Methods used in the lab
In-situ background estimation in fluorescence imaging
Estimating the background of a ROI using its own fluorescence data.
Fluorescence imaging of bulk-stained tissue is a popular technique for monitoring the activities in a large population of cells. However, a precise quantification of such experiments is often compromised by an ambiguity of background estimation. Although, in single-cell-staining experiments, background can be measured from a neighboring nonstained region, such a region often does not exist in bulk-stained tissue.
In contrast to previous methods, we determined the background of a given region of interest (ROI) using the information contained in the temporal dynamics of its individual pixels. Since no information outside the ROI is needed, the method can be used regardless of the staining profile in the surrounding tissue. Moreover, we extend the method to deal with background inhomogeneities within a single ROI, a problem not yet solved by any of the currently available tools.
Demonstration routine for MATLAB
The file contains three m-files. bg_est_demo.m is the main demonstration routine. It simulates intracellular calcium traces for a certain region of interest (via f_generate.m), adds a known constant background value and random noise to all pixels, then calls bg_est_release.m to estimate the background.
For more information:
Chen et al. (2006)