Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 105
fier (PGA) and converted to a numerical value by the analog-to-digital
converter (ADC). To minimize false readings
the signal is restricted to given range of voltages. If the signal
is not within this range the event is suppressed by a window
discriminator (WD). The voltage is converted to a digital
value that is stored as a single event with the measured
time delay. A histogram of the decay is measured by repeating
this process numerous times with a pulsed-light source.
The principle of TCSPC can be understood from the
preceding description. However, at present almost all
TCSPC measurements are performed in the "reverse
mode." The process is the same as described above except
that the emission pulse is used to start the TAC and the excitation
pulse is used to stop the TAC. This procedure is used
because of the high repetition rate of modern pulsed-light
sources. The TAC has to be reset and set to zero before each
start pulse, which takes a finite amount of time. The TAC
can be constantly in reset mode if the start signals arrive too
rapidly. The emission signals occur about 1 per 100 excitation
pulses, and thus much less frequently than the excitation
pulses. These emission pulses are used to start the TAC,
and the next laser pulse is used to stop the TAC.
There are many subtleties in TCSPC that are not obvious
at first examination. Why is the photon counting rate
limited to 1 photon per 100 laser pulses? Present electronics
for TCSPC only allow detection of the first arriving photon.
The dead times range from 10 microseconds in older
systems to about 120 ns with modern TCSPC electronics.
These times are much longer than the fluorescence
decay. The dead time in the electronics prevents
detection of another photon resulting from the same excitation
pulse. Recall that emission is a random event. Following
the excitation pulse, more photons are emitted at
early times than at late times. If all these photons could be
measured, then the histogram of arrival times would represent
the intensity decay. However, if many arrive, and only
the first is counted, then the intensity decay is distorted
to shorter times. This effect is described in more detail in
Section 4.5.6.
Another important feature of TCSPC is the use of the
rising edge of the photoelectron pulse for timing. This
allows phototubes with ns pulse widths to provide subnanosecond
resolution. This is possible because the rising
edge of the single-photon pulses is usually steeper than one
would expect from the time response of the PMT. Also, the
use of a constant fraction discriminator provides improved
time resolution by removing the variability due to the
amplitude of each pulse.
4.3.2. Example of TCSPC Data
Prior to examining these electronic components in more
detail it is valuable to examine the actual data. Intensity
decay for the scintillator 2,5-diphenyl-1,3,4-oxadiazole
(PPD) is shown in Figure 4.9. These data were obtained
with a cavity-dumped R6G dye laser that was cavitydumped
at 1 MHz and frequency-doubled to 300 nm. The
detector was an MCP PMT. There are typically three curves
associated with an intensity decay. These are the measured
data N(t k ), the instrument response function L(t k ), and the
calculated decay N c (t k ). These functions are in terms of discrete
times (t k ) because the counted photons are collected
into channels each with a known time (t k ) and width (∆t).
The instrument response function (IRF) is the response of
the instrument to a zero lifetime sample. This curve is typically
collected using a dilute scattering solution such as
colloidal silica (Ludox) and no emission filter. This decay
represents the shortest time profile that can be measured by
Figure 4.9. TCSPC data for 2,5-diphenyl-1,3,4-oxadiazole (PPD) in
ethanol. The light source was an R6G dye laser, cavity dumped at 1
MHz. The detector was an R2809 MCP PMT (Hamamatsu). The left
side of the residuals (lower panel) show some minor systematic error.
From [15].