Attaching Suspension Cells to Slides for Staining.

Suspension cells can be prepared for staining by several different methods. A simple method for detecting intracellular antigens in cells that grow in suspension is to attach the cells to a solid substrate before fixation. This can be achieved by the use of a cytocentrifuge. For surface staining, suspension cells can be attached to slides by cross-linking with poly-l-lysine. Lysine can be polymerized to any desired length, and poly-l-lysine will bind to most solid supports through its charged side chains. The positively charged polymer will provide a site for binding of cells (which carry an overall negative charge). Although this cross-link is not covalent, it is sufficiently strong for most cell-staining techniques.

Fluorochromes for DNA Staining and Quantitation.

In these last few decades the great explosion of the molecular approaches has casted a little shadow on the DNA quantitative analysis. Nevertheless DNA cytochemistry represented a long piece of history in cell biology since the advent of the Feulgen reaction. This discovery was really the milestone of the emerging quantitative cytochemistry, and scientists from all over the world produced a very large literature on this subject. This first era of quantitation (histochemistry followed by cytochemistry) started by means of absorption measurements (histophotometry and cytophotometry). The successive introduction of fluorescence microscopy gave a great boost to quantitation, making easier and faster the determination of cell components by means of cytofluorometry. The development of flow cytometry further contributed to the importance of quantitative cytochemistry. At its beginning, the mission of flow cytometry was still DNA quantitation. For a decade the Feulgen reaction had been the reference methodology for both conventional and flow cytofluorometry; the advent of Shiff-type reagents contributed to expand the variety of possible fluorochromes excitable in the entire visible spectrum as well as in the ultraviolet region. The fluorescence scenario was progressively enriched by new probes among which are the intercalating dyes which made DNA quantitation simple and fast, thus spreading it worldwide. The final explosion of cytofluorometry was made possible by the availability of a large variety of probes directly binding DNA structure. In addition, immunofluorescence allowed to correlate the cell cycle-related DNA content to other cell markers. In the clinical application of flow cytometry, this promoted the introduction of multiparametric analyses aimed at describing the cytokinetic characteristics of a given cell subpopulation defined by a specific immunophenotype setting.

A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue.

For 3-dimensional (3D) imaging of a tissue, 3 methodological steps are essential and their successful application depends on specific characteristics of the type of tissue. The steps are 1° clearing of the opaque tissue to render it transparent for microscopy, 2° fluorescence labeling of the tissues and 3° 3D imaging. In the past decades, new methodologies were introduced for the clearing steps with their specific advantages and disadvantages. Most clearing techniques have been applied to the central nervous system and other organs that contain relatively low amounts of connective tissue including extracellular matrix. However, tissues that contain large amounts of extracellular matrix such as dermis in skin or gingiva are difficult to clear. The present survey lists methodologies that are available for clearing of tissues for 3D imaging. We report here that the BABB method using a mixture of benzyl alcohol and benzyl benzoate and iDISCO using dibenzylether (DBE) are the most successful methods for clearing connective tissue-rich gingiva and dermis of skin for 3D histochemistry and imaging of fluorescence using light-sheet microscopy.

One Size Fits All: Evaluation of the Transferability of a New "Learning" Histologic Image Analysis Application.

Quantitative analysis of histologic slides is of importance for pathology and also to address surgical questions. Recently, a novel application was developed for the automated quantification of whole-slide images. The aim of this study was to test and validate the underlying image analysis algorithm with respect to user friendliness, accuracy, and transferability to different histologic scenarios. The algorithm splits the images into tiles of a predetermined size and identifies the tissue class of each tile. In the training procedure, the user specifies example tiles of the different tissue classes. In the subsequent analysis procedure, the algorithm classifies each tile into the previously specified classes. User friendliness was evaluated by recording training time and testing reproducibility of the training procedure of users with different background. Accuracy was determined with respect to single and batch analysis. Transferability was demonstrated by analyzing tissue of different organs (rat liver, kidney, small bowel, and spleen) and with different stainings (glutamine synthetase and hematoxylin-eosin). Users of different educational background could apply the program efficiently after a short introduction. When analyzing images with similar properties, accuracy of >90% was reached in single images as well as in batch mode. We demonstrated that the novel application is user friendly and very accurate. With the "training" procedure the application can be adapted to novel image characteristics simply by giving examples of relevant tissue structures. Therefore, it is suitable for the fast and efficient analysis of high numbers of fully digitalized histologic sections, potentially allowing "high-throughput" quantitative "histomic" analysis.

Histochemistry in biology and medicine: a message from the citing journals.

Especially in recent years, biomedical research has taken advantage of the progress in several disciplines, among which microscopy and histochemistry. To assess the influence of histochemistry in the biomedical field, the articles published during the period 2011-2015 have been selected from different databases and grouped by subject categories: as expected, biological and biomedical studies where histochemistry has been used as a major experimental approach include a wide of basic and applied researches on both humans and other animal or plant organisms. To better understand the impact of histochemical publications onto the different biological and medical disciplines, it was useful to look at the journals where the articles published in a multidisciplinary journal of histochemistry have been cited: it was observed that, in the five-years period considered, 20% only of the citations were in histochemical periodicals, the remaining ones being in journals of Cell & Tissue biology,  general and experimental Medicine, Oncology, Biochemistry & Molecular biology, Neurobiology, Anatomy & Morphology, Pharmacology & Toxicology, Reproductive biology, Veterinary sciences, Physiology, Endocrinology, Tissue engineering & Biomaterials,  as well as in multidisciplinary journals.It is easy to foresee that also in the future the histochemical journals will be an attended forum for basic and applied scientists in the biomedical field. It will be crucial that these journals be open to an audience as varied as possible, publishing articles on the application of refined techniques to very different experimental models: this will stimulate non-histochemist scientists to approach histochemistry whose application horizon could expand to novel and possibly exclusive subjects.

[The application of different types of liquid technologies in cytology].

The article discusses the results of comparative study of traditional smears and three different liquid technologies of making ready of cytological preparations (CytoSpin3, E-Prep Processor BD TriPath) using material from 112 patients with pathological processes of such different localizations as exudative liquids (31), soft tissues (22), lymphatic nodes (18), mammal glands (20), saliva glands (7), lungs (9), thyroid gland (5). It is established that under joint application of liquid and traditional cytology the effectiveness of cytological analysis increases up to 3.3%-5% depending on localization of pathologic nidus. The implementation of liquid technologies expands the possibilities of applying immunocitochemistry and molecular genetics to cytological materials.

Needle optical coherence elastography for the measurement of microscale mechanical contrast deep within human breast tissues.

Optical coherence elastography (OCE) is an emerging imaging technique that probes microscale mechanical contrast in tissues with the potential to differentiate healthy and malignant tissues. However, conventional OCE techniques are limited to imaging the first 1 to 2 mm of tissue in depth. We demonstrate, for the first time, OCE measurements deep within human tissues using needle OCE, extending the potential of OCE as a surgical guidance tool. We use needle OCE to detect tissue interfaces based on mechanical contrast in both normal and malignant breast tissues in freshly excised human mastectomy samples, as validated against histopathology. Further, we demonstrate the feasibility of in situ measurements >4 cm from the tissue surface using ultrasound guidance of the OCE needle probe. With further refinement, our method may potentially aid in accurate detection of the boundary of the tumor to help ensure full removal of all malignant tissues, which is critical to the success of breast-conserving surgery.

Mass spectrometry imaging with high resolution in mass and space.

Mass spectrometry (MS) imaging links molecular information and the spatial distribution of analytes within a sample. In contrast to most histochemical techniques, mass spectrometry imaging can differentiate molecular modifications and does not require labeling of targeted compounds. We have recently introduced the first mass spectrometry imaging method that provides highly specific molecular information (high resolution and accuracy in mass) at cellular dimensions (high resolution in space). This method is based on a matrix-assisted laser desorption/ionization (MALDI) imaging source working at atmospheric pressure which is coupled to an orbital trapping mass spectrometer. Here, we present a number of application examples and demonstrate the benefit of 'mass spectrometry imaging with high resolution in mass and space.' Phospholipids, peptides and drug compounds were imaged in a number of tissue samples at a spatial resolution of 5-10 µm. Proteins were analyzed after on-tissue tryptic digestion at 50-µm resolution. Additional applications include the analysis of single cells and of human lung carcinoma tissue as well as the first MALDI imaging measurement of tissue at 3 µm pixel size. MS image analysis for all these experiments showed excellent correlation with histological staining evaluation. The high mass resolution (R = 30,000) and mass accuracy (typically 1 ppm) proved to be essential for specific image generation and reliable identification of analytes in tissue samples. The ability to combine the required high-quality mass analysis with spatial resolution in the range of single cells is a unique feature of our method. With that, it has the potential to supplement classical histochemical protocols and to provide new insights about molecular processes on the cellular level.

Photoacoustic section imaging using an elliptical acoustic mirror and optical detection.

A method is proposed that utilizes the advantages of optical ultrasound detection in two-dimensional photoacoustic section imaging, combining an optical interferometer with an acoustic mirror. The concave mirror has the shape of an elliptical cylinder and concentrates the acoustic wave generated around one focal line in the other one, where an optical beam probes the temporal evolution of acoustic pressure. This yields line projections of the acoustic sources at distances corresponding to the time of flight, which, after rotating the sample about an axis perpendicular to the optical detector, allows reconstruction of a section using the inverse Radon transform. A resolution of 120 [micro sign]m within and 1.5 mm between the sections can be obtained with the setup. Compared to a bare optical probe beam, the signal-to-noise ratio (SNR) is seven times higher with the mirror. Furthermore, the imaging system is tested on a biological sample.

Generation of THz frequency using PANDA ring resonator for THz imaging.

In this study, we have generated terahertz (THz) frequency by a novel design of microring resonators for medical applications. The dense wavelength-division multiplexing can be generated and obtained by using a Gaussian pulse propagating within a modified PANDA ring resonator and an add/drop filter system. Our results show that the THz frequency region can be obtained between 40-50 THz. This area of frequency provides a reliable frequency band for THz pulsed imaging.

Identification of tumor specimens by DNA analysis in a case of histocytological paraffin tissue block swapping.

We report on a patient who was diagnosed with high-grade breast carcinoma by all the pre-surgery clinical evidence of malignancy, but histopathological reports did not reveal any such tumor residue in the post-surgical tissue block. This raised a suspicion that either exchange of block, labeling error, or a technical error took place during gross examination of the tissue. The mastectomy residue was unprocurable to sort out the problem. So, two doubtful paraffin blocks were sent for DNA fingerprinting analysis. The partial DNA profiles (8-9/15 loci) were obtained from histocytological blocks. The random matching probability for both the paraffin blocks and the patient's blood were found to be 1 in 4.43E4, 1.89E6, and 8.83E13, respectively for Asian population. Multiplex short tandem repeat analysis applied in this case determined that the cause of tumor absence was an error in gross examination of the post-surgical tissue. Moreover, the analysis helped in justifying the therapy given to the patient. Thus, with DNA fingerprinting technique, it was concluded that there was no exchange of the blocks between the two patients operated on the same day and the treatment given to the concerned patient was in the right direction.

Endomicroscopy imaging of epithelial structures using tissue autofluorescence.

We explore autofluorescence endomicroscopy as a potential tool for real-time visualization of epithelial tissue microstructure and organization in a clinical setting. The design parameters are explored using two experimental systems--an Olympus Medical Systems Corp. stand-alone clinical prototype probe, and a custom built bench-top rigid fiber conduit prototype. Both systems entail ultraviolet excitation at 266 nm and/or 325 nm using compact laser sources. Preliminary results using ex vivo animal and human tissue specimens suggest that this technology can be translated toward in vivo application to address the need for real-time histology.

Electron microscopy of Paramecium (Ciliata).

Paramecium may be the best known single-celled organism in existence (Hausmann et al., 2003). Today its image often appears on television programs where the producers use it to illustrate a stereotypic microorganism, be it pathogenic or nonpathogenic, prokaryotic or eukaryotic. Paramecium was probably one of the first single-celled organisms observed with a light microscope by the Dutch cloth vendor and amateur lens maker Antoni van Leuwenhoek (1632-1723) (Dobell, 1932), and it is still being investigated in the 21st century in the days of the modern electron microscopes.

Laser-capture microdissection and pressure catapulting for the analysis of gene expression in the renal glomerulus.

Investigation into the molecular mechanisms regulating normal renal physiology and pathophysiology has benefited from the development of microdissection techniques enabling sampling of specific cell populations or structures within the kidney. Laser-capture microdissection and pressure catapulting is a relatively new, entirely non-contact microdissection technique that facilitates the assay of mRNA and protein expression in single nephron segments or populations. Herein, we describe methods for sample preparation, microdissection and collection of glomeruli from archival renal biopsies for later analysis of gene expression using real-time PCR. Microdissection of glomeruli from archival renal biopsy sections was carried out using the PALM Microbeam UV laser system from P.A.L.M. Technologies.

Uso de biomateriales con fines neurorregenerativos. Aplicación en modelo experimental de traumatismo craneoencefálico / Use of biomaterials in neural regeneration. Application in an experimental model of head injury

Objetivos: Evaluar el rendimiento de los biomateriales poliméricos basados en ácido hialurónico y su utilidad en el Sistema Nervioso Central, sirviendo como soporte, para la supervivencia y diferenciación celular. Material y Metodos: Con el fin de evaluar la viabilidad de los soportes poliméricos y acanalados, se realizaron experimetos in vitro e in vivo mediante el implante en corteza cerebral de ratas Wistar. Mediante técnicas inmunocitoquímicas e histológicas se procedió al análisis de la viabilidad de los soportes. Resultados: Tras el cultivo pudimos constatar la viabilidad celular sobre los biomateriales, asi como su potencial utilidad para la regeneración in vivo de estructuras vasculares y neurales. Conclusiones: La posibilidad de regenerar estructuras vasculares y neurales a través del implante de biomateriales basados en ácido hialurónico, constituye un avance en la utilización de biomateriales en el Sistema Nervioso Central (AU)

Objetives: To evaluate the performance of polymeric biomaterials based on hyaluronic acid and their usefulness in the central nervous system as support for cell differentiation and survival. Material and methods: With the purpose of assessing the viability of polymeric cannulated scaffolds, in vitro and in vivo experiments were made involving implantation in the Wistar rate brain cortex. Immunocytochemical and histological techniques were used to analyze scaffold viability. Results: Following culture, cell viability on the biomaterials was confirmed, together with the potential usefulness of the latter for the in vivo regeneration of vascular and neural structures. Conclusions: The possibility of regenerating vascular and neural structures through the implantation of biomaterials based on hyaluronic acid constitutes an advance in the use of biomaterials in the central nervous system (AU)

Fine needle aspiration cytology of thyroid nodules: conventional vs thin layer technique.

OBJECTIVE: Liquid-based cytology using the thin layer technique has recently been introduced in thyroid fine needle aspiration cytology together with or in substitution of direct smears, but its usefulness is still controversial and relatively few studies have been published in this field. The aim of the present study was to compare the results obtained from conventional smears with those from thin layer smears. DESIGN: In 3875 thyroid nodules, a double cytologic sampling was taken in randomized order, to prepare conventional or thin layer smears. MAIN OUTCOME: The diagnoses agreed in 2934 (75.7%) cases and disagreed in 941 (24.3%). The analysis of discordant data showed there were fewer non-diagnostic cases in the thin layer smears (377 vs 541, p<0.001) whereas in conventional smears there were more cases positive for carcinoma (27 vs 4, p<0.001). The cytohistologic correlation was available for 194 cases and showed that conventional smears had a greater capacity for revealing carcinomas (44 vs 31). Finally, diagnoses based on conventional smears were more sensitive than thin layer smears (93.6% vs 65.9%) whereas specificity was constant. CONCLUSIONS: From our experience, the conventional smear offers a greater possibility of diagnosis when suspecting malignancy or diagnosing malignancy cases, whereas thin layer smears significantly reduce the number of non-diagnostic cases. For this reason, we suggest combining the two techniques in routine cytologic diagnosis.

Caracterização citoquímica ultra-estrutural da cercária de Schistosoma mansoni / Cytochemical ultrastructural caracterization of cercaria of Schistosoma mansoni

Uma alternativa para identificar a base de processos críticos necessários ao estabelecimento do parasito no hospedeiro é enfocar o estágio responsável pela invasão primária, ou seja, a estrutura infectante. O principal objetivo do trabalho foi realizar uma caracterização ultra-estrutural da cercária de Schistosoma mansoni, através de técnicas citoquímicas. Para a identificação de lipídios saturados e insaturados foi utilizado, respectivamente, as técnicas de Filipina e do Tampão Imidazol, para localização de proteínas básicas foram empregadas as técnicas do Ácido Fosfotúngstico (PTA) e da Prata Amoniacal. Para localizar sítios de cálcio utilizamos a técnica descrita por Hepler (1980) e para evidenciar grupamentos aniônicos empregamos ferritina cationizada além do tratamento enzimático com tripsina, condroitinase e neuraminidase de Vibrio cholerae. Foi observada a presença de lipídios insaturados de formato globular em toda a região externa da larva infectante, tegumento, glândula da cabeça, glândula pré-acetabular e corpos de inclusão. Utilizando a técnica de filipina, identificamos a presença de colesterol delimitando vários compartimentos da larva e também no tegumento, músculo e membranas de células subtegumentares. Através da técnica de PTA foi identificada a presença de proteínas básicas no tegumento, núcleo e nucléolo de células subtegumentares, corpos de inclusão e glândulas pré-acetabulares. Já na prata amoniacal, identificamos forte marcação em toda larva infectante além de marcações no núcleo das células musculares, tecido muscular circular e glândulas pré-acetabulares. A localização de sítios de cálcio mostrou-se bastante uniforme, demarcando os espaços internos da larva, principalmente, o tecido muscular da larva. As amostras tratadas com ferritina cationizada apresentaram uma forte marcação em nível cuticular. O tratamento das amostras com a neuraminidase não alterou o padrão de marcação dessas partículas na superfície do trematóide. Porém, o tratamento com tripsina ou condroitinase resultou numa ausência de marcação na superfície da larva. O esclarecimento da composição bioquímica da larva infectante de S. mansoni fornece dados para um melhor entendimento a respeito da biologia do parasito bem como a intrigante relação parasitohospedeiro, além de contribuir para um possível controle químico.

Chemical mapping of tumor progression by FT-IR imaging: towards molecular histopathology.

Fourier-transform infrared (FT-IR) spectro-imaging enables global analysis of samples, with resolution close to the cellular level. Recent studies have shown that FT-IR imaging enables determination of the biodistribution of several molecules of interest (carbohydrates, lipids, proteins) for tissue analysis without pre-analytical modification of the sample such as staining. Molecular structure information is also available from the same analysis, notably for protein secondary structure and fatty acyl chain peroxidation level. Thus, several cancer markers can be identified from FT-IR tissue images, enabling accurate discrimination between healthy and tumor areas. FT-IR imaging applications are now able to provide unique chemical and morphological information about tissue status. With the fast image acquisition provided by modern mid-infrared imaging systems, it is now envisaged to analyze cerebral tumor exereses in delays compatible with neurosurgery. Accordingly, we propose to take FT-IR imaging into consideration for the development of new molecular histopathology tools.