Truecolor nanosecond fluorescence imaging

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True-color nanosecond fluorescence imaging for
multiplexed detection of fluorescent labels
C. G. Morgan and A. C. Mitchell
Biomedical Sciences Research Institute, Universuty
of Salford, Salford UK and Photonic Research
Systems Ltd, Salford UK
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FLIM has usually needed an intensified camera to
achieve nanosecond time resolution
Intensifiers are-- expensive noisy easily
damaged
We have investigated the use of a directly
gated CCD camera with a pulsed laser for FLIM
lower noise immune to damage better
resolution excellent red sensitivity wider
dynamic range
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An interline transfer CCD used with a fast
pulsed light source can now achieve nanosecond
time resolution or better This opens up new
possibilities for FLIM
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Pixel
column
Vertical
transfer
register
Charge drain to reset sensor
Horizontal shift
register
output to
framestore
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Timing Diagram for collection of time-resolved
image
Light Source
dt
Trigger
Pulsed
Excitation
Light Source
This fraction of the
emission is transferred
to the
This fraction of the
Storage Region
emission is drained to
the substrate
End of
Transfer
Drain
Pulse
Pulse
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If the vertical transfer register is not read out
between pulses of light it is possible to use it
to integrate charge to improve S/N ratio as shown
in the next slide
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The shielded region can be used to
integrate repetitive signals until a useful
level is achieved
Store
Store
Store
Store
Discard
Discard
Discard
Discard
Light Pulses
Start Exposure
Read Out Signal
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The following images were measured using the
Imagex TGi nanosecond CCD imager in conjunction
with a diode-pumped solid state, actively
Q-switched, frequency-tripled laser emitting
pulses of 1.2 ns at 355nm with a time-averaged
power of 4mW and a repetition rate of 5kHz, The
trigger jitter is quoted as 1.5ns
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Experimental layout for time-gated imaging
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Cuvettes of fluorescein illuminated by pulsed UV
laser
One cuvette holds fluorescein in buffer (4ns)
while the other has a sample of twice the
concentration but quenched with potassium iodide
to match the dilute sample (2ns). The image was
taken with the Imagex camera in steady state mode
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Steady-State Fluorescence image of Fluorescein
Samples with no background light
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Time-Resolved images of Fluorescein Samples
2ns
4ns
6ns
Delay following laser pulse
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The next slide shows the resultant decay-weighted
image when the appropriate pair of time-resolved
images are divided. A pseudo-colour look-up
table is used to show the lifetime
differences. Note the very high signal-to-noise
ratio, which is evident even in the uniform
colour of the pixels representing very weak
signals from reflections at the meniscus and
base of the cuvettes
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N.B. the background speckle is due to the
division of pixel values close to zero
quenched
unquenched
Ratio FLIM Image
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The next slides show the time resolution and high
S/N ratio that can be achieved with the
solid-state FLIM technology. Samples of quinine
sulphate can be progressively quenched down to
subnanosecond lifetime using chloride ion. The
images show a set of quenched samples and a
decay-time weighted image. Also shown is a
matrix image produced by multiplying the decay
image by a normal intensity image. This
encodes fluorescence intensity and decay time
simultaneously and suppresses noise due to
division of pixels with near-zero signal
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FLIM of Quinine
Sulfate
solutions quenched

with
NaCl
0.5 ns
1 ns
1.5 ns
2ns
0 ns delay, 0.8
secs
exposure
pseudo-colour ratio image
8 ns delay, 5
secs
exposure
N.B. the delay refers to the laser trigger,
which precedes the lamp pulse by several
nanoseconds
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Colour-masked CCDs are commercially
available For the first time we demonstrate the
use of a true- colour CCD detector for nanosecond
time-gated imaging The final slide aslo shows
how the time-gated images can be combined to
generate a conventional FLIM image if required.
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Standard interline CCD chips are available with
colour masks for true-colour imaging...thus they
allow TRUE-COLOUR NANOSECOND TIME-GATED IMAGING
as well as conventional FLIM to be implemented
readily
CYMG mask
RGB mask
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True-colour time-gated imaging
delay8ns, exposure 3secs
delay0, exposure 1sec
delay22ns, exposure 7secs
Cuvettes contain aqueous solutions of
sulforhodamine-B (t c.2ns), sodium fluorescein
(t c.4ns) and quinine sulphate (partially
quenched (t c.8ns))
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Colour-TGI can also generate conventional FLIM
images of high quality
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Acknowledgements
Research on novel applications of nanosecond
time-resolved imaging to multiplexed detection is
currently funded by a UK research grant from
BBSRC. Closely related work on time-resolved
sensor technology is similarly funded by EPSRC,
to whom we are also grateful.