Photophysical aspects of two complementary types of probes are discussed in relation to their molecular properties. The general difference in the optical response originates in the way energy is released following absorption of visible light. Thus, analysis will focus on: (1) radiative fluorescence emission from a dynamic chromophore and (2) non-radiative photothermal response from a protein-coated gold nanoparticle (Au NP).

In (1), time-resolved spectroscopy was applied to study the roles of hydrogen bonding, steric constraints, and extension of the π-conjugation on fluorescence emission. Results are consistent with reversible switching motions interconverting an emissive folded and a non-emissive unfolded conformer. Hydrogen bonding stabilized the folded conformer and thus fluorescence emission of the chromophores, while steric constraints favored the unfolded conformer. Extension of the π-conjugation resulted in an increase in the fluorescence emission, which could be explained by the suppression of torsional motions. Further steady-state fluorescence measurements revealed intermolecular interactions through π–π stacking at higher concentrations.

In (2), photothermal heterodyne imaging (PHI) was developed to detect single Au NPs and follow changes in their thermal response as a function of environment. As a proof-of-principle, changes in the PHI signal due to the encapsulation of a nanoparticle by a virus protein coat were measured. A numerical model was devised to study the effect of different physical and chemical factors on the strength and sensitivity of the PHI signal. In particular the importance of the protein surface coverage and hydration were examined and illustrated through experiments done on HIV-1 Gag virus nanoparticles. A comparison of the temperature change observed in simulations shows good agreements with predicted relative signal strengths in PHI experiments. For the first time, an optical method was used in situ as an alternative to TEM for estimating the protein surface coverage of an immature HIV-1 particle model.