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The key to ultrabright fluorescent nanomaterials is the control of dye emission in the aggregated state. Here, lipophilic rhodamine B derivatives are assembled into nanoparticles (NPs) using tetraphenylborate counterions with varied fluorination levels that should tune the short-range dye ordering. Counterion fluorination is found to drastically enhance the emission characteristics of these NPs. Highly fluorinated counterions produce 10-20 nm NPs containing >300 rhodamine dyes with a fluorescence quantum yield of 40-60% and a remarkably narrow emission band (34 nm), whereas, for other counterions, aggregation caused quenching with a weak broad-band emission is observed. NPs with the most fluorinated counterion (48 fluorines) are ∼40-fold brighter than quantum dots (QD585 at 532 nm excitation) in single-molecule microscopy, showing improved photostability and suppressed blinking. Due to exciton diffusion, revealed by fluorescence anisotropy, these NPs are efficient FRET donors to single cyanine-5 acceptors with a light-harvesting antenna effect reaching 200. Finally, NPs with the most fluorinated counterion are rather stable after entry into living cells, in contrast to their less fluorinated analogue. Thus, the present work shows the crucial role of counterion fluorination in achieving high fluorescence brightness and photostability, narrow-band emission, efficient energy transfer and high intracellular stability of nanomaterials for light harvesting and bioimaging applications.

Controlling the emission of bright luminescent nanoparticles by a single molecular recognition event remains a challenge in the design of ultrasensitive probes for biomolecules. Herein, we developed 20‐nm light‐harvesting nanoantenna particles, built of a tailor‐made hydrophobic charged polymer poly(ethyl methacrylate‐co‐methacrylic acid), encapsulating circa 1000 strongly coupled and highly emissive rhodamine dyes with their bulky counterion. Being 87‐fold brighter than quantum dots QDots 605 in single‐particle microscopy (with 550‐nm excitation), these DNA‐functionalized nanoparticles exhibit over 50 % total FRET efficiency to a single hybridized FRET acceptor, a highly photostable dye (ATTO665), leading to circa 250‐fold signal amplification. The obtained FRET nanoprobes enable single‐molecule detection of short DNA and RNA sequences, encoding a cancer marker (survivin), and imaging single hybridization events by an epi‐fluorescence microscope with ultralow excitation irradiance close to that of ambient sunlight. [ABSTRACT FROM AUTHOR]

The key challenge in the field of fluorescent nanoparticles (NPs) for biological applications is to achieve superior brightness for sizes equivalent to single proteins (3-7 nm). We propose a concept of shell-cross-linked fluorescent micelles, in which PEGylated cyanine 3 and 5 bis-azides form a covalently attached corona on micelles of amphiphilic calixarene bearing four alkyne groups. The fluorescence quantum yield of the obtained monodisperse NPs, with a size of 7 nm, is a function of viscosity and reached up to 15 % in glycerol. In the on-state they are circa 2-fold brighter than quantum dots (QD-585), which makes them the smallest PEGylated organic NPs of this high brightness. FRET between cyanine 3 and 5 cross-linkers at the surface of NPs suggests their integrity in physiological media, organic solvents, and living cells, in which the NPs rapidly internalize, showing excellent imaging contrast. Calixarene micelles with a cyanine corona constitute a new platform for the development of protein-sized ultrabright fluorescent NPs. [ABSTRACT FROM AUTHOR]