Spectral Angle Histogram - a Novel Image Analysis Tool for Quantification of Colocalization and Cross-talk
Milan Gavrilovic, Carolina Wählby, Joakim Lindblad, Ewert Bengtsson
Centre for Image Analysis, Uppsala University and Swedish University of Agricultural Sciences, Uppsala, Sweden
In fluorescence microscopy, when analyzing spectral components, it is common to record two (or more) greyscale images. Each greyscale image, referred to as a channel, corresponds to intensities in different wavelength intervals. If each pixel of a two-channel image is plotted in a space spanned by the two intensity channels a conventional scatter-plot is obtained. Single-coloured pixels are distributed along the axes, while colocalized pixels are distributed closer to the diagonal of the scatter-plot, and cross-talk (as well as noise) is observed as deviations of the single-coloured vectors from the axes. Detection of colocalized pixels is often based on a division of this 2D space into different regions by intensity thresholding. We have developed a method for reducing the scatter-plot to a 1D spectral angle histogram through a series of steps that compensate for the quantization noise which is always present in digital image data.
Using the spectral angle histogram, we can quantify colocalization in a fully automated and robust manner. As compared to previous methods for quantification of colocalization, this approach is insensitive to cross-talk. In fact, it can also be employed to quantify and compensate for cross-talk, using either linear unmixing or fuzzy classification by spectral angle, ensuring complete suppression of cross-talk with minimal loss of information. Recently we started investigating how the method can deal with autofluorescence. Initial tests on real image data show that the method may be useful for improved background suppression and amplification of the true signals.
The article “Quantification of colocalization and cross-talk based on spectral angles”, describing the method, is about to be published in the Journal of Microscopy. Authors have also filed a patent application “Pixel classification in image analysis” in 2008.