processed using WMS, August 2009 to March 2013 were histologically re-evaluated. The following parameters were analyzed: histological type, Gleason score, presence of a tumour in the apex, multicentricity, infiltration of the seminal vesicles, presence of positive resection margins and extracapsular extension. Results: There were 191 RP specimens processed with WMS. Histological diagnosis of acinar prostatic adenocarcinoma was made in 97.4% of cases. The most frequent Gleason score was 6 (3 + 3) in 42.9%. In 20.4%, the tumour was multicentric. Invasion of the apex was found in 127 cases. The extracapsular extension was detected in 20.9%. The metastatic infiltration of the lymph nodes was registered in 2.1%. The presence of positive resection margins in the specimen was detected in 36.1%. Infiltration of seminal vesicles was present in 12.0%. Invasion of the neural-vascular bundle was detected in 3.7% and invasion of the striated muscle of the external sphincter in 3.1% of cases. Conclusions: WMS allows precise determination of the disease stage and Gleason score. It also facilitates precise detection of positive surgical margins. The routine use of WMS can be recommended in histopathological diagnostics of prostate cancer due to its obvious benefits for the patient.

• MeSH
• Adenocarcinoma diagnosis   MeSH
• Histology, Comparative * methods   statistics & numerical data   MeSH
• Humans MeSH
• Microtomy * methods   statistics & numerical data   MeSH
• Prostatic Neoplasms diagnosis   MeSH
• Prostatectomy * methods   statistics & numerical data   MeSH
• Neoplasm Staging methods   statistics & numerical data   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

When biological specimens are cut into physical sections for three-dimensional (3D) imaging by confocal laser scanning microscopy, the slices may get distorted or ruptured. For subsequent 3D reconstruction, images from different physical sections need to be spatially aligned by optimization of a function composed of a data fidelity term evaluating similarity between the reference and target images, and a regularization term enforcing transformation smoothness. A regularization term evaluating the total variation (TV), which enables the registration algorithm to account for discontinuities in slice deformation (ruptures), while enforcing smoothness on continuously deformed regions, was proposed previously. The function with TV regularization was optimized using a graph-cut (GC) based iterative solution. However, GC may generate visible registration artifacts, which impair the 3D reconstruction. We present an alternative, multilabel TV optimization algorithm, which in the examined samples prevents the artifacts produced by GC. The algorithm is slower than GC but can be sped up several times when implemented in a multiprocessor computing environment. For image pairs with uneven brightness distribution, we introduce a reformulation of the TV-based registration, in which intensity-based data terms are replaced by comparison of salient features in the reference and target images quantified by local image entropies.

• MeSH
• Algorithms MeSH
• Artifacts MeSH
• Staining and Labeling MeSH
• Embryo, Nonmammalian MeSH
• Embryo, Mammalian MeSH
• Entropy MeSH
• Microscopy, Confocal methods   MeSH
• Rats MeSH
• Chickens MeSH
• Mesonephros chemistry   ultrastructure   MeSH
• Microtomy methods   MeSH
• Reproducibility of Results MeSH
• Sensitivity and Specificity MeSH
• Image Enhancement instrumentation   methods   MeSH
• Paraffin Embedding MeSH
• Turtles MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

A set of methods leading to volume reconstruction of biological specimens larger than the field of view of a confocal laser scanning microscope (CLSM) is presented. Large tissue specimens are cut into thin physical slices and volume data sets are captured from all studied physical slices by CLSM. Overlapping spatial tiles of the same physical slice are stitched in horizontal direction. Image volumes of successive physical slices are linked in axial direction by applying an elastic registration algorithm to compensate for deformations because of cutting the specimen. We present a method enabling us to keep true object morphology using a priori information about the shape and size of the specimen, available from images of the cutting planes captured by a USB light microscope immediately before cutting the specimen by a microtome. The errors introduced by elastic registration are evaluated using a stereological point counting method and the Procrustes distance. Finally, the images are enhanced to compensate for the effect of the light attenuation with depth and visualized by a hardware accelerated volume rendering. Algorithmic steps of the reconstruction, namely elastic registration, object morphology preservation, image enhancement, and volume visualization, are implemented in a new Rapid3D software package. Because confocal microscopes get more and more frequently used in scientific laboratories, the described volume reconstruction may become an easy-to-apply tool to study large biological objects, tissues, and organs in histology, embryology, evolution biology, and developmental biology. In this work, we demonstrate the reconstruction using a postcranial part of a 17-day-old laboratory Wistar rat embryo. (c) 2008 Wiley-Liss, Inc.

• MeSH
• Algorithms MeSH
• Embryo, Mammalian cytology   MeSH
• Financing, Organized MeSH
• Microscopy, Confocal methods   MeSH
• Rats MeSH
• Microtomy methods   MeSH
• Image Processing, Computer-Assisted methods   MeSH
• Elasticity MeSH
• Imaging, Three-Dimensional methods   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH

Electron microscopy in situ hybridization (EM-ISH) represents a powerful method that enables the localization of specific sequences of nucleic acids at high resolution. We provide here an overview of three different nonisotopic EM-ISH approaches that allow the visualization of nucleic acid sequences in cells. A comparison of various methods with respect to their sensitivity and the structural preservation of the sample is presented, with the aim of helping the reader to choose a convenient hybridization procedure. The post-embedding EM-ISH protocol that currently represents the most widely used technique is described in detail, with a special emphasis on the organization of the cell nucleus.

• MeSH
• Chlorocebus aethiops MeSH
• COS Cells MeSH
• Nucleic Acid Denaturation MeSH
• Deoxyribonuclease I MeSH
• DNA genetics   ultrastructure   MeSH
• Cryoelectron Microscopy methods   MeSH
• Microscopy, Electron methods   MeSH
• Endopeptidase K MeSH
• Financing, Organized MeSH
• Fixatives MeSH
• Genes, rRNA MeSH
• HeLa Cells MeSH
• HIV-1 genetics   MeSH
• In Situ Hybridization methods   MeSH
• Humans MeSH
• Microtomy methods   MeSH
• Molecular Probes MeSH
• Ribonuclease, Pancreatic MeSH
• RNA, Viral genetics   ultrastructure   MeSH
• RNA genetics   ultrastructure   MeSH
• Plastic Embedding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Comparative Study MeSH

• MeSH
• Research Support as Topic MeSH
• Cryopreservation methods   MeSH
• Humans MeSH
• Microtomy methods   instrumentation   MeSH
• Tissue and Organ Harvesting methods   MeSH
• Suspensions MeSH
• In Vitro Techniques MeSH
• Tissues metabolism   MeSH
• Tissue Preservation methods   MeSH
• Sample Size MeSH
• Check Tag
• Humans MeSH
• Publication type
• Review MeSH
• Comparative Study MeSH