The association and dissociation of a fluorescent ligand was visualised and
quantified at the single-cell level using a perfusion system as described by
LSM710 confocal microscope. A pressure pump was connected to a closed
perfusion system, which allowed the constant perfusion of fluid from six
reservoirs through an imaging chamber and into a runoff (Figure 2.4). Cells
were grown to near confluence on 32 mm glass coverslips placed into wells of
6-well plates (see Cell culture) one day prior to experimentation. Where
transient transfection of cells with BiFC DNA constructs was required, the cells
were plated out three days prior to experimentation and then prepared as
described above (see Confocal microscopy). On the day of the experiment, the
growth medium was removed from one 6-well plate and the wells were
washed once in pre-warmed (37 °C) imaging buffer (HBSS containing 4.5 mM
D-glucose) and subsequently kept in 2 mL imaging buffer at 37 °C until used
for experimentation (the cells were kept in these conditions for no longer
than 2 hours). To start experimentation, the coverslip was placed into a
specially designed imaging chamber, which was tightly closed and then placed
onto a heated (37 °C) microscope stage where it was connected to tubes on
either side that facilitated the flow of fluid through the imaging chamber.
Following this, the reservoir supplying the imaging buffer was switched on to
perfuse over the cells in a laminar flow imaging chamber that holds a total
L
L M et al., 2010a). Following this, an area of cells was selected using the eyepiece of the microscope and the required laser, emission filters
and time series mode were set up. BODIPY-TMR-CGP was excited using a 543
nm Helium-Neon laser on the Zeiss LSM510 microscope and a 561 nm Helium-
Figure 2.4 The set-up of the closed perfusion system in conjunction with the Zeiss LSM510 confocal microscope. The pump allows the flow of fluid at constant pressure from a reservoir through the imaging chamber (imaging cell) and into a runoff. The entire system is temperature-controlled and kept at 37 °C. Figure taken from May et
560 and 565 nm long-pass filter, respectively; BODIPY630/650-S-PEG8-
propranolol was excited using a 633 nm argon laser and a 650 nm long-pass
filter at both confocal microscopes. Where cells transiently transfected with
BiFC DNA constructs were used, YFP and BODIPY-TMR-CGP were excited using
a 488 nm argon laser and 561 nm Helium-Neon laser, respectively with
emission being captured through a 505-550 nm narrow band-pass and a 565
nm long band-pass filter, respectively (these experiments were carried out
using the Zeiss LSM710 confocal microscope).
Association and dissociation kinetic binding experiments
The association and dissociation kinetics of increasing concentrations (3-100
nM) of BODIPY-TMR-CGP and BODIPY630/650-S-PEG8-propranolol were
investigated. Both fluorescent ligands used in perfusion experiments were
diluted in ddH2O to working concentrations (100x desired final concentrations)
and subsequently kept on ice. When used, 400 µL of the ligand (at working
concentration) was added to 40 mL imaging buffer in a designated reservoir.
First, a 20-30 second baseline fluorescence read was taken in the presence of
only imaging buffer. To initiate association of the fluorescent ligand, the flow
of fluid from the reservoir containing imaging buffer (reservoir 1) was stopped
and the reservoir containing the fluorescent ligand (reservoir 2) was switched
on for a given length of time (e.g. 5 minutes). After that, flow of fluid from
reservoir 2 was stopped and reservoir 1 was again switched on to begin
dissociation of the fluorescent ligand (generally performed for the same
remove the labelled ligand, which is possible due a sharp change in
concentration caused by the selected pressure and flow rate for these
experiments (May et al., 2010a). Throughout the experiment (from baseline
to dissociation read) transmitted light and fluorescence images were captured
every 2-3 seconds (512x512 pixels, 2 averages per images). The first glass
coverslip (slide) was used to establish the optimal microscope settings (laser
power, gain and offset) as described above in Confocal microscopy. These
settings were then kept constant within each experiment and between
CHO 1-CS (used to
measure total binding) and CHO-CS cells (used to measure non-specific
binding levels) to allow for direct comparison of total and non-specific
fluorescent ligand binding levels at the different concentrations tested.
Dissociation kinetic binding experiments in the presence of
unlabelled ligands
All unlabelled ligands were diluted in ddH2O to working concentrations
(10,000x desired final concentration) and 4 µL of the ligand at working
concentration was added to 40 mL imaging buffer in designated reservoir
(reservoir 3). The association of the fluorescent ligand was achieved as
outlined above. Following association, the flow of fluid from reservoir 2
(imaging buffer containing the fluorescent ligand; as defined above) was
stopped and reservoir 3 was switched on, thus initiating the dissociation of
the fluorescent ligand in the presence of an unlabelled ligand (for a given
any unlabelled ligand were performed on each experimental day. Transmitted
light and fluorescence images were captured every 2-3 seconds and the
microscope settings were set using the first slide (as described above) and
then kept constant within each experiment, but adjusted, if necessary,
between experiments on different days. Dissociation kinetic data was
therefore expressed in % fluorescent intensity, allowing experimental data
from different days to be grouped.
Data collection
During each experiment, 8bit images recording the fluorescence intensity of
the fluorescent ligand binding to cells in the perfusion imaging chamber were
captured. To analyse and quantify the association and dissociation kinetics of
the fluorescent ligands, regions of interest (ROIs) were drawn around the
membranes of ten single cells of each imaged slide. Cells containing
oversaturated pixels were identified using the range palette and were
excluded from the analysis. For cells with low fluorescent intensity, the
transmitted light image was used to aid cell selection. The Zeiss software then
provided average pixel intensity values for each ROI (10 ROIs per slide), which
were plotted against time. The changes in fluorescent intensity over time
were then analysed to obtain association and dissociation rates (see Data
analysis) for either each single cell or per slide (i.e. grouped data from 10
single cells). For kinetic data shown in this thesis, an n number of 1 refers to
data obtained from one slide. Within each experiment, each condition (e.g.
Throughout this thesis, the number of different experimental days, in which
the n numbers were acquired, is also stated.