Chemistry Reference
In-Depth Information
Figure 6.3 Time series and particle track
showing movement of a QD
indicated QD
-
EGF
-
EGFR complex (A,
arrowhead) on a filopodium that exhibits
random diffusional movement (black) until the
addition of unlabeled EGF (green box), after
which the complex begins active retrograde
transport (red). (Figure from [3], reproduced by
permission).
EGF
EGFR on an
-
-
A431
eGFP cell. (A) Selected frames from a time
series after binding 5 pMEGF
-
QD (red) followed
by addition of free EGF (50 ng/ml) at 300 s. Bar,
5
-
m. Images are contrast enhanced and were
taken with a Zeiss LSM 510. (B) Trajectory of the
m
receptors suggests that
filopodia function as sensory organelles for the cell, probing
for the presence and concentration of effector molecules distant from the cell body,
and coupling sensing to cellular response via a directed transport of receptors
activated upon achieving a given threshold of ligand [3].
6.4
Single QD
-
EGF
-
EGFR Tracking
Measurements of QD
-
EGF
-
EGFR diffusion using single particle tracking methods
in laser scanning confocal microscopes yielded diffusion constants an order of
magnitude slower than those reported in studies of EGF
-
EGFR based on
uores-
cence recovery after photobleaching (FRAP) or
uorescence correlation spectroscopy
(FCS) [3]. In this chapter, we present new data acquired with a fast emCCD camera of
kinase-inhibited QD
-
EGF
-
EGFR under conditions permitting visualization of
single molecules.
AlthoughQDblinking can be advantageous, i.e. for identifying single particles and
achieving spatial superresolution [8], the phenomenon complicates the analysis of
tracking due to the transient interruption of the signal. We have developed special
data processing routines to take into account the random blinking periods and to
achieve automated identi
cation and tracking of the QD
EGF
EGFR complexes.
-
-
De
nitions and procedures are as follows:
(a) An on QD signal is de
ned as an intensity at least 50%of the running average of
all QD intensities. Intensities below this threshold are considered off.
(b) Positional information is only stored when the QD is on.
