Photoactivation And Photobleaching Properties Of Optical Highlighter Fluorescent Proteins

Optical microscopy is an essential tool for life science research. However due to the diffraction of light, optical microscopes are not capable of imaging biological smaller than 200 nm. The recently reported super resolution microscopic imaging techniques have break though this limit and achieved resolution up to 20 nm. One type of these techniques is Photoacitvated Localization Microscopy (PALM), this technique includes new activation and imaging light path, highly sensitive imaging camera, reconstruction algorithm and a newly reported fluorescent tags—the optical highlighter fluorescent proteins. These proteins experience significant spectral variance under the activation of high energy light. PALM systemwith favorable performance relies on optical highlighter fluorescent proteins with optimized activation features, yet these features are important references for selecting and properly controlling the activation process in practical imaging experiments. However, the study of activation behavior of the optical highlighter fluorescent proteins is significanly affected by the activation condition, this results in the diverse outcomes of activation experiments for a single protein, thus a proper parameter for profiling the activation characteristics of optical highlighter fluorescent proteins is still lacking, hence the absence of instruction for protein selecting and modulation in practical imaging experiments.Based on the characterization method of the photobleaching features of fluorescent proteins, we introduced and applied the parameters of intensity dependent photoactivation and photobleaching rates for characterizing the activation behavior of optical highlighter fluorescent proteins. By this method, we measured and explained the differences of activation rates between these proteins by the photoactivation mechanism. Moreover, by introducing the light dose concept from photodynamic therapy, we obtained the light dose dependent fluorescent expression and calculated the respective florescent curves of these proteins. At last, by studying the activation properties of the optical highlighter fluorescent protein under different activation intensities, we optimized the activation modulation of PALM imaging, the activation time is reduced by 50% without deteriorating the imaging quality.The main results of this thesis are:(1) Introduced the intensity dependent photoactivation and photobleaching rates for characterizing the activation properties of optical highlighter fluorescent proteins. Based on the characterization of the photobleaching rate of the photobleaching research, considered the effect of the primary factor of the activation imaging experiments: activation light intensity on the activation behavior of optical highlighter fluorescent proteins, this thesis introduced a new parameter of intensity dependent photoactivation and photobleaching rate, which is able to profile the inherent nature of optical highlighter fluorescent proteins under photoactivation. The experimentally obtained the intensity dependent photoactivation and photobleaching rates of four typical optical highlighter fluorescent proteins indicates that, the intensity dependent activation rates differs due to the divergences of activation mechanisms of different proteins.(2) Introduced the light dose fluorescence curve for evaluating the photoactivation properties of optical highlighter fluorescent proteins directly and quantitatively, and for straightforward referencing during the experimental photoactivation process. By experiments and theoretical calculation, we obtained the equation of the fluorescence of the optical highlighter fluorescent proteins with the photoactivation light dose during the activation process, discussed the impacts of the imaging condition and the physiological environment on the characterization of photoactivation features of optical highlighter fluorescent proteins, observed the significant difference of photoactivation contrast between the purified and physiological condition.(3) Experimentally evaluated the potential of applying the intensity dependent photoactivation rate to optimize the photoactivation process thus to increase the temporal resolution of the PALM imaging. We then extended this idea to general photoactivation fluorescent imaging techniques and achieved the design and modulation of the photoactivation process of photoactivation fluorescent imaging.