Dye-loaded lipid nano-droplets present an attractive alternative to inorganic nanoparticles, as they are composed of non-toxic biodegradable materials and easy to prepare. However, to achieve high fluorescence brightness, the nano-droplets have to be heavily loaded with the dyes avoiding fluorescence self-quenching and release (leakage) of the encapsulated dyes from the nano-droplets in biological media. In the present work, we have designed highly lipophilic fluorescent derivatives of 3-alkoxyflavone (F888) and Nile Red (NR668) that can be encapsulated in the lipophilic core of stable nano-emulsion droplets at exceptionally high concentrations in the oil core, up to 170 mM and 17 mM, respectively, corresponding to ~ 830 and 80 dyes per 40-nm droplet. Despite this high loading, these dyes keep high fluorescence quantum yield and thus, provide high nano-droplet brightness, probably due to their bulky structure preventing self-quenching. Moreover, simultaneous encapsulation of both dyes at high concentrations in single nano-droplets allows observation of FRET. FRET and fluorescence correlation spectroscopy (FCS) studies showed that NR668 release in the serum-containing medium is very slow, while the reference hydrophobic dye Nile Red leaks immediately. This drastic difference in the leakage profile between NR668 and Nile Red was confirmed by cellular studies as well as by angiography imaging on zebrafish model, where the NR668-loaded nano-droplets remained in the blood circulation, while the parent Nile Red leaked rapidly from the droplets distributing all over the animal body. This study suggests new molecular design strategies for obtaining bright nano-droplets without dye leakage and their use as efficient and stable optical contrast agents and .

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.