An automated method to quantify and visualize colocalized fluorescent signals.
The most commonly used method to analyze colocalization of fluorescent signal in paired images is based on superimposition of images ("merging") and visual inspection. A method based on the comparison of the mean deviation of fluorescent signal intensity has recently been proposed to quantify colocalization within a user-defined area [Li Q, Lau A, Morris TJ, Guo L, Fordyce CB, Stanley EF. A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization. J Neurosci 2004;24:4070-81]. Unfortunately, the latter quantification method does not provide a spatial representation of the correlation between the two fluorescent signals. Here we propose a new method that combines quantification and imaging of colocalization. We describe an algorithm based on edge detection and calculation of signal intensity deviation. The method is illustrated and validated on both simulated images and experimental data. This new and automated method calculates a correlation index (I(corr)) and generates an image of the correlated signals from the two original images. In addition to help in comparing and quantifying colocalization between two fluorescent stainings, this method can be adapted to measure the distribution of ions, proteins, organelles and cells in a large array of techniques.
Jaskolski
Frédéric
F
Laboratoire Physiologie Cellulaire de la Synapse, CNRS UMR 5091, Institut François Magendie, Université Bordeaux 2, Rue C. Saint-Saëns, 33077 Bordeaux Cedex, France. fjaskol@pcs.u-bordeaux2.fr
Mulle
Christophe
C
Manzoni
Olivier J
OJ
D000465:Algorithms; D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D005456:Fluorescent Dyes; D007090:Image Interpretation, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D009482:Neurophysiology / Q000295:instrumentation / Q000379:methods*; D055095:Optics and Photonics / Q000295:instrumentation
D005456:Fluorescent Dyes