Construction Of A Miniaturized Absorption Spectrophotometer For Monitoring Fast Photoactivation Dynamics

The technology of supper resolution microscopy developed these years has broken through the resolution limitation caused by diffraction and become an essential tool to study the micro-world of biomedicine. Among them, a kind of approaches realized through single molecular localization is receiving intensive attention for its feature of cell viability long term maintenance and large field of view. However, this method relies heavily on the photoactivation performance of its probe. For example, as an important upgrade method, fast photoactivation localization microscopy need optical highlighter proteins with high photoactivation speed rates. The development of systems for quantifying photoactivation process is surely of great benefit for evaluating and screening the proteins for application. And the objective of this work exactly is to design and realize a compact fiber-optic absorption measure system which should have the ability of monitoring fast photoactivation process.To achieve the goal, the characters of optical highlighter proteins usually used was summarized and the requirements to the system on wavelength, accuracy, time resolution and so on was analyzed. Then, the core devices of the system was chosen followed the requirements and the system was constructed on a whole based on a miniature fiber spectrometer. The performance of the system was evaluated through the experiment with Dronpa as a representative then, after the optimization. The system can measure the absorption spectra changes of the optical highlighter proteins over time with a time resolution of 10ms and wavelength resolution of 3.8nm in the wavelength region of 440-600nm during the fast photoactivation process at an accuracy of 2.89×10-3. And the quantification results of the photoactivation characteristics achieved by the system is comparable both to the previous work with fluorescence-based and absorption-based methods.What is more, comparing to the fluorescence-based approach, the complex procedure of image processing is avoided and the system is much more simple and stable. Also, the time resolution of this system is much better than other absorption-based approaches and owing to its compact design using fiber, the system occupies much less space. The system would eventually become a powerful tool for the development of fast photoactivation localization microscopy.