How to Measure Separations and Angles Between Intramolecular Fluorescent Markers

Structure and function of an individual biomolecule can be explored with minimum two fluorescent markers of different colors. Since the light of such markers can be spectrally separated and imaged simultaneously, the markers can be colocalized. Here, we describe the method used for such two-color colocalization microscopy. Then we extend it to fluorescent markers with fixed orientations and in intramolecular proximity. Our benchmarking of this extension produced two extra results: (a) we established short double-labeled DNA molecules as probes of 3D orientation of anything to which one can attach them firmly; (b) we established how to map with super-resolution between color-separated channels, which should be useful for all dual-color colocalization measurements with either fixed or freely rotating fluorescent molecules. Throughout, we use only simple means: from each color-separated microscope image in a time-lapse movie, we simultaneously determine both the relative (x,y)-separation of the fluorophores and their individual orientations in space, both with accuracy and precision. The relative positions and orientations of two domains of the same molecule are thus time-resolved. Using short double-stranded DNA (dsDNA) molecules internally labeled with two fixed fluorophores, we (i) demonstrate the accuracy and precision of our localization- and mapping-methods, using the known structure of dsDNA as benchmark; (ii) resolve 10 base pair differences in fluorophore separations; (iii) determine the unique 3D orientation of each DNA molecule.