Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 121
measurements. A number of known single exponential lifetimes
are summarized in Appendix II.
As described in Chapter 2, it is also important to test
for background fluorescence from blank solutions. Autofluorescence
from the sample can result in errors in the intensity
decay, which results in confusion and/or erroneous conclusions.
There is a tendency to collect the time-resolved
data prior to measuring the steady-state spectra of the sample
and controls. This is particularly dangerous for timeresolved
measurements because the data are often collected
through filters, without examining the emission spectra.
Measurements may be performed on an impure sample,
resulting in a corrupted data set. Since the emission spectra
were not recorded the impurity may go undetected. Subsequent
analysis of the data is then unsatisfactory, and the
source of the problem is not known. In our experience,
more time is wasted by not having the spectra than the time
needed to record blank spectra prior to time-resolved data
collection.
Background correction in TCSPC is straightforward.
Data are collected from the blank sample at the same time
and conditions as used for the sample. As a result, the background
can then be subtracted from the sample data. The
source intensity, repetition rate, and other conditions
must be the same. Inner filter effects in the samples can
attenuate the background signal. If the control samples have
a lower optical density, the measured background can be
an overestimation of the actual background. If the number
of background counts is small, there is no need to consider
the additional Poisson noise in the difference data file.
However, if the background level is large, it is necessary to
consider the increased Poisson noise level in the difference
data file.
4.6.6. Timing Effects of Monochromators
As the time resolution of the instrumentation increases one
needs to consider the effects of the various optical components.
Monochromators can introduce wavelength-dependent
time delays and/or broaden the light pulses. 125–126 This
effect is shown in Figure 4.28, which shows the path length
difference for an optical grating with N facets. Monochromators
are usually designed to illuminate the entire grating.
The maximum time delay is given by 127
t d Nmλ
c
(4.19)
Figure 4.28. Path length difference across a monochromator grating.
N is the total number of facets in the grating and ∆x in the path length
difference between adjacent reflections. From [126].
where N is the number of facets, m is the diffraction order
(typically 1), λ is the wavelength, and c is the speed of light.
A typical grating may have 1200 lines/mm and be 60 mm
across. The maximum time delay at 350 nm is thus 84 ps.
While ps and fs laser pulses are not usually passed through
a monochromator, this can be expected to broaden the
pulse. Alternatively, the apparent intensity decay of a shortlived
fluorophore may be broadened by the use of a monochromator
to isolate the emission. These effects can be
avoided by the use of subtractive dispersion monochromators.
128–129
4.7. MULTI-DETECTOR AND MULTI-
DIMENSIONAL TCSPC
In all the preceding examples TCSPC was performed using
a single detector. However, there are many instances where
it would be useful to collect data simultaneously with more
than one detector. This method is called multidimensional
or multichannel TCSPC. These measurements can be
accomplished in several ways. One approach is to use a
number of completely separate electronics for each channel,
130 which unfortunately results in high complexity and
costs. Another approach is to use a single TAC and MCA
and to multiply the input from several detectors. 131–135
Recall that TCSPC is limited to detection of about 1 photon
per 100 excitation pulses. By using several detectors the
overall rate of photon counting can be increased several
fold. The maximum counting rate does not increase in
direct proportion to the number of detectors because of
interference between simultaneously arriving signals, but