STORM Super-resolution Imaging Study Of Mitochondrial Dynamic Processes In Live Cells

Optical microscopy,due to its capability in high-contrast imaging,has been showing extremely of significance contributions to various scientific areas,in particular,it plays critical roles in biomedical fields for the biological diagnosis and treatment because of the noninvasive imaging in vivo,which has attracted increasing interests from the fields of biology and biomedicine.In the past decade,with the emergence of super-resolution microscopy,the last barrier of optical diffraction limit in optical microscopy has been overcome with the imaging spatial resolution up to 20 nm.The strategies depending on different ways of “bypassing” the optical diffraction limit can be mainly categorized into three types: First,the point spread function(PSF)engineering based spatial optical modulation technique,e.g.Stimulated Emission Depletion Microscopy(STED)was initially proposed by Stefan Hell in 1994;Second,Optical frequency domain modulation based superresolution microscopy,such as(Saturated)Structural Illumination Microscopy(SSIM/SIM);Third,Single Molecule Localization Microscopy(SMLM)based superresolution microscopy.In SMLM,the representative microscopies are Photoactivated localization microscopy(PALM)and Stochastic Optical Reconstruction Imaging Microscopy(STORM),which were developed by Shroff and Zhuang in 2006,respectively.Especially for STORM,biological specimen is firstly labeled with fluorescent molecule with stochastically photo-switching under laser illumination.And the photoblinking frames are input into reconstruction algorithm to provide superresolution images with high spatial resolutions.STORM has proved to be a promising tool with the typical features of high imaging resolution,flexible probe design and biological specimen preparations.Mitochondrion,known as the powerhouse of eukaryotic cells for long time,is highly dynamic organelles inside cells.And it plays great roles in maintaining the balance of intracellular ions,synthetic precursors for biomacromolecules,such as lipids,proteins and DNA,etc.,and functions to scavenge metabolic byproducts including ammonias or reactive oxygen species(O2-,1O2,H2O2,and so on).Additionally,mitochondria take part in the modulations in the overall signaling pathway and stress responses.Dynamic changes of mitochondrial morphology and spatial distributions including mitochondrial fusion,division,and tubule interconnections are thought to interfere a lot with biological behaviors of the whole cells,especially leading to metabolic diseases,cancer development,neurodegeneration and aging,etc.The insights into mitochondrial structures are helpful for the deep interpretation of the biological functions.Although traditional optical microscopies have shown the great potential in disclosure of the mitochondrial structural details,they encounter the embarrassment in the imaging of mitochondrial sub-structures beyond optical diffraction limit.Therefore,superresolution imaging of mitochondrial dynamics is of great significance to reveal how mitochondria function in the promotion of human health and occurrence of diseases.STORM is promising for the visualizing ultra-fine mitochondrial structures.In this dissertation,we mainly focus on the study of mitochondrial dynamics using STORM microscopy with the detailed items as follows:1.To improve the STORM superresolution imaging,we developed a novel Cy5 dye for STORM imaging of mitochondria in living cells.The optical behaviors of the new probe in solution involved in absorption and emission spectra,fluorescent blinking performance and photostability were studied and the selective labeling of mitochondria was further verified by fluorescent colocalization experiment.2.An NC-PCA de-noising-K-factor algorithms were developed to pre-process the raw data of photoblinking before being reconstructed by compressed sensing STORM algorithm.The NC-PCA method was able to reduce the noise signals,and the k-factor algorithm was applied to sharpen the image,which can further improve the positioning accuracy based existing localization methods.3.Upon being imaged by above Cy5 probe on home-built STORM equipment,STORM superresolution imaging of dynamics of mitochondria in live cells was achieved to show ultra-fine biological behaviors using pre-processing algorithm of NC-PCA-K-factors.The innovation involved in this thesis are itemed as follows:1.A disulfide-conjugated Cy5 dye was designed for spontaneous in situ photoblinking activation triggered by disulfide reduction inside cells without addition of imaging buffer or extra thiols.The probe was modified to orient preferentially to the mitochondrial membrane,where it formed covalent bonds with cysteine residues on membrane proteins.And it has more excellent results in live cell applications than Alexa 647.2.An NC-PCA algorithms were developed to pre-process the STORM imaging raw data to enhance spatial-resolutions,which successfully improved the positioning accuracy based on the CSSTORM algorithm.3.Using the novel Cy5 probe and pre-processing with the NC-PCA-K-factor algorithms,long-term superresolution imaging of dynamics of mitochondrial outer membrane in live cells was acquired to provide the deep insight into mitochondrial bio-functions.