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High-density superresolution microscopy with an incoherent light source and a conventional epifluorescence microscope setup

By Kirti Prakash

Posted 17 May 2017
bioRxiv DOI: 10.1101/121061

We report that single-molecule superresolution microscopy can be achieved with a conventional epifluorescence microscope setup and a Mercury arc lamp. The configuration termed as Omnipresent Localisation Microscope (OLM), is an extension of Single Molecule Localisation Microscopy (SMLM) techniques and allows single molecules to be switched on and off (a phenomenon termed as “blinking”), detected and localised. The use of a short burst of deep blue excitation (350-380 nm) can be further used to reactivate the blinking, once the blinking process has slowed or stopped. A resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). Finally, we demonstrate that STED and OLM can be performed on the same biological sample using a simple imaging medium. It is hoped that this type of correlative imaging will provide a basis for a further enhanced resolution. Scope of the findings Ten years into its development, superresolution microscopy is still limited to relatively few microscopy and optics groups. This is mainly due to the significant cost of current superresolution microscopes, which require high-quality lasers, high NA objective lenses, very sensitive cameras, and highly precise microscope stages, and to the complexity of post-acquisition data reconstruction and analysis. We present results that demonstrate the possibility of obtaining nanoscale-resolution images using a conventional microscope and an incoherent light source. We describe an easy-to-follow protocol that every biologist can implement in the laboratory. We hope that this finding will help any scientist to generate high-density superresolution images even with a limited budget. Ultimately, the new photophysical observations reported here should pave the way for more in-depth investigations on the processes underlying the excitation, photobleaching and photoactivation of a fluorophore.

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