When considering colocalization, too often composite images of red and green channels are considered sufficient. If I want to show DAPI in green and EGFP as magenta, there is nothing “wrong” about that. These false colors are only useful to tell which channel is which. In the majority of fluorescence microscopy images, all the “colors” of a multi-channel image are captured using a monochromatic detector that doesn’t know what color the photons that hit it are that is determined by the fluorescence emission filters we use. You can find more details about optical resolution and image pixel spacing in the “Notes and precautions” section below. Additionally, the spatial resolution of your image must be sufficient to actually support your hypothesis. The colocalization measurement we make only means anything in relation to the spatial scale we are working at, so it needs to be explicitly stated. We must colocalise at some defined and explicit spatial scale: In our case the optical resolution or image pixel/voxel spacing, whichever is the larger value in nm, micrometers, mm, meters, km, etc. Practically, our situation lies between the two extremes. At the other extreme, a universe of one voxel (not cubic of course) is completely colocalised - everything is inside it.
So, actually nothing is “really” colocalised. The Pauli exclusion principle states that two particles cannot have the same quantum numbers so they cannot be in the same place.
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For this reason, colocalization is most often used to determine if a protein is localizing to an organelle or other well defined cellular structure.įor more information on colocalisation and for how to correctly capture quantitative fluorescence microscopy images suitable for colocalisation analysis, look here: Image Processing Courses at BioDIP, Dresden. Regardless of your microscope, this volume is many, many times greater than the volume of a single protein. Importantly, colocalization results cannot indicate that two proteins/molecules are bound or interacting, only that they are both localized to within a certain volume, and is mostly dependent upon your microscope and its acquisition parameters. It could mean that one signal of one channel is contained within the bounds of another, or that your stains/dyes are typically found separated by a certain distance or are generally clustered, or simply that the signal from both channels overlap each other when imaged at a particular spatial resolution. The specific nature of that correlation, and what it means for your research, can vary quite a bit.
Generally speaking, when we evaluate colocalization, we are usually attempting to demonstrate that a significant, non-random spatial correlation exists between two channels of a dual color image.
First you have to define what you mean by colocalisation, and that is not trivial. Suppose you are given some images by a colleague, or have some images of your own, and you want to measure the amount of colocalisation between two of the dyes or stains in the images.
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