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[Overview] [Experimental Setup] [Protocol] [Data Evaluation] [Examples] [Literature] |
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Further necessary corrections Laser fluctuations, acquisition photobleaching, and fluorescence loss during photobleaching leads to intensity changes during image acquisition. In order to obtain data with a linear relationship between the measured fluorescence intensity and the concentration of fluorescent molecules, the raw data has to be corrected for these changes. One straightforward possibility to do so is to divide the background subtracted fluorescent measurement by the total cell intensity at each time point. If this is not possible, e.g. when only a part of the cell can be imaged, alternative correction methods are available: Acquisition photobleaching can be corrected for by measurement of the fluorescence intensity of neighboring cells, in control experiments or the prebleach series. The fluorescence measurement at each timepoint can be divided by a function representing the acquisition photobleaching: y(n) = exp(-n/x) with n = image number can be easily determined by measuring the total fluorescence intensity of an unbleached neighboring cell or the gradual fluorescence decrease in the prebleach or postbleach images. Laser intensity fluctuations can be compensated for by dividing the fluorescence measurement at each timepoint by the corresponding value of the laser monitor diode or the averaged intensity of the transmission channel outside the cell (corrected for the nonzero offset of the diode or transmission detector, respectively). |
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contact: Stefan Terjung last update: 02/06/04 |