Quantitative Analysis of Single Molecule Microscopy

PROJECT SUMMARY ABSTRACTSingle molecule microscopy is a relatively novel technique that allows individual molecules to be imaged using the optical microscopeIt is of major interest to scientists because it enables the observation of cellular processes at the molecular levela task that is not achievable with classical optical imaging approaches due to the cellular processes being obscured by averaging and bulk effectsSingle molecule tracking and localization based superresolution microscopyPALM STORMare two of the main examples of single molecule imaging experimentsThe improvement of these techniques over classical imaging approaches is directly dependent on the accuracy with which the position of the single molecules in the sample can be estimatedThis leads to two major challengesOnethe design of experiments such that the best possible data can be obtainedTwothe analysis of the data in the most optimal fashionThe technical difficulties of single molecule experiments are to a large extent due to the very low photon count in the presence of significant noise sources such as scattered photons and camera readout noiseThe proposed project aims to implement algorithms to permit estimation tasks such as the localization of a molecule to be carried out with the best possible accuracyAn important part of the project is a Java based software framework for the analysis of single molecule experimental dataAt its core will be a carefully designed software architecture for the analysis algorithms that will allow in a streamlined fashion the incorporation of different models for the image profiles of single moleculesdifferent camera dependent data generation processesdifferent data pre processing stepsand different estimation algorithmsBy employing graphical processing unitsadvantage will be taken of the massively parallel structure of the overall data analysisExploiting our earlier information theoretic resultswe will also develop approaches and supporting software to allow the microscopist to analyze the impact of parameters such as exposure timeexcitation levelmagnificationand pixel sizeand by doing sodesign an experiment that will produce data that is optimized for the subsequent analysisThis will be complemented by methods to analyze the quality of the objective lensthe performance of dichroic filtersthe noise level of camerasetcThe specific aims are Aimto develop a single molecule data analysis platform including modules for single molecule tracking and single molecule superresolution microscopyand Aimto develop a framework for experimental design and instrument characterization in single molecule microscopyThe proposal originates from a spin out company of Texas Aandamp M University that seeks to further develop and commercialize single molecule methodologies developed over more than a decade PROJECT NARRATIVE Single molecule microscopy can reveal major new insights into biological processes at the molecular level in living cellssuch as the interactions of anticancer drugs with cancer cellsHoweverthis new method of microscopy requires complex analysis of the acquired image data to produce the desired resultsThe current proposal aims to provide new methods such as algorithms and software to accomplish this task