Genetic Engineering & Biotechnology News

AUG 2017

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10 | AUGUST 2017 | GENengnews.com | Genetic Engineering & Biotechnology News To overcome the phototoxicity that can occur with fluorophores such as MitoTrack- er or with nuclear stains, live-cell imaging can rely on efficient light capture, incorporating objectives with high numerical apertures and long exposures, using low amounts of exci- tation light from stable LED light sources. Another option is to use label-free imaging with digital phase contrast. GEN Have advances in live-cell imaging made the technology easier to use on non-mammalian cells, such as yeast or bacteria? Dr. Banks Yeast and bacteria are much smaller than mammalian cells. Conse- quently, yeast and bacteria present certain imaging challenges. Although microbes observed with live-cell imaging readily pro- vide details about proliferation, they may withhold information about intracellular processes, which tend to be accelerated in smaller cells. Intracellular imaging of cellular processes requires high-resolution microscopy and, typically, fast frame rates. Along with high resolution comes a limited field of view, which could complicate studies that attempt to gen- erate cell-population statistics. To overcome a limited field of view, investigators may need to deploy expensive cameras or assemble com- posite images. Dr. Appledorn The use of live-cell analysis on non-mammalian cells is not an area that I fol- low closely. I have noticed, however, that over the past few years, many researchers have started using fluorescently labeled microor- ganisms, such as Listeria monocytogenes and hepatitis C virus, to examine how non-mam- malian and mammalian cells interact. Taking a live-cell analytical approach to answering the questions around rates of in- fection and coinfection in multiple cell types is absolutely required to capture the kinetics of the biology under examination. I imagine there are many other uses for innovative live- cell imaging approaches to studying biofilms, yeast behaviors, and a wide variety of micro- organisms, as well as whole organisms such as zebrafish and Caenorhabditis elegans. The applications are limitless. Dr. Shumate Yes, we have many customers taking advantage of the high-magnification capabilities of our instruments, which in- corporate 40×, 60×, and 100× objectives. We have even been able to do automated imaging with oil-immersion objectives over limited areas. Our customers are looking at quorum sensing, biofilm behavior, and bacterial bio- sensors. We also have customers interested in applications such as algal fuel and yeast chemical intermediate production. Often the organisms in these studies grow at high humidity and elevated temperatures; [these are] adverse environments where our mi- croscopes can reside and operate with no problems over extended periods of time. Dr. Schneider Yeast and bacteria present unique challenges for live-cell imaging ef- forts that inform imaging-based analysis. Because these microorganisms are so small, they necessitate the use of newer super- resolution platforms for the visualization of subcellular structures. Smallness also complicates multispectral imaging, which is further hampered by the presence of the cell wall/envelope. To facilitate the live-cell imaging of mi- croorganisms, genetically engineered fluo- rescent strains and small-molecule dyes have been developed. These tools provide alter- natives to bulky antibody-based methods. Studies of host-pathogen interactions, biofilms, and the cell cycle require systems that are not only capable of precise delivery of media and reagents, but are also com- patible with continuous, long-term culture maintenance. Specialized microfluidic- based plates with machine-driven media control, such as the CellASIC ® ONIX2 plat- form, provide an attractive option for such studies. Dr. Boettcher Live-cell imaging is usually performed in aqueous growth media to keep cells as healthy as possible. Non-mammalian cells such as yeast or bacteria are inherently smaller than mammalian cells and require high-magnification and high-resolution im- aging. One of the most important advances within the field of high-content imaging is, therefore, the availability of automated wa- ter-immersion objectives. Water-immersion objectives enable high numerical apertures (NAs) and collect up to seven times the light collected by air objec- tives. Moreover, high-NA objectives provide higher resolution than low-NA objectives, providing more details from yeast or bacteria. As confocality also increases resolution, the best image quality is achieved on instruments which combine both technologies—water immersion and confocality—such as the Op- eretta CLS or the Opera Phenix system. GEN What innovations are coming down the road for live-cell imaging? Dr. Banks In the last few years, instruments have been developed that provide automat- Roundup Continued from page 9 Drug Discovery DEVELOPING AND SUPPLEMENTING CELL CULTURE AND MICROBIAL MEDIA © 2017 BD. BD and the BD Logo are trademarks of Becton, Dickinson and Company. Learn more about BD cell culture media and supplements at bdbiosciences.com/advbio

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