Nanographene-based dyes as high performance probes for super-resolution microscopy

Super-resolution microscopy critically relies on the availability of appropriate fluorophores. For single-molecule localization microscopy (SMLM), fluorophores are needed that stochastically switch between bright (on) and dark (off) states. This phenomenon is also called fluorescence blinking. For stimulated emission depletion microscopy (STED) the dyes must be photo-switchable.

Nowadays, four classes of fluorophores are used in such applications: small molecule organic dyes, photo-activatable/switchable fluorescent protein, inorganic quantum dots (QDs) and carbon dots (CDs).

Small molecule organic dyes, e.g., ThermoFisher Scientific Alexa Fluor® 647, have excellent blinking properties and emit high number of photons, but require specialized buffers for their use. This restricts their use to the imaging of fixed biological samples.

Photo-activatable/switchable fluorescent proteins have good excellent target molecule specificity but have the issue of low photon numbers resulting in a lower localization precision compared to organic dyes.

The size of QDs (~10 nm) and their unfavorable blinking properties limit their function as reliable molecular probes. Furthermore, QDs contain heavy metals, which limits their applicability in biomedicine due to toxicity.

CDs show environment-independent blinking properties, but their undefined amorphous carbon structure makes the specific coupling to biological samples difficult and leads to unfavorable broad emission spectra.

Recently graphene quantum dots (GQDs) obtained by means of "top-down" synthesis methods were proposed as another class of superior photo-switchable fluorophores due to their small size (<10 nm) and low toxicity. However, the emission spectra of GQDs are typically very broad due to their undefined chemical structures, and their blinking properties are also not ideal for SMLM applications.

Here nanographene structures are introduced as a new class of fluorophores for super-resolution fluorescent microscopy that overcome the limitations described above. These nanographene structures are large polycyclic aromatic hydrocarbons obtained by means of organic "bottom-up" synthesis methods. They perform like hybrids of organic dyes and QDs. They have excellent environment-independent blinking properties, emit high photon numbers, display high photostability, possess low toxicity, have a molecular size below 2 nm, and have narrow excitation and emission spectra. These features make nanographenes ideal candidates for SMLM. but also for STED microscopy.

^a in air; ^b in polystyrene

The “bottom-up” method allows the synthesis of nanographenes with well-defined absorption and emission spectra due to their precise chemical structure, enables the introduction of various functional groups for binding to specific ligands, reacting to specific target molecules or allowing water solubility.

In particular DBOV-Mes has very narrow absorption and emission spectra compared to commonly used organic dyes while being water soluble, making this nanographene ideal for multi-color imaging.

• MI0903-5658: "Use of a substituted or unsubstituted polycyclic aromatic hydro-carbon compounds for high-resolution microscopy", EP priority application filed in October 2018, PCT application filed in October 2019.
• MI0903-5696: "Hydrophilic and particularly water soluble DBOV-derivatives", EP priority application filed in October 2018, PCT application filed in October 2019.

X. Liu, S-Y Chen, Q. Chen, X. Yao, M. Gelléri, S. Ritz, S. Kumar, C. Cremer, K. Landfester, K. Müllen, S. Parekh, A. Narita, M. Bonn: "Nanographenes: ultrastable, switchable, and bright probes for super-resolution microscopy", Angewandte Chemie, Published online 28 October 2019, https://doi.org/10.1002/ange.201909220