Quantification of colocalization and cross-talk based on spectral angles
Speaker: Carolina Wahlby , Broad Institute
Date: February 25 2010
Time: 3:00PM to 4:00PM
Location: 32-D507
Host: Polina Golland, CSAIL
Contact: Polina Golland, x38005, polina@csail.mit.edu
Relevant URL: Highly specific staining methods and fluorescent biological markers
emitting light at different wavelengths together with fluorescence
microscopy allow for detailed studies of the spatial distribution,
localization, and interaction of biomolecules.
Quantification of colocalization of biomolecules includes not only
calculating a global measure of the degree of colocalization within an
image, but also a classification of each image pixel as showing
colocalized signals or not.
Common methods for automated quantification of colocalization require
images where cross-talk has been eliminated by pre-processing, as they are
based on intensity thresholding. Such pre-processing typically requires
manual input.
I will present a novel, automated method for quantification of
colocalization and classification of image pixels based on hue rather than
intensity. The hue distribution is presented as an angle histogram created
by a series of steps that compensate for the quantization noise always
present in digital image data. Classification rules are thereafter based on
the shape of the angle histogram, and detection of colocalized signals is
thus only dependent on hue, making it possible to classify also
low-intensity objects in noisy images, and decouple image segmentation from
detection of colocalization.
Cross-talk will show up as shifts of the peaks of the histogram, and thus a
shift of the classification rules, making the method essentially
insensitive to cross-talk. The method can also be used to quantify and
compensate for crosstalk, independent of the microscope hardware.
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