An optical imaging chamber for viewing living plant cells and tissues at high resolution for extended periods
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METHODOLOGY
PLANT METHODS
Open Access
An optical imaging chamber for viewing living plant cells and tissues at high resolution for extended periods Grant Calder, Chris Hindle, Jordi Chan and Peter Shaw*
Abstract Background: Recent developments in both microscopy and fluorescent protein technologies have made live imaging a powerful tool for the study of plant cells. However, the complications of keeping plant material alive during a long duration experiment while maintaining maximum resolution has limited the use of these methods. Results: Here, we describe an imaging chamber designed to overcome these limitations, which is flexible enough to support a range of sizes of plant materials. We were able use confocal microscopy to follow growth and development of plant cells and tissues over several days. The chamber design is based on a perfusion system, so that the addition of drugs and other experimental treatments are also possible. Conclusions: In this article we present a design of imaging chamber that makes it possible to image plant material with high resolution for extended periods of time. Keywords: Arabidopsis, Imaging chamber, Time-lapse microscopy, Perfusion, Live cell imaging, Plant tissue
Background The progress in optical microscopy technology (faster acquisition, more sensitive cameras, specialised immersion objectives, confocal, multi photon etc.) combined with direct labelling of target proteins using fluorescent protein markers has made live cell imaging a powerful tool for investigating dynamic processes in plants [1-5] This technology enables us to follow dynamic events in live experiments, rather than taking snapshot images of chemically fixed material. This allows many biological processes to be studied from start to finish. For example, the processes involved in the formation of a tissue or organ are regulated by developmental cues or circadian rhythms. To understand these developmentally regulated patterns it is necessary to follow cells in a developing tissue throughout organ formation, which can take from hours to days [6]. Short term imaging of living samples is relatively easy and the sample preparation can be as simple as putting the sample onto a slide in a suitable medium with a cover glass on top. However, successful high resolution * Correspondence: [email protected] Department of Cell & Developmental Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK
imaging for a long period of time is more difficult. Three key factors need to be addressed: a constant environment surrounding the sample; stably holding the sample’s position within the imaging volume; and, for larger samples, imaging a large enough volume. To track a sample for long durations it is necessary to make specialised sample holders or chambers to address these issues [7]. In general plant material is very robust to large variations in many environmental conditions. However, plant samples are highly susceptible to decreasing levels of nutrients and oxygen (anoxia) during live cell imaging experime
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