Chemistry Reference
In-Depth Information
(c) To avoid losing the QD during the off period and switching the track to another
on QD, we compare the newly found track position against the probability of a
jump distance equal to or greater than the observed jump distance, r, by diffusion
in the time
e
r
2
=4DDt
where D is an assumed diffusion constant.
If this probability is less than 5%, we search future time-frames for an on
position with higher probability. If one is found, we mark the QD as off until
tracking continues at the future time-fame. If no higher probability events are
found, we accept the unlikely jump position.
D
t, P
ð
r
; D
t
Þ¼
EGFR on the cell body
and
filopodia of cells treated with the kinase inhibitor PD153035. As shown in
Figure 6.4, analysis of the trajectories from single QDs yield mean square displace-
ment (MSD) curves that could be
fit to retrieve diffusion constants [9]. The MSD
calculated from the trajectory in Figure 6.4
ts well to a corralled diffusion
model MSD
Using this algorithm we imaged at video rates QD
EGF
-
-
2
e
Dt=T
where L is the diameter of the con
nement zone,
T is the time required to explore the zone, D
L
3
ðD
t
Þ¼
offset
þ
L
2
/(12T) and the offset accounts for
localization error. The parameters derived from this
t were: D
¼
m
2
/s;
¼
0.05
m
T
m. A histogram of the values derived from these data is
shown in Figure 6.4e. The mean diffusion constant of QD
-
EGF
-
EGFR was
0.021
¼
0.39 s and L
¼
0.48
m
m
2
/s on
filopodia; i.e. there
were no discernable differences in the motion at short time scales for the two cases.
The MSD plots indicate that in the presence of kinase inhibitor, the receptors
underwent corralled diffusion, and not transport. A complete analysis of single
molecule diffusion of the EGFR on the cell body as well as on
filopodia has
been conducted and compared with data obtained by FRAP of the unliganded
receptor (K. A. Lidke et al., unpublished data).
m
2
/s on the cell body and 0.015
0.022
m
0.013
m
6.5
Programmable Array Microscopy
Dual pass programmable array microscopes (PAMs) are de
ned by the use of a
spatial light modulator (SLM) in a primary image plane of a standard
uorescence
microscope. The SLM provides for structured illumination as well as conjugate
descanning of the image to achieve optical sectioning. The major advantages of the
PAMare: (i) simple, inexpensive design with nomoving parts; (ii) speed-up in optical
sectioning due to an illumination duty cycle for each pixel of up to 50%; (iii) optimal
detection sensitivity, e.g. using emCCD cameras; (iv) continuously programmable,
arbitrary, and adaptive optical sectioning modes between or within images using
libraries of dot, line, or pseudo-random (Sylvester) sequence patterns; (v) ef
cient
and sensitive optical sectioning due to simultaneous detection and processing of both
conjugate and non-conjugate light; (vi) compatibility with polarization, hyperspec-
tral, lifetime-resolved, and other imaging modes; and (vii) minimal photobleaching.
Our initial implementation of the PAM [10
-
13] used a digital micromirror device
(DMD) for optical sectioning or transmissive liquid crystal SLMs for imaging
