Chemistry Reference
In-Depth Information
Technology). Fast tracking of QDs was performed with 20-ms exposure times at 30-
ms intervals with the emCCD using 655QD
-
EGF conjugates.
6.A.6
PAM
The new generation programmable array microscope (PAM) is a prototype
developed in collaboration with Cairn Research Ltd. (Faversham, Kent, UK). The
stand-alone module, including light source(s) and detector(s), features an inno-
vative catadioptric design and a ferroelectric liquid-crystal-on-silicon (LCoS, SXGA-
R2D, Forth Dimension Displays, Dunfermline, Scotland) SLM instead of the
original DMD used in the
first PAM design. The LCoS-based PAM can be attached
toacameraportofanyunmodi
ed
fluorescencemicroscope. The prototype system
currently operated at the Max Planck Institute for Biophysical Chemistry
(Gottingen, Germany) incorporates a 6-position high-intensity LED illuminator
as well as modulated laser light sources (both diode lasers and AOM-modulated
argon-ion lasers), and an Andor iXon emCCD camera. The system is mounted on
an Olympus IX71 inverted microscope with 60
-
150
objectives, a high precision
X,Y,Z stage (Nanoscan Z), and high speed
filter wheels (Prior Scienti
c, Cam-
bridge, UK).
6.A.7
Hyperspectral Imaging
Hyperspectral imaging was performed with an Applied Spectral Imaging (Migdal
Haemek, Israel) SpectraCube imaging spectrograph equipped with a VDS cool-
1300 camera and mounted on the IX71microscope. QD samples were imaged with
a 150
1.45 NA objective. Excitation was with a Hg arc lamp and the epi-
illumination
filter set consisted of 435-nm narrow bandpass excitation, 505-nm
dichroic, and 510-nm longpass emission
filters. Streptavidin-coated QDs emitting
at 585, 605, or 655 nm were diluted from the original stock concentration to
nal
concentrations of
1 pM (QD655, PEG coated, QD585, and QD605) in PBS.
Approximately 25
l of the diluted solution was pipetted onto an Ibidi (Munich,
Germany) 18-well, poly-L lysine-coated slide and allowed to bind to the surface for
5min. The samples were washed once with water and maintained in water. A
mixture of all three QD types was also prepared. Hyperspectral images in the range
500
-
800 nm were acquired in 185 s, using 128 frames (1-s exposure) and 45
interferometer steps between each frame. After Hanning windowed-Fourier
transformation, single QDs were marked and classi
ed with the ASI SpectraView
software. The edge of each full
field image was selected as background, and the
spectrum of this area was subtracted from the individual QD spectra. For cellular
experiments not discussed in this chapter [7], a 1 : 1 mixture of 200 pM EGF-
QD605 and 200 pM EGF
-
QD655 were incubated with F1-4 CHO cells expressing
EGFR eGFP. These cells were not treated with kinase inhibitor as in the single
m
