CONICET | Buscador de Institutos y Recursos Humanos

The subcellular localization of proteins and cellular structures is crucial for their function and regulation and can be assessed by fluorescence labeling and microscopy. The colocalization of two or more markers within the cell is defined as an overlap in the physical distribution of the molecular populations within two or three-dimensional space, which may be complete or partial. Analysis tools do not necessarily fit all circumstances as cells contain a plethora of structures of multiple morphologies, from linear elements of the cytoskeleton, punctuate and isotropic compartments such as vesicles, endosomes or vacuoles, to more complex forms such as Golgi and the network-like endoplasmic reticulum. Analysis should be able to discriminate real colocalization from the random localization of two free molecules in the same compartment. The certainty of the physical dimensions and location of small objects in the two-dimensional and even more in the three-dimensional space is subject to the limits of resolution in optical microscopy. Whether two fluorochromes are located on the same physical structure or on two distinct structures is sometimes hard to estimate. Indeed, it is difficult to endeavor whether a protein is localized to mitochondria or Golgi due to the small size of these organelles, particularly when the protein has a widespread distribution within the cell. We introduce a novel statistical approach that quantifies colocalization in any region of an image without the bias of visual interpretation in a variety of cellular systems with different colocalization extent. With this method we were able to infer different mechanisms that govern key processes in neuronal pathology as Parkinsons disease and in neuronal growth and survival as mediated by NGF receptors, by accurately determining the localization of key molecules in definite organelles.