2015 Fiscal Year Research-status Report
Hybrid Raman-bioluminescence imaging for the study of proteome-metabolome interaction
| Project/Area Number |
15K20950
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| Research Institution | The University of Tokyo |
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Principal Investigator |
邱 亮達 東京大学, 理学(系)研究科(研究院), 助教 (80648220)
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| Project Period (FY) |
2015-04-01 – 2017-03-31
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| Keywords | Metabolome analysis / Raman spectroscopy / Anti-Stokes fluorescence / Differentiation / メタボローム解析 / ラマン分光法 / アンチストークス蛍光 / 分化 |
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| Outline of Annual Research Achievements |
The purpose of this proposal was to establish a platform for the study of proteome-metabolome interaction directly inside living cells. Here, I am glad to report that a significant part of the major goal has already been achieved in the first-year term.
Now, I have completed not only the hardware for the hybrid fluorescence-Raman imaging system, but also the data analysis software to interpret the hybrid spectral image data together with my collaborators. In the application part, I successfully applied the hybrid imaging system for the chemical analysis of individual organelles in living HeLa cells, and the analysis of subtle chemical changes in embryonic stem cells between the presence and abscence of Oct4 expression during differentiation. The stem cells study specifically demonstrated how the hybrid imaging setup can elucidate the relationship between the metabolome profile and the protein expression pattern of a cell. The manuscript of the study will be submitted soon, with myself as a co-corresponding author.
In addition to the upcoming paper submission, I have already presented the work in an international conference (Focus of Microscopy 2016) and has started several collaboration projects based on the hybrid imaging setup. The next step of the project is to change the incidence wavelength for the implementation of bioluminescence detection, so that we can overcome the photobleaching problem of the fluorescence signal during the detection.
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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
In terms of the major goal of the proposal, i.e. visualising proteome-metabolome interaction, the progress is actually much better than initially planned.
However, one unexpected problem that occured during the study was that the combination of bioluminescence and Raman signal detection was more difficult than previously expected, because I did not expect that the bioluminescence substrate would be photobleached by the Raman excitation laser, such that no bioluminescence signal can be detected together with Raman scattering signals.
Although now I have partly overcame the issue by using fluorescence instead of bioluminescence for proteome detection, bioluminescence still has its advantage in its photobleach-free nature, so next I will focus on finding the method for bioluminescence implementation to the hybrid imaging system.
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| Strategy for Future Research Activity |
The major remaining task for this project is to achieve the simultaneous detection of bioluminescence and Raman signals for hybrid bioluminescence-Raman imaging. Although the major goal of the proposal has been achieved by using fluorescence instead of bioluminescence, still, bioluminescence has advantages in terms of its photobleach-free nature, so it is more suitable for time-lapse detection.
The plan for the implementation of simultaneous bioluminescence-Raman detection is to change the excitation laser from 532 nm to wavelengths above 632 nm. By increasing the spectral gap between the luminescence emission spectrum and excitation laser, I hope to overcome the photobleaching problem of the bioluminescence substrate.
If the hybrid bioluminescence-Raman imaging system can be established, I will challenge the time-lapse visualisation of chemical state fluction according to protein expression pattern in living cells. Even if the hybrid bioluminescence-Raman imaging system cannot be realised, I am already having a lot of collaborators that hopes to use the hybrid fluorescence-Raman imaging system to tackle biological problems, and I expect that in the end the project will have a higher impact than originally expected.
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