Continuous live cell imaging of cellulose attachment by microbes under anaerobic and thermophilic conditions using confocal microscopy |
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Authors: | Zhi-Wu Wang Seung-Hwan Lee James G Elkins Yongchao Li Scott Hamilton-Brehm and Jennifer L Morrell-Falvey |
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Institution: | 1. BioEnergy Science Center, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA ;College of Food, Agricultural and Environmental Sciences, The Ohio State University,1328 Dover Rd, Wooster, OH 44691, USA 2. National Institute of Advanced Industrial Science and Technology, Biomass Refinery Research Center, Hiroshima, Japan ;Department of Forest Biomaterials Engineering, College of Forest and Environmental Sciences,Kangwon National University, Chuncheon 200-701, Korea 3. BioEnergy Science Center, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA |
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Abstract: | Live cell imaging methods provide important insights into the dynamics of cellular processes that cannot be derived easily from population-averaged datasets. In the bioenergy field, much research is focused on fermentation of cellulosic biomass by thermophilic microbes to produce biofuels; however, little effort is dedicated to the development of imaging tools to monitor this dynamic biological process. This is, in part, due to the experimental challenges of imaging cells under both anaerobic and thermophilic conditions. Here an imaging system is described that integrates confocal microscopy, a flow cell device, and a lipophilic dye to visualize cells. Solutions to technical obstacles regarding suitable fluorescent markers, photodamage during imaging, and maintenance of environmental conditions during imaging are presented. This system was utilized to observe cellulose colonization by Clostridium thermocellum under anaerobic conditions at 60°C. This method enables live cell imaging of bacterial growth under anaerobic and thermophilic conditions and should be widely applicable to visualizing different cell types or processes in real time. |
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Keywords: | thermophile lipophilic dye cellulose biofuel confocal laser scanning microscopy biofilm |
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