Endogenous Labeling for Light Microscopy during HIV-1 Immune Responses.

New approaches in single molecule spectroscopy and microscopy are able to resolve the spatial and temporal resolution of T cell receptor signaling in the context of immune responses to HIV-1 infection. These approaches need to be complemented with novel techniques that endogenously tag the protein or proteins of interest, yet avoid overexpression, to image protein dynamics under physiological conditions.

Varying practices in tumor regression grading of gastrointestinal carcinomas after neoadjuvant therapy: results of an international survey.

Tumor regression grading is routinely performed on neoadjuvantly treated gastrointestinal cancer resections. Challenges in tumor regression grading include grossing standards, multiple grading systems, and difficulty interpreting therapy-induced changes. We surveyed gastrointestinal pathologists around the world for their practices in handling neoadjuvantly treated gastrointestinal cancer specimens and reporting tumor regression using a 23-question online survey. Topics addressed grossing, histologic work-up, tumor regression grading systems, and degree of difficulty identifying and estimating residual cancer within treatment effect. Two-hundred three responses were received, including 173 participants who completed the entire questionnaire. Fifty percent of the participants were from Europe, 29% from North America, 10% from Australia, and 11% from other continents. Ninety-five percent routinely report a tumor regression grade and 92% have standardized grossing and histologic work-up: 27% always completely embed the entire tumor bed, 54% embed the complete tumor site if not a grossly apparent, large mass. Fifty-nine percent use hematoxylin & eosin alone for assessment; the remaining use additional stains. In North America and Australia, the American Joint Committee on Cancer (AJCC)/College of American Pathologists (CAP)/Ryan system is routinely used for gastroesophageal (71%) and rectal carcinomas (77%). In Europe, the Mandard system is common (36%) for gastroesophageal tumors, followed by AJCC/CAP/Ryan (22%), and Becker (10%); for rectal CA, the Dworak system (30%) is followed by AJCC/CAP/Ryan (24%) and Mandard (14%). This regional differences were significant (p < 0.001 each). Fifty-one percent prefer a four-tiered system. Sixty-six percent think that regressive changes in lymph nodes should be part of a regression grade. Sixty-nine percent consider identifying residual tumor straight-forward, but estimating therapy-induced fibrosis difficult (57%). Free comments raised issues of costs for work-up and clinical relevance. In conclusion, this multinational survey provides a comprehensive overview of grossing and histologic work-up with regards to tumor regression grading in gastrointestinal cancers with partly significant regional differences particularly between North America and Europe.

Digital mucous cyst: Altered epidermal mucin as a clue to diagnosis.

BACKGROUND: Digital mucous cyst (DMC) is histopathologically characterized by accumulation of mucin in the dermis. Some cases of DMC also show epidermal mucin, the histopathologic appearance and staining properties of which have not been described in detail. METHODS: A total of 24 cases of DMC were investigated by routine hematoxylin-eosin (H&E) and Alcian blue stains in addition to AE1/AE3 immunohistochemistry. RESULTS: Nine out of the 24 cases of DMC showed epidermal mucin. As the epidermal mucin migrates upward within the epidermis, it transforms from a flocculent granular substance into one or several solid horizontal plugs with a more homogeneous appearance and incorporates cytoplasmic fragments of keratinocytes/corneocytes. The homogeneous mucin plugs stain eosinophilic or amphophilic with an H&E formulation using hematoxylin 7212 and basophilic with Gill 3 or Harris's hematoxylin. The eosinophilic staining is enhanced when the eosin solution contains phloxine. CONCLUSIONS: The variably eosinophilic, amphophilic, or basophilic staining of epidermal mucin can be explained by its composition of basophilic mucin and eosinophilic debris from cytoplasmic fragments. The eosinophilic staining of mucin has not been reported before and can be diagnostically important because it may be mistaken for serum exudate.

Strategic labelling approaches for RNA single-molecule spectroscopy.

Most single-molecule techniques observing RNA in vitro or in vivo require fluorescent labels that have to be connected to the RNA of interest. In recent years, a plethora of methods has been developed to achieve site-specific labelling, in many cases under near-native conditions. Here, we review chemical as well as enzymatic labelling methods that are compatible with single-molecule fluorescence spectroscopy or microscopy and show how these can be combined to offer a large variety of options to site-specifically place one or more labels in an RNA of interest. By either chemically forming a covalent bond or non-covalent hybridization, these techniques are prerequisites to perform state-of-the-art single-molecule experiments.

Waltzing around Sacred Cows on the Way to the Future.

Our mostly manual, agar-based clinical microbiology laboratory is slowly but steadily being redefined by automation and innovation. Ironically, the oldest test, the Gram stain test, is still manually read and interpreted by trained personnel. In a proof-of-concept study, Smith et al. (J. Clin. Microbiol. 56:e01521-17, 2018, https://doi.org/10.1128/JCM.01521-17) used computer imaging with a deep convolutional neural network to examine and interpret Gram-stained slides from positive blood culture bottles. In light of the shortage of medical technologists/microbiologists and the need for results from positive blood culture bottles 24/7, this paper paves the way for the next innovations for the clinical microbiology laboratory of the future.

Recent Advances and Future Prospects of Aggregation-induced Emission Carbohydrate Polymers.

Aggregation-induced emission (AIE) is an abnormal phenomenon that has sparked great attention for diverse applications in different fields. In particular, the fabrication and biological imaging applications of AIE-active fluorescent organic nanoparticles (FONs) have become a focus in the emerging and promising fields. A large number of AIE-active polymeric nanoprobes have recently been fabricated through different strategies. The advances and progress in this direction have also recently been summarized by some groups. However, the fabrication and biomedical applications of AIE-active FONs based on carbohydrate polymers and AIE-active dyes are quite rare and limited. In this feature article, the recently reported AIE-active FONs with different structures and applications based on AIE-active dyes and carbohydrate polymers are highlighted, and the major current limitations and development tendencies are also discussed.

Intein applications: from protein purification and labeling to metabolic control methods.

The discovery of inteins in the early 1990s opened the door to a wide variety of new technologies. Early engineered inteins from various sources allowed the development of self-cleaving affinity tags and new methods for joining protein segments through expressed protein ligation. Some applications were developed around native and engineered split inteins, which allow protein segments expressed separately to be spliced together in vitro. More recently, these early applications have been expanded and optimized through the discovery of highly efficient trans-splicing and trans-cleaving inteins. These new inteins have enabled a wide variety of applications in metabolic engineering, protein labeling, biomaterials construction, protein cyclization, and protein purification.

Labelling of mammalian cells for visualisation by MRI.

Through labelling of cells with magnetic contrast agents it is possible to follow the fate of transplanted cells in vivo with magnetic resonance imaging (MRI) as has been demonstrated in animal studies as well as in a clinical setting. A large variety of labelling strategies are available that allow for prolonged and sensitive detection of the labelled cells with MRI. The various protocols each harbour specific advantages and disadvantages. In choosing a particular labelling strategy it is also important to ascertain that the labelling procedure does not negatively influence cell functionality, for which a large variety of assays are available. In order to overcome the challenges still faced in fully exploiting the benefits of in vivo cell tracking by MRI a good understanding and standardisation of the procedures and assays used will be crucial.

Micro-staining microbes: An alternative to traditional staining of microbiological specimens using microliter volumes of reagents.

Microbial staining techniques are widely employed in clinical and academic laboratories for classifying and identifying microorganisms derived from clinical, food and environmental samples. Staining allows for the rapid visualization and determination of many morphological characteristics of microorganisms, used for their identification and classification. Over the past century, staining techniques such as the Gram stain, the Capsule stain, the Acid-fast stain and the Endospore stain, have seen few advances, and manual staining remains the gold standard. Typical instructions for these staining procedures recommend 'flooding' glass slides with milliliter volumes of dye, resulting in large volumes of hazardous waste. Here we present micro-staining, a simple alternative to flooding that utilizes microliter volumes of dye. Micro-staining minimizes the volume of waste generated, leads to significant cost savings for the laboratory, requires limited training, and produces results with equivalent quality to traditional stains.

Migration, fate and in vivo imaging of adult stem cells in the CNS.

Adult stem cells have been intensively studied for their potential use in cell therapies for neurodegenerative diseases, ischemia and traumatic injuries. One of the most promising cell sources for autologous cell transplantation is bone marrow, containing a heterogenous cell population that can be roughly divided into hematopoietic stem and progenitor cells and mesenchymal stem cells (MSCs). MSCs are multipotent progenitor cells that, in the case of severe tissue ischemia or damage, can be attracted to the lesion site, where they can secrete bioactive molecules, either naturally or through genetic engineering. They can also serve as vehicles for delivering therapeutic agents. Mobilized from the marrow, sorted or expanded in culture, MSCs can be delivered to the damaged site by direct or systemic application. In addition, MSCs can be labeled with superparamagnetic nanoparticles that allow in vivo cell imaging. Magnetic resonance imaging (MRI) is thus a suitable method for in vivo cell tracking of transplanted cells in the host organism. This review will focus on cell labeling for MRI and the use of MSCs in experimental and clinical studies for the treatment of brain and spinal cord injuries.

Congo red and protein aggregation in neurodegenerative diseases.

Congo red is a commonly used histological dye for amyloid detection. The specificity of this staining results from Congo red's affinity for binding to fibril proteins enriched in beta-sheet conformation. Unexpectedly, recent investigations indicate that the dye also possesses the capacity to interfere with processes of protein misfolding and aggregation, stabilizing native protein monomers or partially folded intermediates, while reducing concentration of more toxic protein oligomers. Inhibitory effects of Congo red upon amyloid toxicity may also range from blockade of channel formation and interference with glycosaminoglycans binding or immune functions, to the modulation of gene expression. Particularly, Congo red exhibits ameliorative effect in models of neurodegenerative disorders, such as Alzheimer's, Parkinson's, Huntington's and prion diseases. Another interesting application of Congo red analogues is the development of imaging probes. Based on their small molecular size and penetrability through blood-brain barrier, Congo red congeners can be used for both antemortem and in vivo visualization and quantification of brain amyloids. Therefore, understanding mechanisms involved in dye-amyloidal fibril binding and inhibition of aggregation will provide instructive guides for the design of future compounds, potentially useful for monitoring and treating neurodegenerative diseases.

Brain histology.

In this article we summarise nervous system histology in health and disease and acquaint the reader with developments in the staining techniques that are in current use, particularly immunostains. Although clinicians do not need to know the details of stain appearances, some familiarity with these aspects of neuropathology is invaluable in interpreting the reports they receive from the laboratory, as well as reminding them of the amazing beauty of the central nervous system's microscopic structure.

MIAQuant, a novel system for automatic segmentation, measurement, and localization comparison of different biomarkers from serialized histological slices.

In the clinical practice, automatic image analysis methods quickly quantizing histological results by objective and replicable methods are getting more and more necessary and widespread. Despite several commercial software products are available for this task, they are very little flexible, and provided as black boxes without modifiable source code. To overcome the aforementioned problems, we employed the commonly used MATLAB platform to develop an automatic method, MIAQuant, for the analysis of histochemical and immunohistochemical images, stained with various methods and acquired by different tools. It automatically extracts and quantifies markers characterized by various colors and shapes; furthermore, it aligns contiguous tissue slices stained by different markers and overlaps them with differing colors for visual comparison of their localization. Application of MIAQuant for clinical research fields, such as oncology and cardiovascular disease studies, has proven its efficacy, robustness and flexibility with respect to various problems; we highlight that, the flexibility of MIAQuant makes it an important tool to be exploited for basic researches where needs are constantly changing. MIAQuant software and its user manual are freely available for clinical studies, pathological research, and diagnosis.

Nonlinear microscopy: new techniques and applications.

Nonlinear microscopy, a general term that embraces any microscopy technique based on nonlinear optics, is further establishing itself as an important tool in neurobiology. Recent advances in labels, labeling techniques, and the use of native or genetically encoded contrast agents have bolstered the capacity of nonlinear microscopes to image the structure and function of not just single cells but of entire networks of cells. Along with novel strategies to image over exceptionally long durations and with increased depth penetration in living brains, these advances are opening new opportunities in neurobiology that were previously unavailable.

Labeling neurons in vivo for morphological and functional studies.

Increasingly sophisticated strategies for labeling cells in vivo are providing unprecedented opportunities to study neurons in living animals. Transgenic expression of genetically encoded reporters enables us to monitor changes in neuronal activity in response to sensory stimuli, and the labeling of single neurons with fluorescent proteins allows the dynamics of neuronal connectivity to be observed in transgenic animals over periods ranging from minutes to months. Advances in transient labeling techniques such as viral infection and electroporation provide a rapid means by which to analyze neuronal gene function in vivo. These new approaches to labeling, manipulating and imaging neurons in intact organisms are transforming the way in which the nervous system is studied.

News from the Biological Stain Commission, No. 17.

In the 17(th) issue of News from the Biological Stain Commission (BSC) under the heading of Regulatory affairs, the Biological Stain Commission's International Affairs Committee presents information from the 20(th) meeting of ISO/TC 212 Clinical laboratory testing and in vitro diagnostic test systems held on October 15 - 17, 2014 in Toronto, Canada, and from the 29(th) meeting of CEN/TC 140 In vitro diagnostic medical devices held on February 3, 2015 in Berlin, Germany.