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We demonstrate a spectroscopic imaging based super-resolution approach by separating the overlapping diffraction spots into several detectors during a single scanning period and taking advantage of the size-dependent emission wavelength in nanoparticles. This approach has been tested using off-the-shelf quantum dots (Invitrogen Qdot) and in-house novel ultra-small (~3 nm) Ge QDs. Furthermore, we developed a method-specific Gaussian fitting and maximum likelihood estimation based on a Matlab algorithm for fast QD localisation. This methodology results in a three-fold improvement in the number of localised QDs compared to non-spectroscopic images. With the addition of advanced ultra-small Ge probes, the number can be improved even further, giving at least 1.5 times improvement when compared to Qdots. Using a standard scanning confocal microscope we achieved a data acquisition rate of 200 ms per image frame. This is an improvement on single molecule localisation super-resolution microscopy where repeated image capture limits the imaging speed, and the size of fluorescence probes limits the possible theoretical localisation resolution. We show that our spectral deconvolution approach has a potential to deliver data acquisition rates on the ms scale thus providing super-resolution in live systems.

Spectroscopic super-resolution fluorescence cell imaging using ultra-small Ge quantum dots