Chemistry Reference
In-Depth Information
In this chapter we feature (i) the retrograde transport of activated EGFR; (ii) the
diffusional behavior of non-activated, kinase-inhibited single receptors on the cell
body and
filopodia using a high speed and high sensitivity electron multiplying CCD
(emCCD) camera; and (iii) the rapid, light ef
cient three-dimensional (3D) imaging of
the early binding events of individual QD-EGF ligands with a commercial prototype of
a new generation, optically sectioning programmable array microscope (PAM).
6.2
Single QD Imaging
The excitation and emission spectra of commercially available QDs from Invitrogen
are shown in Figure 6.1. Due to their high absorption cross-section in the low visible
and UV range and emission in the red to far red, QDs are ideal emitters for signal
acquisition by CCD cameras. However, the relatively long lifetimes (10
-
20 ns)
implies that their maximal rate of
fluorescence emission (in terms of photon
ux)
is somewhat limited [5]. Imaging single QDs in raster scanning systems, particularly
with PMT detectors, requires long pixel dwell times, and high laser powers. Such
systems are not ideally suited for observing diffusion rates or rapid, live processes. On
the other hand, CCD cameras have high sensitivity at long wavelengths, and
emCCDs in particular have superior sensitivity for short acquisition times at low
light levels [6]. These features were exploited in the experiments presented here. In
addition, a versatile optical sectioningmicroscope, the PAM, was used tomeasure the
initial steps of QD-EGF binding and EGFR activation, and the diffusion of individual
(mono-liganded) QD-EGFR complexes in three dimensions.
In order to take full advantage of the potential afforded by the unique character-
istics of QDs, wide-
eld imaging systems generating continuous emission spectra at
every pixel position are also highly desirable. Fourier encoding and CCD detection
combines very ef
cient detection and spectral reconstruction of low-light level
images. To record single QD spectra, we used a commercial Fourier interferometric
spectrograph, the SpectraCube manufactured by Applied Spectral Imaging (Migdal
Haemek, Israel). With the very high magni
cation afforded by a unique Olympus
150
1.45 NA objective mounted in an IX71 microscope, we obtained very distinct
signals attributable to individual QDs either attached to a surface transpore or in
cells [7] (Figures 6.1b and c).
6.3
Retrograde Transport of Activated EGFR Dimers
The epidermal growth factor receptor (EGFR) is a single chain integral transmem-
brane protein with an ectodomain encompassing the EGF peptide binding site. The
receptor is distributed on the cellular plasma membrane, including the
lopodia
(Figure 6.2). Filopodia are
fine processes extending from the cell body with a core of
actin bundles, the
filaments of which have pointed ends oriented towards the cell
