ABSTRACT
Ultrabright fluorescent nanoparticles (NPs) hold great promise for demanding bioimaging applications. Recently, extremely bright molecular crystals of cationic fluorophores were obtained by hierarchical coassembly with cyanostar anion-receptor complexes. These small-molecule ionic isolation lattices (SMILES) ensure spatial and electronic isolation to prohibit aggregation quenching of dyes. We report a simple, one-step supramolecular approach to formulate SMILES materials into NPs. Rhodamine-based SMILES NPs stabilized by glycol amphiphiles show high fluorescence quantum yield (30 %) and brightness per volume (5000â M-1 cm-1 /nm3 ) with 400 dye molecules packed into 16-nm particles, corresponding to a particle absorption coefficient of 4×107 â M-1 cm-1 . UV excitation of the cyanostar component leads to higher brightness (>6000â M-1 cm-1 / nm3 ) by energy transfer to rhodamine emitters. Coated NPs stain cells and are thus promising for bioimaging.
Subject(s)
Fluorescent Dyes/chemistry , Nanoparticles/chemistry , Rhodamines/chemistry , Small Molecule Libraries/chemistry , HEK293 Cells , Humans , Molecular Structure , Optical Imaging , Particle Size , Ultraviolet RaysABSTRACT
Intrinsic fluorescence of biological material, also called auto-fluorescence, is a well-known phenomenon and has in recent years been used for imaging, diagnostics and cell viability studies. Here we show that in addition to commonly observed auto-fluorescence, intrinsic anti-Stokes emission can also be observed under 560 nm or 633 nm excitation. The anti-Stokes emission is shown to be spatially located on/in the mitochondria. The findings presented here show that sensitive imaging experiments e.g. single molecule experiments or two-photon excitation imaging can be compromised if intracellular anti-Stokes emission is not accounted for. On the other hand, we suggest that this anti-Stokes emission could be exploited as an additional modality for mitochondria visualization and cell viability investigation even in systems that are already labeled with commonly used fluorophores that rely on normal Stokes-based detection.
Subject(s)
Cell Tracking/methods , Fluorescence , Fluorescent Dyes/pharmacology , Cell Survival/drug effects , Fluorescent Dyes/chemistry , HeLa Cells , Humans , Microscopy, Fluorescence , PhotonsABSTRACT
Three series of ionic self-assembled materials based on anionic azo-dyes and cationic benzalkonium surfactants were synthesized and thin films were prepared by spin-casting. These thin films appear isotropic when investigated with polarized optical microscopy, although they are highly anisotropic. Here, three series of homologous materials were studied to rationalize this observation. Investigating thin films of ordered molecular materials relies to a large extent on advanced experimental methods and large research infrastructure. A statement that in particular is true for thin films with nanoscopic order, where X-ray reflectometry, X-ray and neutron scattering, electron microscopy and atom force microscopy (AFM) has to be used to elucidate film morphology and the underlying molecular structure. Here, the thin films were investigated using AFM, optical microscopy and polarized absorption spectroscopy. It was shown that by using numerical method for treating the polarized absorption spectroscopy data, the molecular structure can be elucidated. Further, it was shown that polarized optical spectroscopy is a general tool that allows determination of the molecular order in thin films. Finally, it was found that full control of thermal history and rigorous control of the ionic self-assembly conditions are required to reproducibly make these materials of high nanoscopic order. Similarly, the conditions for spin-casting are shown to be determining for the overall thin film morphology, while molecular order is maintained.
