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\section{Discussion}  The most commonly quoted hydrodynamic radius of BSA is 3.48~nm,\cite{Axelsson1978} and our measured radius 3.49~nm is in excellent agreement with this.  The measured anisotropies are clearly double-exponential: besides the component corresponding to the protein rotation, there is an additional fast component. This component cannot be attributed to non-spherical shape of the molecule; in case of an ellisoid, the anisotropy decay is three-exponential, but the three exponentials are linked, and the fast component is too fast to fit this model. While it is too fast to be measured accurately with this method due to the long lifetime of the dye, the \textit{Compare all  resultsindicate a size comparable to  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, indicating that the proteins are approximately spherical in shape. other literature values.}  The fitted rotational correlation times follow a linear increase with viscosity up measured anisotropies are clearly double-exponential: besides the component corresponding  to viscosity of $\sim$20 cP ($\sim$70\% volume fraction glycerol).\cite{Suhling2004} Glycerol the protein rotation, there  is known an additional fast component. This component cannot be attributed  to cause preferential hydration non-spherical shape  ofproteins,\cite{Gekko1981} and at higher concentrations  the rotational correlation times do not follow molecule; in case of an ellipsoid,  the linear model.  The Stokes-Einstein-Debye equation \cite{VanHolde1998} assumes that anisotropy decay is three-exponential, but  the solvent containing three exponentials are linked, and  the rotating particles fast component  is homogenous and continuous. Although too fast to fit  this may not always model. While it is too fast to  be measured accurately with this method due to the long lifetime of  the case, dye,  the approximation seems results indicate a size comparable  to be valid for 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, indicating that the  proteinswhich  are generally much larger than the solvent molecules.\cite{Weber1953} approximately spherical in shape.  The fitted rotational correlation times follow a linear increase with viscosity up to viscosity of $\sim$20 cP ($\sim$70\% volume fraction glycerol).\cite{Suhling2004} Glycerol is known to cause preferential hydration of proteins,\cite{Gekko1981} and at higher concentrations the rotational correlation times do not follow the linear model.  The most commonly quoted hydrodynamic radius of BSA Stokes-Einstein-Debye equation \cite{VanHolde1998} assumes that the solvent containing the rotating particles  is 3.48~nm,\cite{Axelsson1978} although reports in literature range from 3.3 homogeneous and continuous. Although this may not always be the case, the approximation seems  to 3.4~nm.[insert references] Our measured radius 3.07~nm is slightly smaller, but is in good agreement with be valid for proteins which are generally much larger than  the radius of 3.04~nm calculated from MW using a formula by Wilkins \textit{et.\ al}.\cite{Wilkins1999} solvent molecules.\cite{Weber1953}  The measurement time interval of 5 $\mu$s used in these experiments is ideal for measuring the rotational correlation times of these proteins with this Ru dye at low viscosities. However, with the long lifetime of the dye, long pixel dwell times are needed, making scanning-based data acquisition slow. 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, might benefit the size measurement of similar or higher MW drugs.  \begin{itemize}  \item The initial anisotropy is $<$0.15 instead of 0.4 - comment on this.  \item Compare \textit{Compare  to DLS, SAXS and SANS.  \item Ophthalmological implications?  \end{itemize} SANS.}