A wide range of fluorophores can be visualized in this way, from special GFP mutants to organic dyes such as Cy5. For umcompromised results, it is paramount to achieve highest system sensitivity and precise optical sectioning. To this end, Carl Zeiss has combined its proven stands and software with a novel laser TIRF illumination concept and the latest EM-CCD technology.

Photoactivated localization microscopy lets you see single fluorescent molecules switching between an “on” and “off” state online, leading to imaging resolutions of ~20-30 nm. Suitable fluorophores are already plentiful and still expanding, including fluorescent proteins as well as organic dyes. Every point-like object is imaged as an extended spot – the so-called point spread function (PSF) in a light microscope. If two such objects come close enough, their PSFs will overlap heavily, making it impossible to determine their precise localization, let alone see them as separate entities.

But imagine you could view one at a time. Suddenly you would be able to determine the centers of the PSFs, which can be localized to a much higher precision than the PSFs themselves (see above figure). That’s all that is done in PAL-M. Fluorescent molecules are illuminated in such a way that only a few are activated, ensuring that their PSFs do not overlap. After registration these molecules are switched off, while new ones are activated and so it continues. The plot of all the localized molecules represents a superresolution image.



TIRF image (left) and PAL-M image (right) of antibody staining for tubulin in a cultured cell. Specimen: S. Niwa, University of Tokyo, Japan.