Articulo
Confined displacement algorithm determines true and random colocalization in fluorescence microscopy
JOURNAL OF MICROSCOPY (PRINT);
J. Microsc..
Registro en:
0
D07I1019
D07I1019
WOS:000280994900001
0022-2720
Autor
Couve-Correa, Andrés
Rojas-Moraleda, Rodrigo
Hartel-Grundler, Steffen
García-Acevedo, Alejandra
Ramírez, O.
Institución
Resumen
P>The quantification of colocalizing signals in multichannel fluorescence microscopy images depends on the reliable segmentation of corresponding regions of interest, on the selection of appropriate colocalization coefficients, and on a robust statistical criterion to discriminate true from random colocalization. Here, we introduce a confined displacement algorithm based on image correlation spectroscopy in combination with Manders colocalization coefficients M1(ROI) and M2(ROI) to quantify true and random colocalization of a given florescence pattern. We show that existing algorithms based on block scrambling exaggerate the randomization of fluorescent patterns with resulting inappropriately narrow probability density functions and false significance of true colocalization in terms of p values. We further confine our approach to subcellular compartments and show that true and random colocalization can be analysed for model dendrites and for GABA(B) receptor subunits GABA(B)R1/2 in cultured hippocampal neurons. Together, we demonstrate that the confined displacement algorithm detects true colocalization of specific fluorescence patterns down to subcellular levels. The authors thank D. Diaz-Espinoza and M. Osorio-Reich for the ImageJ plugin, N. Contreras from Area Kreativa for support with the figures and J. Cowan for insightful comments on the paper. SCIAN-Lab is a selected member of the German-Chilean Center of Excellence Initiative for Medical Informatics (DAAD). Research in SCIAN-Lab (SH) is funded by FONDECYT 1090246, FONDEF D07I1019 and PBCT ACT47 (CONICYT, Chile). A.G. is funded by D07I1019. O.R. is funded by FONDECYT 1090246. A.C. is funded by FONDECYT 1071001. S.H. and A.C. are members of NEMO, ICM-P04-068-F and the Advanced Imaging & Bioinformatics Initiative (AIBI) (www.aibi.cl). 19 FONDEF shartel@med.uchile.cl FONDECYT [1090246, 1071001]; FONDEF [D07I1019]; PBCT [ACT47] 3 FONDEF 239