2020 Fiscal Year Research-status Report
Mechanical stability of microtubules investigated under unidirectional strain using combined fluorescence microscopy and high-speed atomic force microscopy
| Project/Area Number |
20K03889
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| Research Institution | Center for Novel Science Initatives, National Institutes of Natural Sciences |
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Principal Investigator |
Ganser Christian 大学共同利用機関法人自然科学研究機構(新分野創成センター、アストロバイオロジーセンター、生命創成探究, 生命創成探究センター, 特任助教 (50846095)
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| Project Period (FY) |
2020-04-01 – 2023-03-31
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| Keywords | high-speed AFM / microtubules / mechanical properties / correlated imaging |
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| Outline of Annual Research Achievements |
A basic mechanism that allows stretching of the substrate was successfully tested and it was confirmed that nanometer scale effects could be observed. Furthermore, the sensitivity of the atomic force microscope was improved significantly to allow for undisturbed long-time observation of fragile samples such as microtubules. It was successfully demonstrated that the tip-scan high-speed atomic force microscope can take images simultaneously with the fluorescence microscope and that both images are spatially and temporally correlated.
The force mapping of the high-speed atomic force microscope was improved so that the invasiveness was minimized further to allow undisturbed mechanical mapping of microtubules under strain.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The initial state of the tip-scan high-speed atomic force microscope system allowed for imaging microtubules, however, the invasiveness was considerable and caused microtubules to depolymerize during force mapping as well as during imaging. To overcome this problem, the system has been improved by optimizing the alignment of the read-out laser, including rigorous blocking of reflected light with an optical isolator. The scanner has been balanced to remove unwanted resonance frequencies in order to scan faster and more stable. In addition the optical fluorescence microscope has been fully synchronized with the atomic force microscope which finally allowed correlated observations.
All these improvements lead to a solid basis for continued research on strained microtubules.
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| Strategy for Future Research Activity |
In the next steps, the mechanical properties of microtubules under different conditions (strain, nucleotide state, stabilizing agents) will be investigated using the improved combined atomic force/fluorescence microscope.
Further along the timeline, the mechanism to stretch the substrate will be improved to eventually allow in-situ straining while observing with the high-speed atomic force microscope. This will require a complex tracking algorithm to consistently follow the region of interest during strain application.
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Research Products
(3 results)
