Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 125
Figure 4.33. Decay time measurements using gated detection in the
pulse sampling method. Revised from [10].
measured. In fact, the first time-domain lifetime instruments
used gated detection to sample the intensity decay. 44
Gated detection can be accomplished in two ways. One
method is to turn on or gate the gain of the detector for a
short period during the intensity decay. 145–146 Surprisingly,
this can be accomplished on a timescale adequate for measurement
of nanosecond lifetimes. Alternatively, the detector
can be on during the entire decay, and the electrical pulse
measured with a sampling oscilloscope. 147–148 Such devices
can sample electrical signals with a resolution of tens of
picoseconds.
While such methods seem direct, they have been mostly
abandoned due to difficulties with systematic errors. The
flashlamps and N 2 lasers generate RF signals that can be
picked up by the detection electronics. This difficulty is
avoided in TCSPC because low-amplitude noise pulses are
rejected, and only the higher-amplitude pulses due to primary
photoelectrons are counted. Also, in TCSPC the standard
deviation of each channel can be estimated from Poisson
statistics. There are no methods to directly estimate the
uncertainties with stroboscopic measurements. Hence,
TCSPC became the method of choice due to its high sensitivity
and low degree of systematic errors, when the goal of
the experiments was resolution of complex intensity
decays.
Recent years have witnessed reintroduction of gated
detection methods. 149–150 The time resolution can be good
but not comparable to a laser source and an MCP PMT.
Typical instrument response functions are close to 3 ns
wide. An advantage of this method is that one can detect
many photons per lamp pulse, which can be an advantage in
clinical applications when the decays must be collected rap-
Figure 4.34. Time-resolved instrument using a high-speed oscilloscope
for measuring of time-resolved decays of skin of human skin.
Revised from [150].
idly. Gated detection can be used with low-repetition-rate
nitrogen lasers, which can also be used to pump dye lasers.
An example of directly recorded intensity decay is shown in
Figure 4.34. The instrument was designed for studies of
skin and tissue using this autofluorescence, so the excitation
light was delivered to the sample via an optical fiber. 150
The excitation source was a nitrogen laser excitation source
with a repetition rate near 10 Hz. The emission was detected
with an APD and digitized on a high-speed oscilloscope.
The intensity decay of human skin was collected in about 1
s by averaging of several transients. The IRF is about 5 ns
wide but the intensity decay of skin autofluorescence could
be recorded. The wide IRF shows why TCSPC is used
rather than direct transient recording. At this time it is not
possible to directly record nanosecond decays with the
accuracy needed for most biochemical studies.
4.8.2. Streak Cameras
Streak cameras can provide time resolution of several
ps, 151–160 and some streak cameras have instrument response
functions of 400 fs, considerably faster than TCSPC with an
MCP PMT. Streak cameras operate by dispersing the photoelectrons
across an imaging screen. This can be accom-