Application of super-resolution imaging technique as an analytical tool

Abstract

Far-field microsocpy provide great advances to investigate structural and dynamic information of various sample system with low sample damage and high imaging contrast. Especially, Super-resolution technique is known as one of the innovative techniques in life sciences and material sciences because it enable to visualize nanoscopic structure of biological samples and materials. Since the sub-nanostructure of the sample determines their properties, developing the methods to analyze nanoscale structure directly is very important. Therefore, we try to establish a methodology to accurately measure biological molecules at a single unit level.<br/> A combination of DNA stretching method and super-resolution nanoscopy allows an accurate and precise measurement of the length of DNA fragments ranging widely in size from 117 bp to 23,130 bp. BstEII- and HindIII-treated λDNA fragments were stained with an intercalating dye and then linearly stretched on a coverslip by dynamic molecular combing. The image of individual DNA fragments was obtained by stimulated emission depletion nanoscopy. For DNA fragments longer than ~1,000 bp, the measured lengths of DNA fragments were consistently within ~0.5 to 1.0% of the reference values, raising the possibility of this method in a wide range of applications including facile detection for copy number variations and trinucleotide repeat disorder.<br/> Mitochondria play a crucial role in maintaining cellular energy metabolism and various cellular function. Moreover, mitochondrial dysfunction is closely related with cellular senescence that may ultimately contribute to aging. Since the sub-structure of mitochondria can reflect mitochondrial activity in various cellular environment, we apply super-resolution STED nanoscopy to show the morphological dispersion of mitochondria under the diffraction limit both in sub-cultured human primary skin fibroblasts and mouse skin tissues. In the sub-cultured fibroblasts, confocal microscopy provides overall morphology of mitochondrial membrane and indiscerptible TFAM location within diffraction limit. However, using super-resolution STED nanoscopy, we can resolve nanoscale distribution of TOM20 clusters and analyze quantitatively accurate number of nucleoids per cell in each sample condition. Similar results were shown for TOM20 distribution analysis in the mouse tissues. Our results suggested that the super-resolution technique based on fluorescence imaging can be useful to analyze structural difference of mitochondrial sub-structure in cells and tissues.<br/> We report a water-soluble turn-on type diarylethene SO3-BPDBTEO that reversibly switches on/off its fluorescence upon light stimuli. We note that two sodium sulfonate groups enable SO3-BPDBTEO to exhibit excellent photoswithching properties in aqueous condition over a wide pH range between 3 and 10. It is demonstrated that SO3-BPDBTEO can be applied for the conventional fluorescence imaging of mammalian cell line such as HeLa cells at various pH conditions with low cytotoxicity. We also examine its feasibility for super-resolution resolution fluorescence imaging as a photoswitching probe.<br/> Application of BALM (binding activated localization microcopy) was shown to allow facile imaging of amyloid fibrils with a typical diameter of ~14 nm FWHM. We also observed a twisted ribbon-like substructure of mutant amyloid fibrils and even what appear to be toxic amyloid oligomers with their characteristic morphological features consistent with TEM images. Use of an easily available staining dye in this method greatly enhances the prospect of addressing amyloid-related diseases in their diagnosis and drug tests by allowing facile in situ and in vivo detection by optical imaging.