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\section{Discussion}  The radii obtained by anisotropy measurements are in the expected range and consistent with calculations based on MW, see Fig~\ref{fig:theor_radii}. Fig~\ref{fig:theor_radii}, and our measured radius of 3.49$\pm$0.03~nm for the well-characterised protein BSA is in excellent agreement with the accepted value of 3.48~nm.\cite{Axelsson1978}  While calculations based on the empirical formulas obtained from the analysis of thousands of proteins can give a good indication of the expected size, they cannot take into account the properties of specific molecules relating to surface roughness, shape, and the ionic charge which all affect the diffusion of the molecule. Our measured radius of 3.49$\pm$0.03~nm for the well-characterised protein BSA is in excellent agreement with With anisotropy measurements,  theaccepted value of 3.48~nm.\cite{Axelsson1978} The  cubic dependence of the radius on the rotational correlation time means that this technique is more sensitive to small changes in radius than methods where the radius depends on the measurement linearly, e.g.\ fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). Anisotropy measurements can also give information about the shape of the molecule. The measured anisotropies are clearly double-exponential: besides the component corresponding to the rotation of the drug molecule, there is an additional fast component. This component cannot be attributed to non-spherical shape of the molecule; in case of an ellipsoid, the anisotropy decay is three-exponential, but the three exponentials are linked, and the fast component is too fast to fit this model. model.\cite{Lakowicz2006}  While it is too fast to be measured accurately with this method due to the long lifetime of the dye, the results indicate a size comparable to the dye molecule, and this component is most likely caused by the rotation of the dye molecule on its bond. Wilkins \textit{et al.}\ report a similar component.\cite{Wilkins1999} Double-exponential fit taking this component into account produces excellent fit results for our experimental data, indicating that the proteins are approximately spherical in shape. DLS, based on the measurement of fluctuating scattered light intensity due to Brownian motion of the particles, is a well established method for determining the size of small particles in solution, including macromolecular drugs. Wen \textit{et al.}\ \cite{Wen2013} report hydrodynamic radii of 4.2~nm for ranibizumab and 6.3~nm for bevacizumab using DLS, and 5.4~nm for BSA measured as a control, and Li \textit{et al.}\ \cite{Li2011} 4.1~nm, 6.5~nm and 4.8~nm for the same molecules, respectively. These results are slightly higher than estimates based on MW, which they suspect could be due to aggregation. DLS is limited to very low solute concentrations (generally $\lesssim$1~mg/ml).  SAXS and SANS are also popular methods for the size measurement of macromolecules. While they can be used with higher particle concentrations than DLS ($\sim$1-100~mg/ml) and are applicable to a large MW range from few kDa to hundreds of MDa, they have low resolution, and structural information can only be obtained through complex model building.\cite{Pecora_1985} Similar to DLS, SAXS and SANS measure scattering from unlabelled molecules which simplifies sample preparation but makes the results susceptible to artefacts arising from dust and other contamination in the sample solution, and makes these techniques impossible to be used with scattering media, such as tissue.  The measurement time interval of 5~$\mu$s used in these experiments is ideal for measuring the rotational correlation times of these drugs with this ruthenium-based dye at low viscosities. However, if this approach is combined with imaging, to perform Phosphorescence Lifetime Imaging (PLIM) \cite{Baggaley2015} the long lifetime of the dye will require long pixel dwell times, making scanning-based data acquisition slow. We have recently developed wide-field lifetime imaging approaches \cite{Hirvonen2014_ol, Hirvonen2015_njp} that are ideally suited for measuring lifetimes at this time scale, and could image several wells containing different drugs and/or different viscosity solutions simultaneously. If these microsecond-resolution wide-field time-correlated single photon counting approaches were to be combined with polarization-resolved excitation and detection, one could perform time-resolved anisotropy imaging \cite{Suhling2004} on a microsecond time scale. This would benefit the measurement of similar or higher MW drugs in several wells of a multiwell plate simultaneoulsly, simultaneously,  and also enable imaging these drugs \textit{in vitro}.