Biology Reference
In-Depth Information
10
5
cells/well, and transfected after 1 day using jetPEI according to the in-
structions provided by the manufacturer (Ozyme, Saint-Quentin-en-Yvelines,
France). After 24 h and at least 2 h before imaging, the medium is replaced
two times by PBS and then by DCC-DMEM:F12. pH is stabilized by 10 mM
HEPES buffer (Gibco, Paisley, Scotland). Cells are then kept at 37
C before
imaging.
3
2.1.6.2.2
Fluorescence cross-correlation microscopy
FCCS measurements are conducted using a setup based on a dual-channel ISS Alba
fluorescence correlation detector (ISS, Champaign IL) with avalanche photodiodes
and a Zeiss Axiovert 200 microscope (Zeiss, Jena, Germany). The sample is excited
by means of a Mai Tai HP fs IR tunable laser (Spectra-Physics, Newport, Mountain
View, CA) tuned to 935 or 950 nm. The excitation light is focused into the sample by
a Zeiss Apochromat 63X oil immersion objective (numerical aperture 1.4), and an
E700 SP 2P dichroic filter (Chroma Technology Corporation, Rockingham, VT)
is used to eliminate the contribution of the IR excitation light in the detected signal.
A 505 nm dichroic mirror is used to split the detected light onto two channels, and
additional 653
50 nm band pass filters are set before channels 1 and 2,
respectively (Chroma Technology Corporation, Rockingham, VT). The excitation
power is set at
<
10 mW at the scope entrance in all FCCS measurements. This power
is determined to be a threshold, one at which the autocorrelation traces became ex-
citation power-independent, in order to avoid excitation saturation effects (
Nagy,
Wu, & Berland, 2005a,2005b
) and photobleaching (
Petrasek & Schwille, 2008
).
The microscope is equipped with a Piezoelectric stage (Mad City, Madison, WI)
for 3D imaging.
The two ERs are expressed in COS-7 cells as N-terminal fusions with the blue
FP, cerulean (cer-ER
a
and mCherry-ER
b
). More importantly, previous results
(
Savatier, Jalaguier, Ferguson, Cavailles, & Royer, 2010
) for expression of cer-
ER
a
alone showed no bleed-through from the blue detection channel into the
red channel. This is important, since such bleed-through is 100% cross-correlated
and, hence, would introduce a substantial artifact into the FCCS measurements.
Reasonable detection levels for both cerulean and mCherry are achieved with
two-photon excitation at 950 nm, using
50 and 455
10 mW at the microscope entrance
to limit photobleaching. Under these excitation conditions, cer-ER
a
exhibits a
molecular brightness of 3000 cpspm (dimer); that of cer-ER
b
is slightly higher,
4500 cpspm (dimer).
<
2.1.6.2.3
Data analysis
Three curves are generated by time correlation of the fluorescence intensity
fluctuations detected, two autocorrelation functions arising from the blue and red
fluorophores
G
B
(
t
) and
G
R
(
t
), and one cross-correlation function, which accounts
for the molecules in which the two fluorophores diffuse together
G
x
(
t
). The
