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Liisa Hirvonen edited Method.tex
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The 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 up to $\sim$70\% volume fraction glycerol. A drop of each mixture was placed in a multiwell plate with \#1.5 coverslip glass bottom. The refractive index of each solution was measured with a refractometer (Bellingham+Stanley, U.K.) before and after the fluorescence measurement and converted to viscosity using a function fitted to a conversion chart.\cite{Glycerine1963}
A simplified diagram of the experimental setup is shown in Fig~\ref{fig:setup}a. The anisotropy measurements were performed with a Leica TCS SP2, a standard confocal inverted
microscope. A microscope, equipped with a pulsed diode laser (PLP-10 470, Hamamatsu, Japan; optical pulse width 90~ps)
was used that served as the excitation source
at 200~kHz (200~kHz repetition
rate (5~$\mu$s rate, 5~$\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:
\begin{equation}
y = A_1\cdot e^{-\frac{t}{\phi_1}} + A_2\cdot e^{-\frac{t}{\phi_2}} \label{eq:2expfit}
\end{equation}