2023 Fiscal Year Annual Research Report
Pump field probe magnetic field effect fluorescence microscopy for time-resolved radical pair detection in biological systems
| Project/Area Number |
23H01919
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| Allocation Type | Single-year Grants |
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| Research Institution | The University of Tokyo |
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Principal Investigator |
ウッドワード ジョナサン 東京大学, 大学院総合文化研究科, 教授 (80526054)
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| Project Period (FY) |
2023-04-01 – 2026-03-31
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| Keywords | Time-resolved imaging / Pulsed magnetic field / Fluorescence microscopy / Radical pairs / Photochemical kinetics |
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| Outline of Annual Research Achievements |
The main aim of the first year of this project was to build and optimize the Pump Field Probe microscope and develop the analysis methods needed to extract magnetic and kinetic parameters from the different pulse schemes. This was completed successfully.
In particular, the following achievements were made:
1) Construction and optimisation of an efficient single colour two pulse laser system. It was possible to achieve near complete sample excitation with laser pulses of 30ns. 2)Calibration of rapidly switched magnetic field magnitudes. A new measurement schemes was created to allow efficient calibration.3) Construction of custom made electronic hardware to precisely control and synchronise laser pulses, switched field pulses, electromagnetic field switching and camera control. This device controls the entire instrument. 4) Creation of software providing a wide array of different pulse programs and measurements scheme which controls the experimental hardware and has an easy to use user interface. The software has two components: one that runs on a control PC and the other which runs on the custom electronic control system.5) Complete theoretical modelling of the results of the different pulse programs for 3- and 4-state radical pair models.6) Detailed experimental measurements using the various pulse programs and full theoretical analysis for a range of flavin-based radical pair systems (each chosen to demonstrate particular characteristics), to compare directly with existing techniques and studies and highlight the advantages of this new instrument.
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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
All of the planned work for the first year of the project was completed on schedule and the PFP microscope is working very effectively and producing high quality experimental data with sensitivity exceeding expectations.
A sophisticated custom built control system was also constructed from scratch and has proven robust and simple to use across an extensive number of measurements. Detailed theoretical analysis of radical pair kinetic models have demonstrated the information that can be extracted from the different pulse schemes at a level of detail beyond any previous publications and reveal the versatility of the new technique.
An extensive and detailed characterisation of the instrument's capabilities and sensitivity has been completed and the journal article presenting this study is almost complete and ready for submission.
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| Strategy for Future Research Activity |
In the next stage of this project, the instrument will be directly applied to the study of photochemically generated radical pairs in living cells. Experimental schemes invoking optimised pulse programs will be tested and tuned to minimise the effects of photodegradation and to extract the most effective information for a single experiment on a single cell. In addition, for some measurements it will be possible to combine data from measurements on different individual cells and the limits of this approach will be investigated theoretically and experimentally.
Cell based measurements are planned using single cell approaches for HeLa and similar cell types and with multicellular measurements using E. coli, which will allow for multisampled data for hundreds of cells simultaneously.
We aim to design and build a new pulsed magnetic field circuit with integrated PCB based magnetic field coils to optimise the physical arrangement of the experiment and to increase the maximum magnetic field strength and also enable new measurements involving multiple magnetic field pulses. We have already designed and manufactured some preliminary coil prototypes.
We wish to add additional photoexcitation laser wavelengths to the instrument and also to test a new beam combination technique to simplify instrument alignment.
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