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Nanosecond Fluorescence Lifetime Imaging using a directly gated interline transfer CCD detector by Photonic Research Systems Ltd. Web: www.prsbio.com. Material(c) Photonic Research Systems Ltd. 2005. Experimental Details. Sensor: 2/3” format monochrome interline CCD sensor,

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slide1

Nanosecond Fluorescence Lifetime

Imaging using a directly gated interline

transfer CCD detector

by

Photonic Research Systems Ltd

Web: www.prsbio.com

Material(c) Photonic Research Systems Ltd. 2005

slide2

Experimental Details

Sensor: 2/3” format monochrome interline CCD sensor,

peltier cooled to -18oC

Light Source: Diode pumped, frequency tripled solid-state

pulsed laser with active Q-Switching. Pulse repetition rate

was 5kHz, wavelength 355nm, pulse width at half maximum

was 1.2 nsec, laser jitter was <1nsec.

Samples: Two solutions of fluorescein were prepared one

having twice the emission intensity of the other. The brighter

sample was then quenched with potassium iodide until its

intensity matched that of the lower intensity sample

Web: www.prsbio.com

Material(c) Photonic Research Systems Ltd. 2005

slide3

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). This image was taken with the Imagex camera in steady state mode under laser illumination and with some ambient light.

Web: www.prsbio.com

Material(c) Photonic Research Systems Ltd. 2005

slide4

Steady-State Fluorescence image of Fluorescein

samples with no background light

Web: www.prsbio.com

Material(c) Photonic Research Systems Ltd. 2005

slide5

Time-Resolved images of

Fluorescein Samples

quenched

unquenched

quenched

unquenched

quenched

unquenched

2ns delay

4ns delay

6ns delay

Delay following laser pulse

Web: www.prsbio.com

Material(c) Photonic Research Systems Ltd. 2005

slide6

The next slide shows the resultant decay-weighted image

when the appropriate pair of time-resolved images are

divided. A psuedo-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

quenched

unquenched

Web: www.prsbio.com

Material(c) Photonic Research Systems Ltd. 2005

slide7

Ratio ‘FLIM’ image of Fluorescein

Samples

N.B. the background ‘noise’ is due to the division of pixel

values close to zero

Notice how the lifetime ratios are clear even where

the source signal is very weak. The low noise of the Imagex NanoCCD gives excellent dynamic range

quenched

unquenched

Web: www.prsbio.com

Material(c) Photonic Research Systems Ltd. 2005

slide8

Combining Intensity and Lifetime

information in a single image

Intensity Image

Intensity/FLIM Image

quenched

unquenched

‘Flim’ (Ratio) Image

The Imagex Matrix Dialog allows the user

to select 2 source images and adjust

their contrast settings. The images are

then combined into a single ‘Matrix’ image

Web: www.prsbio.com

Material(c) Photonic Research Systems Ltd. 2005

slide9

Intensity-Lifetime ‘Matrix’ image

of Fluorescein Samples

Combining the Intensity and Flim Images

in this way helps to suppress artifacts created by performing

ratio operations on areas of very low intensity.

quenched

unquenched

quenched

unquenched

Web: www.prsbio.com

Material(c) Photonic Research Systems Ltd. 2005

slide10

Future Work

Investigation of smaller format and higher resolution

CCD sensors

Investigate Improved DPSS Light Source:

0.8 nsec FWHM with jitter <0.5nsec now available.

Mount System on Microscope for real biological

applications!

Web: www.prsbio.com

Material(c) Photonic Research Systems Ltd. 2005