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Relative Timing Modes 11

Other applications of the absolute times in TCSPC data are multi-parameter single-molecule

spectroscopy [19, 25, 31], burst-integrated lifetime (BIFL) experiments, and fluorescence in-

tensity and lifetime distribution analysis (FILDA) [23].

Imaging

The DPC-230 can be used for imaging applications. The sample is scanned by a focused laser

beam, the photons are detected with their absolute or relative times, and the image is built up

from the recorded data.

The imaging modes of the DPC-230 use the parallel architecture of the DPC module to record

both the photons and the synchronisation pulses from the scanner. Typically three scan syn-

chronisation signals are used. A ‘Frame Clock’ edge indicates the start of a new frame, a ‘Line

Clock’ edge the start of a new line, and a ‘Pixel Clock’ the transition to a new pixel. The clock

edges are recorded in parallel with the photons, and with their absolute times. By identifying

the clock edges in the data stream every photon is assigned to the pixel the scanner was on in

the moment when the photon was detected. The method is similar to the FIFO Imaging mode

of multi-dimensional TCSPC, a technique routinely used for fluorescence lifetime imaging

(FLIM) with laser scanning microscopes [1, 2]. The principle is illustrated in Fig. 14.

Frame Sync

Line Sync

Pixel Clock

from Laser

Detector

Built up in computer memory

Microscope

Scan

head

Ti:Sa or ps diode

laser

Light

pixels

Photon Distribution

n (x, y, t)

Time in

Photon times

Laser Times

Frame start times

Line start times

Pixel start times

decay curve

Location

scan area

DPC-230

Reference

Fig. 14: DPC-230 Imaging: FLIM with laser scanning microscope

The laser scanning microscope scans the sample with a pulsed laser beam. The fluorescence

light is fed to a photon counting detector via a suitable optical port of the microscope. The

single-photon pulses of the detector are connected to one channel of the DPC-230. A second

channel records reference pulses from the laser. The relative photon times, i.e. the differences

of the photon times recorded in the photon and laser channel, are the times of the photons

within the fluorescence decay.

The frame clock, line clock, and pixel clock pulses from the scanner are connected to three

other channels of the DPC-230. By analysing the events recorded in these channels, the loca-

tion of the laser beam in the scan area is obtained for each individual photon.

The fluorescence lifetime image is built up in the memory of the computer. A data array con-

tains memory space for all pixels of the scan, and, within each pixel, memory space for a large

number of time channels within the fluorescence decay. The software extracts the relative

photon times and the locations from the data stream, and builds up a photon distribution over

these parameters.

Please note that the principle not only works for slow scanning with piezo stages but also for

fast scanning by galvanometer mirrors. In the typical fast-scan applications the pixel dwell

time is on the order of a few microseconds or less. The pixel rate is then higher than the pho-

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