Photoconversion of DNA binding dyes and its application in super-resolution microscopy

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).

Figure 1. A scheme of a single molecule localisation microscopy experiment. The investigated biological specimen is stained with a chosen fluorescent dye. There are many single molecules of the dye piled together and fluorescing simultaneously. By employing specific environment and illumination only a few fluorescent molecules become fluorescent at a time. Images of stochastically appearing fluorescing single molecules are registered until a sufficient number of single molecules is collected in order to reconstruct a super-resolution image of an investigated specimen. Positions of single molecules are localised with high precision, and later a super-resolution image is reconstructed.

Video 1. Schematic video showing stochastically appearing fluorescing single molecules and reconstruction of the image.

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.

Figure 2. Single molecule fluorescent bursts. Single molecule fluorescent bursts de- tected in green-yellow emission range (585 – 675 nm) during a typical experiment using high intensity single wavelength excitation (50 mW, 491 nm). The green-emitting molecules of VdcV (conc. 500 nM) are reversibly bleached and stochastically reappear in the detection channel of the SPDM microscope. Note that some of the molecules appear bright with much longer lifetime than the integration time of the camera.

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).

Figure 3. Super-resolution image of an optical slice of a nucleus. A – Super- resolution image of an optical slice of a nucleus of a Vero-B4 cell stained with 1 μM Vybrant® DyeCycleTM Violet, B – an image of the same nucleus acquired in a widefield microscope (exc. 405 nm, 450 μW, em. 450 – 490 nm).


1. Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe. Żurek-Biesiada D, Szczurek AT, Prakash K, Best G, Mohana GK, Lee HK, Roignant JY, Dobrucki JW, Cremer C, Birk U. Data Brief. 2016 Jan 29;7:157-71. doi: 10.1016/j.dib.2016.01.041. eCollection 2016 Jun. PMID: 27054149 (link)

2. Localization microscopy of DNA in situ using Vybrant(®) DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution. Żurek-Biesiada D, Szczurek AT, Prakash K, Mohana GK, Lee HK, Roignant JY, Birk UJ, Dobrucki JW, Cremer C. Exp Cell Res. 2016 May 1;343(2):97-106. doi: 10.1016/j.yexcr.2015.08.020. Epub 2015 Sep 1. PMID: 26341267 (link)

3. Single molecule localization microscopy of the distribution of chromatin using Hoechst and DAPI fluorescent probes. Szczurek AT, Prakash K, Lee HK, Zurek-Biesiada DJ, Best G, Hagmann M, Dobrucki JW, Cremer C, Birk U. Nucleus. 2014 Jul-Aug;5(4):331-40. doi: 10.4161/nucl.29564. PMID: 25482122 (link)

4. UV-induced spectral shift and protonation of DNA fluorescent dye Hoechst 33258. Żurek-Biesiada D, Waligórski P, Dobrucki JW. J Fluoresc. 2014 Nov;24(6):1791-801. doi: 10.1007/s10895-014-1468-y. Epub 2014 Oct 14. PMID: 25312832 (link)

5. UV-activated conversion of Hoechst 33258, DAPI, and Vybrant DyeCycle fluorescent dyes into blue-excited, green-emitting protonated forms. Zurek-Biesiada D, Kędracka-Krok S, Dobrucki JW. Cytometry A. 2013 May;83(5):441-51. doi: 10.1002/cyto.a.22260. Epub 2013 Feb 15. PMID: 23418106 (link)