  Nanoparticles (NPs) are promising therapeutic delivery agents, yet it is
increasingly apparent that the number and manner of presentation of cell
binding ligands on the NP can affect the eventual fate of the therapeutic.
Whenever NPs are conjugated with biomolecules, a heterogenous population of
decorated NPs will be produced and the details of the subpopulations of
particle-ligand structures needs to be characterised for a reliable
interpretation of NP-based data. We report an optical microscopy method to
quantitatively evaluate the conjugation on a single particle basis in samples
consisting of gold NPs (GNPs) decorated with human holo-transferrin
fluorescently labelled with Alexa647 (Tf). We employed widefield fluorescence
and extinction microscopy on NP-ligand constructs sparesly deposited onto a
glass surface, alongside a correlative analysis which spatially co-localises
diffraction-limited sources of fluorescence with the optical extinction by
individual GNPs. A photobleaching step analysis of the fluorescence emission
was employed to estimate the number of fluorophores contributing to the
detected emission rate. The method quantifies the number of fluorescent
biomolecules attached per GNP, the numbers of unconjugated GNPs and unbound Tf
present within the mixed population, and the size and intraparticle clustering
propensity of conjugated GNPs. We found a high variability in the number of Tf
ligands per GNP within the GNP population, when analysed at the single-particle
level, unraveling a non-trivial statistical distribution not accessible in
ensemble averaged approaches
