deletions | additions       diff --git a/Method.tex b/Method.tex  index 2c4e641..259a7c9 100644  --- a/Method.tex  +++ b/Method.tex 
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\subsection{Sample preparation}  All protein solutions were diluted with sterile phosphate buffered saline (PBS; Sigma; pH~7.4) to 5mg/mL and then dialyzed 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 confirmed by standard SDS-PAGE. The proteins were then conjugated to the ruthenium dye. This was achieved by reacting the succinimidyl ester-modified dye, Bis(2,2′-bipyridine)-4′-methyl-4-carboxybipyridine-ruthenium N-succinimidyl 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. excess (2-fold and 3.5-fold for proteins >60~kD and ranibizumab, respectively).  The reaction was stopped after two hours and conjugated proteins were separated from remaining free dye by size exclusion chromatography (two times via 7~kD cut-off Zeba\textregistered spin columns (Thermo Fisher, UK)). Using this method we generated ranibizumab the various proteins  conjugated to dye with a the fluorophore in the following  dye:protein ratio of 1.1:1 and ratios: ranibizumab 1.1:1; BSA 1.2:1; aflibercept 1:1.8;  bevacizumabwith a dye:protein ratio of  1.3:1. Azide (2.0~mM) was added to all dye-conjugated drugs to protect them from microbial deterioration.*** Add Eylea and BSA ***  \subsection{Anisotropy measurements} 
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A simplified diagram of the experimental setup is shown in Fig~\ref{fig:setup}a. The anisotropy measurements were performed with a Leica TCS SP2, a standard confocal inverted microscope, equipped with a pulsed diode laser (PLP-10 470, Hamamatsu, Japan; optical pulse width 90~ps) that served as the excitation source (200~kHz repetition rate, 5~$\mu$s interval 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 hybrid detector (Becker \& Hickl GmbH, Berlin, Germany) connected to a time-correlated single photon counting (TCSPC) acquisition card (SPC 150, Becker\&Hickl GmbH, Berlin, Germany). The measurement time window was 5~$\mu$s, with 4096 time channels and 1.22 ns/ch, and total data acquisition time of 30-60~min per data set.  \subsection{Calculation of hydrodynamic radii}  The anisotropies were calculated from the phosphorescence intensity decays measured in parallel and perpendicular polarisation directions with eq~\ref{eq:anisotropy} (see Fig~\ref{fig:setup}b,c). The anisotropies contain a fast component in addition of the expected longer component and were fitted with gnuplot V4.6 \cite{gnuplot} to a double-exponential function: