deletions | additions       diff --git a/Method.tex b/Method.tex  index 263b4d2..d4db501 100644  --- a/Method.tex  +++ b/Method.tex 
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\section{Method}  \subsection{Reagents}  ***This subsection needs to be checked (+Eylea)*** 
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***This subsection needs to be checked (+Eylea).***  Ranibizumab and bevacizumab solutions weretwice  dialysed twice  against phosphate buffered saline (PBS)  pH 7.4(PBS)  using D-Tube Midi Dialyzer units from Novagen (6-8~kD cut-off). Subsequently, the purity of the dialyzed dialysed  proteins was checked by standard SDS-PAGE. The proteins were then conjugated to the fluorophore Bis(2,2′-bipyridine)-4′-methyl-4-carboxybipyridine-ruthenium bis(hexafluorophosphate) (synonym, N-succinimidyl ester-Ru(bpy)2(mcbpy-O-Su-ester)(PF6)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 (7kD (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. \subsection{Tr-FAIM measurements} 
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A simplified diagram of the data acquisition setup is shown in Fig~\ref{fig:setup}a. The anisotropy experiments were performed with Leica 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. 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, 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}  The anisotropies were calculated from the intensity time 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 to a double-exponential function: 
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R_h=\sqrt[3]{\frac{3kT}{4\pi}\frac{\phi}{\eta}} \label{eq:R_h}  \end{equation}  where $k$ is the Boltzmann constant, $T$ is the absolute temperature and $\phi/\eta$ is the gradient of the straight line.