Fluorescent Nanodiamonds as Fiducial Markers for Microscopy

The invention relates to fluorescent nanodiamonds (FNDs) and their uses as fiducial markers for microscopy. FNDs are bright fluorescent probes that do not blink or bleach and have broad fluorescence excitation and emission peaks. The fluorescence intensity can be readily controlled by the size of the FND, the number of fluorescent centers produced in the nanodiamonds, or in situ through the application of a weak magnetic field. The particular advantage of the FND compositions of this invention are that they are particularly useful for extended imaging of a single sample over time periods that can be as long as a week or more. In an exemplary embodiment, FNDs are immobilized in a substrate that are coated with an inert top coating, like silicon dioxide, or transparent polymer (e.g. poly-L-lysine, poly-L-arginine, or siloxanes). Generally, any suitable methods known for surface functionalization of the substrate can be used to make the composition. In another aspect of this invention, the inventors designed software for super-resolution imaging correction method is employed to precisely determine the position coordinates of each of a set of FNDs in a plurality of images by using Gaussian fitting of the point spread function comprises each of the FNDs in the plurality of images. The calculated correction is then used to displace each image to align the coordinates of the FNDs. The positions of the FNDs can be tracked with sub-nanometer precision and residual drift can be reduced to the nanometer scale over hundreds of hours of tracking.