Method for Deconvolving Single-Molecule Intensity Distributions for Quantitative Biological Measurements

Introduction Fluorescence microscopy images of cells often contain puncta, or clusters of fluorescence signal. To measure the concentration of labeled proteins in a puncta, calibration typically needs to be performed for each fluorescence species. Traditional calibration techniques only provide average values rather than exact numbers which can lead to large uncertainties in the actual number of the measured proteins. This problem is especially acute when dealing with proteins that are of low to intermediate abundance. To count fluorescent molecules present at one or a few copies, one approach is to use sequential single-molecule photobleaching. However, it is difficult to apply this method to count molecules that are present at more than a few copies. The ability to count both the absolute number and the variation in the number of molecules present in these puncta will provide the opportunity to study a biological system at a level of detail that is inaccessible to traditional biochemical techniques. Technology description Researchers at the UW have invented a method for quantifying fluorescent puncta. This approach utilizes not only the average intensity value to calibrate fluorescence intensities, but the shape of the intensity distribution about the mean value to deconvolve the measured intensities. This method is effective for counting proteins or other molecules over a wide dynamic range and has the advantages of single-molecule measurements, providing both the mean and variation in molecules per puncta. Business opportunity The ability to measure precise quantitative information has great value in analytical research, particularly in systems biology and in the computational modeling of cellular function. Intellectual property position The UW has a US patent pending on this technology. Related Publication(s)
Mutch SA et al, Deconvolving single-molecule intensity distributions for quantitative microscopy measurements, Biophysical Journal. 2007 Apr 15; 92(8): 2926-43.

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