One of the most important parameters describing the quality of a microscopic image is its resolution. Many modifications to standard fluorescence methods have been introduced in order to improve the resolution of images. Consequently, among others an optical microscopy (super-resolution) technique called Single Molecule Localisation Microscopy (SMLM) has been introduced. The term super-resolution may be applied to any optical microscopy technique that allows increasing the resolution beyond the theoretical Abbe’s limit. Conventional microscopy techniques (e.g. Confocal Laser Scanning Microscopy) are limited by the imposed 180-nanometre resolution limit, while super-resolution techniques circumvent this restriction. For SMLM positions of single fluorescent molecules are identified and registered in order to obtain high quality images (see Figure 1, Figure 2 and Video 1).
By using photoswitchable, photoconvertible, or photoactivable dyes/probes one can activate the fluorescence of only a few molecules at a time, making it possible to apply SMLM, and obtain images of cell structures with resolution greater than 180 nm. The current choice of fluorescent dyes/probes eligible for single molecule localisation microscopy is rather broad, and the research based on these fluorophores is constantly developing. Nevertheless, so far, the majority of the conducted research has been focused on cell structures that are easily accessible to the dyes/probes, like microtubules or actin filaments (e.g. by applying low molecular weight dyes (like e.g. Alexa Fluor 488) conjugated to antibodies). One very important field that seeks further investigation is research on DNA structure. DNA, due to its extremely dense packing in a cell nucleus, forms complicated structures. There are not many fluorescent dyes/probes eligible for super-resolution imaging of DNA. Even if the indirect forms of labelling are considered (e.g. fusions with proteins, immunolabelling) and the direct forms of labelling (e.g. using small DNA binding dyes), the choice of eligible fluorophores is still very limited. In my PhD thesis I have focused on investigation of photoconvertible dyes that are directly binding to DNA, i.e. DAPI, Hoechst 33258/33342, and Vybrant® DyeCycle™ Violet. Even though DAPI and Hoechst dyes have been known for many years now, their exploitation in super-resolution microscopy has not been described so far, as their blinking behaviour has not been reported.
The key to the successful exploitation of the abovementioned dyes in super-resolution microscopy is the process of their photoconversion, and generation of the green-emitting photoproducts. DAPI and Hoechst dyes bind to DNA by means of minor groove binding, which is an undeniable advantage, as the dyes themselves do not significantly perturb the chromatin structure. Additionally, all of the proposed dyes enable direct and dense labelling of DNA. They are of low molecular weight and they penetrate into the chromatin structures much more easily than antibodies or big fluorescent proteins. In my thesis I have investigated photophysical properties of the above mentioned dyes and applied those dyes in single molecule localisation microscopy, to obtain high quality images of DNA structures (Figure 3).