Liisa Hirvonen edited Method.tex  over 8 years ago

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\section{Method}  \subsection{Reagents}  Ranibizumab was purchased from Novartis (Frimley, UK) (Lucentis 10~mg/ml) and bevacizumab from Roche (Welwyn Garden City, UK) (Avastin 25~mg/ml). All other reagents were of the highest available purity and were either from Sigma (Poole, UK), VWR (Lutterworth, UK), or Merck (Hoddesdon, UK) unless otherwise specified. 

*** Add Eylea and BSA ***  \subsection{Tr-FAIM \subsection{Anisotropy  measurements} The Ru-labelled molecules in buffer were mixed with glycerol in different proportions to produce solutions with different viscosities. 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 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.  \begin{figure}[tbp]  \centerline{\includegraphics[width=1\columnwidth]{setup}}  \caption{\label{fig:setup} Schematic diagram of the anisotropy measurements. (a) Simplified diagram of the experimental setup. The sample solution was placed on a confocal microscope stage and excited with a polarised, pulsed laser. The fluorescence from the sample was reflected from a dichroic mirror (DM) and recorded with a PMT. (b) Schematic of the fluorescence decays in parallel and perpendicular polarisation directions, from which the anisotropy (c) was calculated with eq~\ref{eq:anisotropy}.}  \end{figure}  \subsection{Calculation of hydrodynamic radii} 

\end{equation}  where $k$ is the Boltzmann constant, $T$ is the absolute temperature and $\phi/\eta$ is the gradient of the straight line.