Using photo-instability to quantify fluorophores and achieve super-resolution imaging

single molecule;acetylcholine receptors;bungarotoxin;photobleaching;super-resolution;image analysis;microtubules;Deoxyribonucleic Acid (DNA);photobleaching and intermittency localization microscopy (PhILM);transient labeling

Simonson, Paul D.

关键词: single molecule;    acetylcholine receptors;    bungarotoxin;    photobleaching;    super-resolution;    image analysis;    microtubules;    Deoxyribonucleic Acid (DNA);    photobleaching and intermittency localization microscopy (PhILM);    transient labeling;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/18590/Simonson_Paul.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

This dissertation presents techniques for localizing and quantifying fluorophores in biological imaging applications.Fluorophore photobleaching, blinking, binding, etc., is exploited to quantify and localize single fluorophores, even when their fluorescent images overlap those of nearby fluorophores.In the first technique, we image single, membrane-bound receptors labeled with fluorophores and count the stepwise drops in fluorescence intensity to determine the number of ligand binding sites.Results from single α7 and neuromuscular junction nicotinic acetylcholine receptors in mammalian cell membranes are shown.The results indicate that there are two bungarotoxin binding sites in neuromuscular junction (NMJ) receptors, as expected, and five in α7 receptors, clarifying previous uncertainty.The other techniques are associated with super resolution imaging.Super-resolution imaging is achieved by localizing diffraction-limited spots corresponding to single fluorophores with high accuracy.In photobleaching and intermittency localization microscopy (PhILM), fluorophore transitions between dark and bright states (compatible with binding, photobleaching, photo-activation, blinking, etc.) are localized.We show that standard photobleaching and blinking movies can be used to create super-resolution images.We also explain how PhILM can be combined with another technique to image chromosomal DNA inside cells.In PAINT (point accumulation for imaging in nanoscale topography), the accumulated, stochastic binding events of fluorescent labels to an imaging target are localized.Combining PhILM and PAINT results in a robust microscopy that is faster than PAINT alone, requires less optimization, and corrects for cell autofluorescence.We used nanomolar concentrations of SYTO (which shows >40x fluorescence enhancement upon binding to DNA) to image chromosomal DNA in fixed cells.We found an average single-fluorophore localization error of 24 nm.We similarly imaged microtubules using fluorescent paclitaxel and streptavidin-based labeling to find 10 and 18 nm errors, respectively.Future work will involve simultaneous imaging of DNA, microtubules, and other proteins to answer important biological questions.