The Developdment Of RFP And Super-resolution Imaging

The development and application of super-resolution imaging made it possible todefine the accurate location of biological molecules at nanometer level, andsuper-resolution technology has become hot issues in recent years in the field ofimaging. Fluorescent proteins have become indispensable imaging tools for biomedicalresearch. Fluorescent proteins can be used to observe the activity of the cells, to labelprotein expression and also can be used in the proteomics experiments.The existingfluorescent proteins include not only green fluorescent proteins but also red， yellow,and orange fluorescent proteins, which have proven to be excellent tools for live-cellimaging.The red fluorescent protein DsRed was first cloned from corals and can emitred fluorescence under the UV irradiation. Although red fluorescent protein DsRedhave been widely used, there are some suboptimal key characteristics such asoligomerization state, maturation rate and cytotoxicity which impeded theirapplication.So the development and optimization of red fluorescent proteins isnecessary.In the second chapter, based on the commomly used RFP in the field ofbio-imaging and biomedical: mCherry, mOrange, rsTagRFP, mOrange2, mKate2andmApple we constructed the prokaryotic expression plasmid and transformed into E.coli for expression and purification.To test the oligomerization state of these RFPs weuse the sedimentation velocity to analyze the purified proteins, to confirm the resultwe choose the method of sedimentation equilibrium by which we can get the Kd ofthese RFPs then inferred the oligomerization state of these RFPs. According to theresult of both sedimentation velocity and sedimentation equilibrium we get thedetermined oligomerization state of RFPs: mCherry, mOrange, rsTagRFP, mOrange2,mKate2and mApple.In the third chapter, according to the oligomerization state of RFP we got from thechapter two, we fused these RFP with cell membrane protein Orai1and tried todetermine their optical properties by confocal microscopy imaging. According to theimaging result we found the strange optical property of Orai1-mCherry which is not inaccordance with its oligomerization state. Further imaging experiments demonstratedthat some clusters showed in lysosomal then we excluded the possibility of autophagyby colocalization Orai1-mCherry with lysosomal marker. After comparing the aminosequence of mOrange2and mCherry we desigined some mutants based on the deference. With confocal microscopy imaging we defined the optical properties of themutant Orai1-mCherry and finally get one mutant which has good optical properties.In the fourth chapter, we focused on the study of Super-resolution microscopy.First of all by fusing mEos3.1and mEos3.2with β-actin and α-actin we studied thepossibility of them being used in the sigal molecular imaging.We reconstructed theresult after them being imaged by (F)PALM/STORM and found that they can be usedas a new tool of signal molecular imaging with a better result than the common usedfluorescent protein.Thus we provided a new kind of imaging tool for super resulotion.Besides we studied the buffer used in STORM and tried to replace into ascorbic acid(AA). AA is a very commom chemical and it is intoxic. We replaced STORM bufferinto AA and try to do STORM experiment under the same condition and got a goodresult.