Principles of Fluorescence Spectroscopy

116 TIME-DOMAIN LIFETIME MEASUREMENTS
tichannel analyzer (MCA). There were some initial concerns
about the linearity of the TAC on the computer boards
as compared to the NIM bin systems. However, the problem
has been solved and there is no longer a need for NIM bin
electronics for TCSPC.
4.5.4. Multichannel Analyzer
In the older systems for TCSPC with separate components
there was a multichannel analyzer (MCA). When a separate
MCA is present its function is to measure the voltages from
the TAC and sort the values according to counts at each particular
voltage (time). The MCA first performs an analogto-digital
conversion (ADC), which typically takes about
1–10 :s, during which time the MCA is unable to read
another voltage from the TAC. The histogram of the number
of counts at each voltage (time) is displayed on a screen.
This histogram represents the measured intensity decay.
MCAs typically have 2048 to 8192 channels, which can be
subdivided into smaller segments. This allows several
experiments to be stored in the MCA prior to data transfer
and analysis. This ability to store several histograms is particularly
important for measurement of anisotropy decays.
In this case one needs to measure the two polarized intensity
decays, as well as one or two lamp profiles. In the modern
systems the MCA is replaced by the ADC with direct
transfer of the data into computer memory.
4.5.5. Delay Lines
Delay lines, or a way to introduce time delays, are incorporated
into all TCSPC instruments. The need for delay lines
is easily understood by recognizing that there are significant
time delays in all components of the instrument. A
photoelectron pulse may take 20 ns to exit a PMT. Electrical
signals in a cable travel a foot in about 1 ns. It would be
difficult to match all these delays within a couple of
nanoseconds in the start and stop detector channels without
a way to adjust the delays. The need for matching delays
through the components is avoided by the use of calibrated
delay lines. Such delays are part of the NIM bin electronics.
However, lengths of coaxial cable are prone to picking up
RF interference.
Calibrated delay lines are also useful for calibration of
the time axis of the MCA. This is accomplished by providing
the same input signal to the start and stop channels of
the TAC. The preferred approach is to split an electrical signal,
typically from the start detector, and direct this signal
Figure 4.23. Effect of the pulse count rate on a single exponential
decay. The numbers are the number of arriving photons per excitation
pulse. For a rate of 0.01 or 1% the curve overlaps the actual decay (0).
From [4].
to both inputs of the TAC. Since the pulses arrive with a
constant time difference, one observes a single peak in the
MCA. One then switches the time delay in the start or stop
channel by a known amount, and finds the peak shift on the
MCA display. By repeating this process for several delay
times, the TAC and MCA can be calibrated.
4.5.6. Pulse Pile-Up
In TCSPC only one photon from the sample is counted for
every 50 to 100 excitation pulses. What errors occur if the
average number of detected photons is larger? If more than
one photon arrives, how does this affect the measured intensity
decay? These questions cannot be answered directly
because the electronics limit the experiment to detecting the
first arriving photon. Simulations are shown in Figure 4.23
for a single exponential decay with larger numbers of arriving
photons. The numbers in the figure refer to the number
of observed photons per excitation pulse, not the percentage.
The apparent decay time becomes shorter and the
decay becomes non-exponential as the number of arriving
photons increases. The apparent decay is more rapid
because the TAC is stopped by the first arriving photon.
Since emission is a random event, the first photon arrives at
earlier times for a larger number of arriving photons. Methods
to correct for pulse pileup have been proposed, 71–72 but
most laboratories avoid pulse pileup by using a low counting
rate, typically near 1%. However, this is probably being
overcautious, 9 as the measured lifetimes decrease by less
than 1% with count rates up to 10%. The intensity decay is
only changed by a modest amount for a 30% count rate
(Figure 4.23). At present the concerns about pulse pileups