Photophysics of fluorescent nanoparticles based on organic dyes - challenges and design principles.

Fluorescent nanoparticles have become attractive for bioanalysis and imaging, due to their high brightness and photostability. Many different optical materials have been applied in fluorescent nanoparticles with a broad range of properties and characteristics. One appealing approach is the incorporation of molecular organic fluorophores in nanoparticles with the intention of transferring their known attractive solution-state properties directly to the nanoparticles. However, as molecular dyes are packed closely together in the nanoparticles their interactions most often result in fluorescence quenching and change in spectral properties making this approach challenging. In this perspective we will first discuss the origins of quenching and spectral shifts observed in dye based nanoparticles. On this background, we will then describe various designs of dye based NPs and how they address the challenges of dye-dye interactions and quenching. Our aim is to provide a general framework for understanding the supramolecular mechanisms that determine the photophysics of dye based nanoparticles. This framework of molecular photophysics and its relation to the internal structure of dye based nanoparticles can hopefully serve to assist rational design and optimization of new and improved dye based nanoparticles.

Control of the fluorescence lifetime in dye based nanoparticles.

Fluorescent dye based nanoparticles (NPs) have received increased interest due to their high brightness and stability. In fluorescence microscopy and assays, high signal to background ratios and multiple channels of detection are highly coveted. To this end, time-resolved imaging offers suppression of background and temporal separation of spectrally overlapping signals. Although dye based NPs and time-resolved imaging are widely used individually, the combination of the two is uncommon. This is likely due to that dye based NPs in general display shortened and non-mono-exponential lifetimes. The lower quality of the lifetime signal from dyes in NPs is caused by aggregation caused quenching (ACQ) and energy migration to dark states in NPs. Here, we report a solution to this problem by the use of the small-molecule ionic isolation lattices (SMILES) concept to prevent ACQ. Additionally, incorporation of FRET pairs of dyes locks the exciton on the FRET acceptor providing control of the fluorescence lifetime. We demonstrate how SMILES NPs with a few percent rhodamine and diazaoxatriangulenium FRET acceptors imbedded with a cyanine donor dye give identical emission spectra and high quantum yields but very different fluorescence lifetimes of 3 ns and 26 ns, respectively. The two spectrally identical NPs are easily distinguished at the single particle level in fluorescence lifetime imaging. The doping approach for dye based NPs provides predictable fluorescence lifetimes and allows for these bright imaging reagents to be used in time-resolved imaging detection modalities.

Cellular Visualization of G-Quadruplex RNA via Fluorescence- Lifetime Imaging Microscopy.

Over the past decade, appreciation of the roles of G-quadruplex (G4) structures in cellular regulation and maintenance has rapidly grown, making the establishment of robust methods to visualize G4s increasingly important. Fluorescent probes are commonly used for G4 detection in vitro; however, achieving sufficient selectivity to detect G4s in a dense and structurally diverse cellular environment is challenging. The use of fluorescent probes for G4 detection is further complicated by variations of probe uptake into cells, which may affect fluorescence intensity independently of G4 abundance. In this work, we report an alternative small-molecule approach to visualize G4s that does not rely on fluorescence intensity switch-on and, thus, does not require the use of molecules with exclusive G4 binding selectivity. Specifically, we have developed a novel thiazole orange derivative, TOR-G4, that exhibits a unique fluorescence lifetime when bound to G4s compared to other structures, allowing G4 binding to be sensitively distinguished from non-G4 binding, independent of the local probe concentration. Furthermore, TOR-G4 primarily colocalizes with RNA in the cytoplasm and nucleoli of cells, making it the first lifetime-based probe validated for exploring the emerging roles of RNA G4s in cellulo.

Universal Concept for Bright, Organic, Solid-State Emitters─Doping of Small-Molecule Ionic Isolation Lattices with FRET Acceptors.

Brightly fluorescent solid-state materials are highly desirable for bioimaging, optoelectronic applications, and energy harvesting. However, the close contact between π-systems most often leads to quenching. Recently, we developed small-molecule ionic isolation lattices (SMILES) that efficiently isolate fluorophores while ensuring very high densities of the dyes. Nevertheless, efficient Förster resonance energy transfer (FRET) energy migration in such dense systems is inevitable. While attractive for energy harvesting applications, FRET also significantly compromises quantum yields of fluorescent solids by funneling the excitation energy to dark trap states. Here, we investigate the underlying property of FRET and exploit it to our favor by intentionally introducing fluorescent dopants into SMILES materials, acting as FRET acceptors with favorable photophysical properties. This doping is shown to outcompete energy migration to dark trap states while also ruling out reabsorption effects in dense SMILES materials, resulting in universal fluorescent solid-state materials (thin films, powders, and crystals) with superior properties. These include emission quantum yields reaching as high as 50-65%, programmable fluorescence lifetimes with mono-exponential decay, and independent selection of absorption and emission maxima. The volume normalized brightness of these FRET-based SMILES now reach values up to 32,200 M-1 cm-1 nm-3 and can deliver freely tunable spectroscopic properties for the fabrication of super-bright advanced optical materials. It is found that SMILES prohibit PET quenching between donor and acceptor dyes that is observed for non-SMILES mixtures of the same dyes. This allows a very broad selection of donor and acceptor dyes for use in FRET SMILES.