Efficient Analysis of Single Molecule Spectroscopic Data via MATLAB
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Efficient Analysis of Single Molecule Spectroscopic Data via MATLAB Caleb M. Hill and Shanlin Pan The University of Alabama, Box 870336, Tuscaloosa, AL 35487 ABSTRACT A MATLAB program is developed to help analyze single molecule spectroscopic data obtained with a standard inverted optical microscope. The described software has provisions to mitigate the effects of high background signals present in such data sets and greatly streamlines their analysis. Efficient single molecule image analysis and statistical blinking analysis are enabled with these programs to support single molecule imaging in an inverted configuration. INTRODUCTION Single molecule fluorescence spectroscopy has emerged as a powerful tool for fundamental photophysical studies and visualizing single molecule recognition events in biological systems. This is due to its ability to probe heterogeneities present at the single molecule level in spectra [1-3], electrochemical activity [4-7], and other kinetic processes [8-11] on the ms to s timescale that are usually obscured in conventional ensemble averaging measurements. As a result, much more data typically needs to be acquired to reach statistically significant conclusions in the single molecule regime. For single molecule kinetic studies, this often means hundreds upon hundreds of trajectories. With a high quantum efficiency chargecoupled device (CCD) camera coupled to an optical microscope, this amount of information can be acquired in under an hour; the data analysis is often the bottleneck in carrying out these studies. A total internal reflection optical configuration is frequently employed for imaging single molecule fluorescence due to its minimized background fluorescence/scattering. However, a traditional inverted optical microscope in an epifluorescence configuration can be also be used, provided the background fluorescence/scattering can be adequately compensated for. Although there already exists many software tools available for high-throughput image analysis [12], most are designed for the analysis of still frame images and do not include considerations for efficiently recording and analyzing the time evolution of hundreds of features as required for single molecule kinetic studies. Outlined here are MATLAB programs (which do not utilize any optional add-ins such as the Image Processing Toolbox) designed to assist with this, and their application is demonstrated for example single-molecule data sets obtained with a standard inverted optical microscope for investigating the photophysical characteristics and charge transfer dynamics of single molecules. EXPERIMENT The example data presented here were obtained from samples of poly [2-methoxy-5-(2ethylhexyloxy)-1, 4-phenylenevinylene] (MEH-PPV) prepared by spincoating a dilute solution in CHCl3 (5 nM) onto a microscope coverglass substrate at 1000 RPM. This was followed by spincoating with a 1 mg/mL solution of poly [methyl methacrylate] (PMMA) in acetone. Samples were illuminated with 488 nm laser excitation at 190 W/cm2 through a 100x, NA=
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