The advent of technology that allows the detection of single molecules in a cellular environment holds the promise that significant new insights can be gained into molecular processes that occur in a cell. While the promise is great so are the technical problems. This is primarily because the signal emitted by a single molecule is low in comparison to the significant noise sources in the sample and in the detection system. Therefore any quantitative analysis of single molecule experiments is challenging and requires carefully developed tools. The overall goal of this proposal is to add to the available methods for the analysis of single molecule experiments. In particular, we address fundamental problems using methods that are novel to single molecule microscopy but have been successfully used in other engineering disciplines. We will analyze performance limits of single molecule microscopy from a modern point of view.
Our specific aims are: 1.) To develop novel analytical tools to investigate the accuracy with which single molecules can be localized. 2.) To investigate existing algorithms and to develop novel algorithms for the localization of single molecules. 3.) To develop novel resolution criteria for single molecule detection and algorithms for the determination of the distance between two single molecules. 4.) To develop software modules for our existing Matlab based microscope image processing toolbox. We will use approaches based on the Fisher information matrix to obtain novel expressions for the accuracy with which a single molecule can be localized and for the accuracy with which the distance between two single molecules can be measured. Of particular importance is the determination to what extent experimental conditions such as cellular background and noise in the detection system deteriorate the localization and resolution accuracy. Existing algorithms will be evaluated to establish how close their performance is to the performance limit. Novel algorithms will be designed to meet the performance measures. A further criterion will be to assess the sensitivity of the algorithms to experimental artifacts such as outliers in the data. All proposed methods will be tested on immunological systems.
| Ward, E Sally; Ober, Raimund J (2018) Targeting FcRn to Generate Antibody-Based Therapeutics. Trends Pharmacol Sci 39:892-904 |
| Ward, E Sally; Devanaboyina, Siva Charan; Ober, Raimund J (2015) Targeting FcRn for the modulation of antibody dynamics. Mol Immunol 67:131-41 |
| Chao, Jerry; Ram, Sripad; Ward, E Sally et al. (2013) Two approximations for the geometric model of signal amplification in an electron-multiplying charge-coupled device detector. Proc SPIE Int Soc Opt Eng 8589:858905 |
| Chao, Jerry; Ward, E Sally; Ober, Raimund J (2012) Fisher information matrix for branching processes with application to electron-multiplying charge-coupled devices. Multidimens Syst Signal Process 23:349-379 |
| Chao, Jerry; Ward, E Sally; Ober, Raimund J (2012) Localization accuracy in single molecule microscopy using electron-multiplying charge-coupled device cameras. Proc SPIE Int Soc Opt Eng 8227: |
| Wong, Yau; Lin, Zhiping; Ober, Raimund J (2011) Limit of the Accuracy of Parameter Estimation for Moving Single Molecules Imaged by Fluorescence Microscopy. IEEE Trans Signal Process 59:895-911 |
| Chao, Jerry; Ward, E Sally; Ober, Raimund J (2010) FISHER INFORMATION FOR EMCCD IMAGING WITH APPLICATION TO SINGLE MOLECULE MICROSCOPY. Conf Rec Asilomar Conf Signals Syst Comput :1085-1089 |
| Chao, Jerry; Ward, E Sally; Ober, Raimund J (2010) A software framework for the analysis of complex microscopy image data. IEEE Trans Inf Technol Biomed 14:1075-87 |
| Abraham, Anish V; Ram, Sripad; Chao, Jerry et al. (2010) Comparison of estimation algorithms in single-molecule localization. Proc SPIE Int Soc Opt Eng 7570:757004 |
| Ram, Sripad; Prabhat, Prashant; Ward, E Sally et al. (2009) Improved single particle localization accuracy with dual objective multifocal plane microscopy. Opt Express 17:6881-98 |
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