title: New algorithm to determine true colocalization in combination with image restoration and time-lapse confocal microscopy to MAP kinases in mitochondria.
abstract: The subcellular localization and physiological functions of biomolecules are closely related and thus it is crucial to precisely determine the distribution of different molecules inside the intracellular structures. This is frequently accomplished by fluorescence microscopy with well-characterized markers and posterior evaluation of the signal colocalization. Rigorous study of colocalization requires statistical analysis of the data, albeit yet no single technique has been established as a standard method. Indeed, the few methods currently available are only accurate in images with particular characteristics. Here, we introduce a new algorithm to automatically obtain the true colocalization between images that is suitable for a wide variety of biological situations. To proceed, the algorithm contemplates the individual contribution of each pixel's fluorescence intensity in a pair of images to the overall Pearsońs correlation and Manders' overlap coefficients. The accuracy and reliability of the algorithm was validated on both simulated and real images that reflected the characteristics of a range of biological samples. We used this algorithm in combination with image restoration by deconvolution and time-lapse confocal microscopy to address the localization of MEK1 in the mitochondria of different cell lines. Appraising the previously described behavior of Akt1 corroborated the reliability of the combined use of these techniques. Together, the present work provides a novel statistical approach to accurately and reliably determine the colocalization in a variety of biological images.
authors.affiliation: Centro de Microscopías Avanzadas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors.forename: Jorge Ignacio
authors.initials: JI
authors.lastname: Villalta
authors.forename: Soledad
authors.initials: S
authors.lastname: Galli
authors.forename: María Florencia
authors.initials: MF
authors.lastname: Iacaruso
authors.forename: Valeria Gabriela
authors.initials: VG
authors.lastname: Antico Arciuch
authors.forename: Juan José
authors.initials: JJ
authors.lastname: Poderoso
authors.forename: Elizabeth Andrea
authors.initials: EA
authors.lastname: Jares-Erijman
authors.forename: Lía Isabel
authors.initials: LI
authors.lastname: Pietrasanta
mesh_terms: D000465:Algorithms; D000818:Animals; D015415:Biomarkers; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D008928:Mitochondria / Q000378:metabolism*; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000378:metabolism; D013997:Time Factors
chemical_list: D015415:Biomarkers; D049452:Green Fluorescent Proteins