Abstract
A centrifuge microscope (CMS) functionally consists of a centrifuge producing a centrifugal force (hypergravity condition) and a microscope making an enlarged image of an object. This combination of equipment allows live-cell imaging during centrifugation. We have developed a new CMS (NSK Ltd.) to observe movements of the plant organelles such as amyloplasts, under hypergravity conditions (Toyota et al., 2013). This CMS is distinct from previously designed CMSs in terms of spatio-temporal resolution, ease of use and compactness. Here, we show a quick protocol to prepare a specimen of Arabidopsis inflorescence stem, use the CMS, obtain imaging data and analyze them using a single tracking method.
Materials and Reagents
Equipment
Software
Procedure
Representative data
Figure 3. Representative tracking image (Toyota et al., 2013). Movement of an amyloplast (arrow head) is successfully traced by the G-Track spot-tracking software during centrifugation. Table 1. Mean square desplacement (MSD) of the amylplast for 1 sec of centrifugation. MSD of the amyloplast traced in Figure 3 is automatically calculated by the tracking program. X, Y and 2D denote movement in the horizontal (10 x g) and vertical directions and in a two-dimensional (2D) plane, respectively. For downloading data, please click the image below.
Recipes
Acknowledgments
We thank Professor T. Mimura (Kobe University), Professor Y. Yoshimoto (Kansai Medical University) and Professor T. Shimmen (University of Hyogo) for valuable information about centrifuge microscopes and Y. Ishibashi for technical assistance. This work was supported in part by TOYOBO Biotechnology Foundation (to M. Toyota); Grant-in-Aid for JSPS Fellows (to M. Toyota), for JSPS Fellows for Research Abroad (to M. Toyota) and for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (16085205 to M.T.M.); and grants from the Bioarchitect Project of RIKEN (to M.T.M.) and PREST (to M. Toyota and M.T.M.).
References
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