Characterization Of Photophysical Properties Of Photoactivatable Fluorescent Proteins For Super-resolution Microscopy And Studies On The Mapping From Chemotaxis Signaling To Swimming Behavior Of Escherichia Coli

Since the 16th century,microscopy has been widely developed to explore the mysteries of microscopic worlds.With the increase of resolution,various kinds of microscopes have been applied to different areas,especially in scientific studies.For the observation of biological microstructures,the noninvasive light microscopes are more preferred due to the relatively slight damage to the sample.In recent years,the developments of fluorescent probes,either small-molecule dyes or fluorescent proteins,have provided a variety of methods to resolve the biological microstructures by imaging.Moreover,photoswitchable fluorophores and proteins have been extensively used for ultra-structural imaging to break the resolution limit set by the diffraction of light,especially in photoactivated localization microscopy(PALM)and stochastic optical reconstruction microscopy(STORM).In this article,the fluorescence microscopy was combined with other experimental techniques to observe a typical prokaryote,Escherichia coli(E.coli).E.coli,which is a kind of enterobacteria,has been widely studied with a series of advantages,such as fast reproduction,easy cultivation and low pathogenicity.The first chapter is an introduction of the background,motive and objective of this article as well as the relevant studies.Two main study subjects were E.coli and photoactivatable fluorescent proteins(PA-FPs),which were studied with PALM experiments.Therefore,we detailedly described the origin and development of fluorescent proteins and super-resolution microscopy,as well as the physiological and motile characteristics of E.coli.In the second chapter,we briefly summarized the experimental methods used in this article.First,we introduced the purpose,preparation process and storage conditions of the experimental reagents.Next,we described the preparatory work before data acquisition,including the principles and procedures of constructing and transforming recombinant plasmids,as well as the routine cell culture of E.coli.The last part of this chapter is the cleaning process of the coverslips and slides which hold the cell samples.We described the first research work in the third chapter.By fusing Dendra2,a common type of PA-FPs,to the motor protein FliM and FliG of E.coli,we recorded the intracellular fluorescence frame by frame,and extracted the time trace of emission intensity of a single fused Dendra2 using several criteria.From the distribution of the characteristic times,the kinetic rates and other parameters in the model of Dendra2 photophysics could be obtained.The blinking properties of Dendra2 in fixed E.coli cells were appreciably different from those in vitro,confiming the potential effects from sample preparation and other experimental conditions.This sensitive dependence of the photophysical properties of PA-FPs on the experimental conditions highlight the importance of characterizing the PA-FP properties under the same experimental conditions as the subsequent PALM imaging or molecular counting experiments.Our simple strategy developed here could be applied to that.The fourth chapter is a description of the second research work in this aritcle.In prior studies,the mechanism of bacterial chemotaxis has been mainly elucidated at the single motor level.We aimed to characterize the missing links between the intracellular signal response and the terminal output of the chemotaxis network,that is,the swimming behaviors of E.coli cells.By constructing an instrument combining optical tweezers,brightfield and fluorescence microscopy,and a sealed chamber,the swimming behavior of an individual E.coli could be recorded for a long time.Moreover,we fused a monomer EGFP molecule to the response regulator protein,CheY.The calibration from cell fluorescence to the intracellular concentration of Phospho-CheY(CheY-P)was established by immunoblotting and lyophilization with the same experimental conditions.By this means,we mapped the relationship between the intracellular concentration of CheY-P and the characteristics of swimming behaviors of individual E.coli cells.Our conclusions developed here would be helpful to understand the chemotaxis network globally.We also fitted our experimental data to the stochastic model,and the fitting parameters could be applied to numerical simulation in future studies.In the last chapter,we summarized the studies of this article,and discussed the possible future work.