Chemistry Reference
In-Depth Information
degradation of the acquired confocal images: lack of a suitable, easy to use video
interface for scanning pattern de
nition, diffraction effects, limited VGA format,
and a low micromirror tilt angle. In addition, the imaging of conjugate (in-focus
light from on pixels) and non-conjugate (out-of-focus light from off pixels) on a
single camera was not feasible.
We have developed a new PAM in collaboration with Cairn Research Ltd.
(Faversham, UK) based on a ferroelectric LCoS microdisplay (Forth Dimension
Displays, Dunfermline, Scotland). This microdisplay has several favorable char-
acteristics for use in a PAM: SXGA resolution, high
fill factor (93%), good contrast
(400 : 1), and a fast
s). When used for video
projection, this microdisplay operates in a 24-bit color-sequential mode; each video
frame is broken into 24 adjustable-length bitplanes, eight each for red, green and
blue. For PAM operation, the color-sequential mode is disabled and each of the 24
bitplanes is displayed for the same length of time, refreshing at 1.44 kHz. Scanning
patterns are de
ned by simple display of a 24-color Windows bitmap
file via a
standard computer DVI video interface. Possible scanning patterns include those
based on dot lattices, line arrays, or pseudorandom (Sylvester) sequences or
arrays [10, 13, 20]. An on pixel is one in which plane polarized light is re
ected
with a 90-degree rotation in the plane of polarization. Off pixels re
ect light with
no change in polarization state. Light sources (LEDs, lasers, lamps) must thus be
linearly polarized, either intrinsically, or by means of a polarizing beam splitter
cube. Light of both linear polarization states (horizontal and vertical) is projected
onto the microdisplay. Since both on and off pixels are re
ected along the optical
axis, an image splitter arrangement allows the conjugate (in-focus) and non-
conjugate (out-of-focus) light to be imaged on a common detector, in our case an
emCCD (Ixon DV885 or DV887, Andor Technology, Belfast, Northern Ireland).
Spinning disk confocal microscopes also generate conjugate images; these are
optically sectioned images with an offset due to cross-talk between the many
pinholes. The PAM has the advantage that a scaled subtraction can be performed
using both the conjugate and non-conjugate images to generate a confocal image
with no DC offset [13, 21]. In addition, the sectioning capability is fully program-
mable and can achieve very high duty cycles. To generate the
liquid crystal switch time (40
m
final image, the
conjugate and non-conjugate images are registered and subtracted, with scaling
factors applied to both images that depend on the pattern used [13]. Image
registration, scaling, and if desired, background subtraction,
filtering, and other
image processing operations are performed in real time (processing and display
require 10ms per image) using a workstation-class graphics card and BrookGPU, a
graphics processing unit
(GPU) programming library freely available on the
Internet [22].
To demonstrate the sensitivity and high speed optical sectioning ability of the
PAM, we imaged single QDs bound to A431 cells expressing the EGFR. The QDs
were added at a concentration of 200 pM, allowed to bind for 3min at room
temperature, then removed by washing with Tyrodes buffer. Imaging was started
immediately at a rate of
6Hz using an exposure time of 33ms. Three selected
frames of a 300-frame
lmare shown in Figure 6.5. Binding is similar to that shown
in Figure 6.2.
