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

Abstract

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 (’blinking’), detected and localised. The use of a short burst of deep blue excitation 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, for the first time, we demonstrate that STED and OLM can be performed on the same biological sample using a simple imaging buffer. It is hoped that such a correlative imaging will provide a basis for a further enhanced resolution.Scope of the findingsDespite ten years of development, superresolution microscopy is still limited to relatively few microscopy and optics groups. This is mainly due to the significant cost of the superresolution microscopes which require high-quality lasers, high NA objective lens, a very sensitive camera, a highly precise microscope stage, and a complex post-acquisition data reconstruction and analysis. We present results that demonstrate the possibility to obtain nanoscale resolution images using a conventional microscope and an incoherent light source. We show an easyto-follow protocol that every biologist can implement in the laboratory. We hope that this finding will help any scientist to generate high-density super-resolution images even with limited budget. Lastly, the new photophysical observations reported here will pave the way for more in-depth investigations on excitation, photobleaching and photoactivation of a fluorophore.