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Summary of the November 2015 issue of BioTechniques

The November 2015 issue of BioTechniques will feature a variety of new methods and techniques:

  • an enhanced protocol for 3-D microtissue imaging
  • a novel approach to imaging heart tissues
  • a software development for performing quantitative analysis on image datasets
  • a new platform tool that enables the batch analysis of imaging files
  • ultrasensitive immunoassays for insulin detection
  • a plate-based assay for analyzing viral encapsidation

This issue will also feature a Tech News article focused on the latest techniques being used for large-scale genome analysis and genotyping.

November article summaries:

Three-dimensional (3D) in vitro platforms have been shown to closely recapitulate human physiology. Despite the benefits of 3D cell culture, limitations in visualization and imaging of 3D microtissues still present significant challenges. In an article slated for the November issue of BioTechniques, researchers from Brown University refined histology and microscopy techniques to overcome the constraints of 3D imaging. For morphological assessment of 3D microtissues of several cell types, a two-step glycol methacrylate embedding protocol improved resolution of nuclear and cellular histopathology. Immunohistochemistry, immunofluorescence and in situ immunostaining techniques were also successfully adapted to microtissues, and enhanced by optical clearing. These refined techniques provide new methods for evaluating disease pathogenesis, delineating toxicity pathways, and enhancing the versatility of 3D imaging applications. Keywords: Clearing tissues; 3D imaging; immunohistochemistry; fluorescence; microscopy; image analysis

The ability to image and render the entire adult mouse heart at high resolution in three-dimensions provides enormous advantages when studying heart disease. However, a comprehensive technique to reveal nuclear/cellular detail as well as the entire heart in 3D with minimal effort is lacking. To address this, researchers from the University of Illinois describe their modification to the Benzyl Alcohol:Benzyl Benzoate clearing technique by labeling hearts with periodic acid Schiff (PAS) and the using a combination of two-photon fluorescence microscopy and automated tile-scan imaging/stitching. By taking advantage of the differential spectral properties of PAS, muscle and nuclear compartments in the heart could be distinguished. The combination of labeling, clearing, and two-photon microscopy together with tiling, helped obviate laborious and time-consuming physical sectioning, alignment and 3D reconstruction.
Keywords: 3D image reconstruction; cardiac disease; heart biology; two-photon microscopy; tile-scan imaging

Quantitative image analysis plays an important role in contemporary biomedical research. Squassh is a method for automatic detection, segmentation, and quantification of subcellular structures and their colocalization analysis. In the November issue, researchers from Switzerland describe Squassh3C and SquasshAnalyst, enhancements to the original Squassh methodology. Squassh3C extends the functionality of Squassh to three fluorescent channels and enables live cell movie analysis, while SquasshAnalyst is an interactive web interface for the analysis of Squassh3C object data. Keywords: automated image analysis; quantitative image analysis; microscopy; live cell imaging

Most imaging studies in the biological sciences rely on analyses that are relatively simple. However, manual repetition of analysis tasks across multiple regions in many images can complicate even the simplest analysis, making record keeping difficult, increasing the potential for error, and limiting reproducibility. In a Report slated for November, scientists from Emory University introduces a new image analysis platform called Slide Set, which provides a framework for reproducible image analysis and batch processing with ImageJ. Slide Set organizes experimental imaging data into tables, associating image files with regions of interest and other relevant information to make image analysis faster.
Keywords: batch image analysis; ImageJ; experimental data organization/storage; microscopy

To decrease patient suffering in clinical settings, the collection of blood samples for the diagnosis and monitoring of disease should be minimized. When the secretional capacity and resistance to insulin in diabetes mellitus (DM) patients is tested, increases immunosensitivity to insulin directly reduce the necessary sample volume. In this issue of BioTechniques, researchers from Japan describe a newly developed ultrasensitive ELISA that is coupled with a thio-NAD cycling which can be employed to measure immunoreactive insulin in blood serum using WHO international standard insulin or its equivalent products as a reference. The increased sensitivity of this new assay means less sample is needed for testing.
Keywords: ELISA, immunoreactivity; insulin analysis; ultrasensitive assays

Hepatitis B virus (HBV) employs its pregenomic RNA (pgRNA) as a template to replicate its DNA genome by reverse transcription, following encapsidation, a packaging step of the pgRNA into viral nucleocapsids. To date, there are only two encapsidation detection methods eligible to evaluate pgRNA levels packaged into nucleocapsids; i) RNase protection assay and ii) native agarose gel electrophoresis assay. In the November 2015 issue, researchers from Korea report on a novel HBV encapsidation assay in 96-well plate format using nucleocapsids capture plates, coated with anti-HBV core antibody, usually used for enzyme-linked immunosorbent assay, to immobilize viral nucleocapsids followed by the detection of viral pgRNA via quantitative real-time RT-PCR. The author’s strategy supports fast, convenient and quantitative analysis of multiple viral RNA samples to evaluate encapsidation inhibitors.
Keywords: qRT-PCR; hepatitis B virus; antibodies; viral RNA detection

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