New microscopy modalities for diffraction unlimited optical imaging.

Despite the advanced microscopes that are available today, zooming into the small compartments of life remains one of the great challenges of science due to the diffraction limit stemming directly from the wave-particle duality of light itself. At present fluorescence microscopy has evolved to the point where single molecules can be reliably detected and followed. Single-molecule fluorescence spectroscopy (SMFS) now also offers a way to avoid the diffraction limit entirely and this modality is known as photoactivation-localization microscopy (PALM). PALM relies on the insight that individual, well-isolated, emitters can be localized with nanometer precision simply by determining the centroid of the emission distribution. One can construct an image of the sample with nanometer resolution derived from the calculated position of all the emitters in a sample, under the premise that one is looking at the emission of single isolated emitters. To be able to use samples containing many fluorophores in nanometer proximity one has to apply this localization procedure to single emission events separated in time, so that only a single fluorophore emits within a diffraction-limited region at any instant. PALM is exploiting the photochemical processes of photo-activation/switching of new fluorescent probes, as the occurrence of these photochemical processes is stochastic.

Keywords:nanoscopy, superresolution, diffraction limit, fluorescence, Microscopy, PALM microscopy

Disciplines:Inorganic chemistry, Organic chemistry, Theoretical and computational chemistry, Other chemical sciences