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This analysis only concerns microscopes equipped with a motorized Z stage.
The co-registration accuracy is the ability of a fluorescence microscope to record images of a multi-labeled object without introducing additional shifts that are not related to the object itself.
For any fluorescence microscope, knowledge of the co-registration accuracy, both in the lateral and axial directions, is important when color information in an image is to be used. For co-localization quantification in images of biological samples stained with several labels, the co-registration accuracy between the different channels should be determined, and corrected for, to prevent misinterpretation.
The “accuracy of co-registration” analysis provides the lateral and axial shifts between two channels, that are due to the translation-only contribution to the co-registration inaccuracy of the system, within a limited area of the field of view.
The “accuracy of co-registration” analysis is associated with any “multi-labeled object” (see Figure 1).
Figure 1: Example images of a “multi-labeled object”, acquired on two different channels, fulfilling the acquisition recommendations. Left: GFP channel. Right: RFP channel.