Study On STED Super-resolution Imaging Of Mitochondrial Membrane Using A New Cyanine Dye

Optical microscopy has been as an important imaging tool in biomedical research for a long time,due to its marked advantages of non-contact and non-damage.Since 1873,the highest imaging resolution of optical microscopes was defined to beca.200 nm because of Ernst Abbe's optical diffraction limit;the biological targets less than 200 nm in size cannot be clearly resolved.With the emergence of super resolution imaging microscopy,the prominent achievement has been made in the field of optical imaging microscopy in this century,which can be used as a powerful tool for biological researching.Super resolution optical imaging(Super-resolution Optical Microscopy)technology is an advanced imaging technique capable of overcoming the optical diffraction limit.Two major strategies are employed to achieve the current super resolution imaging: PSF engineering imaging method,such as Stimulated Emission of radiation Microscopy(Stimulated Emission Depletion Microscopy,STED);and single molecule localized microscopy such as light activated Localization Microscopy(Photo activation Localization Microscopy,PALM)and Stochastically Optical Reconstruction Imaging Microscopy(STORM)etc.Mitochondrial membrane,especially inner mitochondrial membrane plays a critical role in physiological processes of live cell mitochondria,which is meaningful to study the microstructure and function of mitochondrial membrane.Fluorescent imaging has recently received a lot of research interests from various fields because of its high sensitivity,simple processing and low cost etc.And fluorescent dyes are considered as a key factor in biological imaging.Conventional fluorescent dyes,such as Rhodamine123 and JCT-1 etc.,are proved to be mitochondrial matrix targeting probes owing to their molecular structure and charge distribution.However,the dyes with long emission wavelength selectively targeting for mitochondrial membrane and concurrently capable of super-resolution imaging are rarely reported,which is great in demandto develop novel fluorescent probes for mitochondrial membrane imaging allowing super resolution imaging study.Cyanine dyes(Cy5,Alexa-647 etc.)as a long wavelength dyes [2] are usually thought to use for STORM super-resolution imaging.However,such dyes have some disadvantages need to be overcome,such as poor photo stability and low fluorescence quantum yield etc.Comparably,squaraine dyes is a kind of cyanine dyes with typical spectral properties,but electrical zwitterion in structure and poor water solubility,which lack of live cell imaging specificities.Therefore,the novel fluorescent dyes with improved optical behaviors should be developed for mitochondrial membrane targeting and super-resolution imaging.In present dissertation,we hereafter mainly focused on the construction of a STED super-resolution imaging microscope,the development of a new type of fluorescent probe 1 for mitochondrial orientation and its STED super-resolution imaging for mitochondrial membrane,involved in three points as follows:1.Construction of STED super-resolution microscope,and verification of optical system.Key parameter calibration of STED system by applying commercially fluorescent micro particles and commercial STED systems,respectively.2.Synthesis of cyanine dyes(hereinafter called probe 1),and spectral investigations,including in absorption,emission spectra,fluorescence quantum yield,cell staining experiments,etc.3.Study of fluorescent imaging of mitochondrial membrane and its STED super-resolution imaging.The innovation of the master thesis mainly includes the following two points:1.A new cyanine dye based fluorescent probe capable of labeling vicinal dithiol proteins was developed to demonstrate much better binding specificity to the mitochondrial membrane than that of the Mito-tracker(Rhodanmine 123)commercial available.2.A set of STED super-resolution imaging system was constructed to achieve the super resolution imaging of the probe in the mitochondrial membrane of RAW-7 cells,by which the resolution was improved markedly,compared to that of the confocal laser imaging.