deletions | additions       diff --git a/Method.tex b/Method.tex  index 106a380..d12db34 100644  --- a/Method.tex  +++ b/Method.tex 
...
\subsection{Sample preparation}  Ranibizumab and bevacizumab solutions were dialysed twice against phosphate buffered saline (PBS) pH 7.4 using D-Tube Midi Dialyzer units from Novagen (6-8~kD cut-off). Subsequently, the purity of the dialysed proteins was checked by standard SDS-PAGE. The proteins were then conjugated to the fluorophore Bis(2,2′-bipyridine)-4′-methyl-4-carboxybipyridine-rutheniumbis(hexafluorophosphate) (synonym,  N-succinimidyl ester-Ru(bpy)2(mcbpy-O-Su-ester)(PF6)2)(Sigma-Aldrich) ester-bis(hexafluorophosphate) (synonym, Ru(bpy)$_2$(mcbpy-O-Su-ester)(PF$_6$)$_2$, Sigma-Aldrich)  by using a succinimidyl ester-modified fluorophore with a short linker (Invitrogen, F6130). Conjugation reactions were performed in PBS adjusted with bicine buffer to pH 8.6 at 2~mg/ml protein concentration with the activated fluorophore ester being used in excess. The reaction was stopped after two hours and conjugated proteins were separated from remaining free dye by size exclusion chromatography (7~kD cut-off; Thermo Fisher). Using this method we generated ranibizumab conjugated to dye with a dye:protein ratio of 1.1:1 and bevacizumab with a dye:protein ratio of 1.3:1. Azide (2.0~mM) was added to all fluorophore-conjugated drugs to protect them from microbial deterioration. *** Add Eylea and BSA ***  \subsection{Anisotropy measurements}  The Ru-labelled molecules Ru(bpy)$_2$(mcbpy-O-Su-ester)(PF$_6$)$_2$-labelled drugs  in buffer were mixed with glycerol in different proportions to produce solutions with different viscosities. viscosities up to $\sim$70\% volume fraction glycerol. Glycerol is known to cause preferential hydration of proteins,\cite{Gekko1981} and at higher concentrations the rotational correlation times do not follow the linear model.  A drop of each mixture was placed in a multiwell plate with \#1.5 coverslip glass bottom. The refractive index of each solution was measured with a refractometer (Bellingham+Stanley, U.K.) before and after the fluorescence measurement and converted to viscosity using a function fitted to a conversion chart.\cite{Glycerine1963} A simplified diagram of the data acquisition experimental  setup is shown in Fig~\ref{fig:setup}a. The anisotropy experiments measurements  were performed with a  Leica TCS  SP2, a standard confocal inverted microscope. A pulsed diode laser (PLP-10 470, Hamamatsu, Japan; optical pulse width 90~ps) was used as the excitation source at 200~kHz repetition rate. rate (5~$\mu$s between pulses).  The beam was focused in the middle of the well containing the sample solution with a 20$\times$ NA0.5 air objective (Leica HC PL Fluotar). The emission was collected with the same objective through a 550~nm long-pass emission filter. A polariser was inserted in the emission path and parallel and perpendicular polarisation components of the fluorescence emission were recorded sequentially with a photomultiplier tube (PCM-100, Hamamatsu, Japan) connected to a time-correlated single photon counting (TCSPC) acquisition card (SPC 830, 150,  Becker\&Hickl GmbH, Berlin, Germany). The measurement time window was 5~$\mu$s, with data acquisition time of 30-60~min per data set. \subsection{Calculation of hydrodynamic radii}