Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 119
Figure 4.25. Transit time spread for an MCP PMT (R3809U) and a
miniature PMT (R7400 series in a H7422 module). Revised from [90].
anywhere on the photocathode are detected. In contrast, the
active area of an avalanche photodiode is usually less than
1 mm 2 , and less than 10 :m x 10 :m for a high-speed APD.
It is therefore difficult to focus the fluorescence onto the
APD, so the sensitivity is too low for most measurements.
Another disadvantage is the relatively long tail following
each pulse, the extent of which depends on wavelength. The
presence of a wavelength-dependent tail can create problems
in data analysis since the instrument response function
will depend on wavelength. Methods have been developed
to actively quench the tail. Values of the full width at half
Figure 4.26. Compact PMTs and modules containing the PMTs. The
diameter of the PMT is 16 mm. The width of the PMT modules is 25
nm. Courtesy of William Cieslik, Hamamatsu Photonics Systems.
maxima have been reported from 20 to 400 ps, and APDs
have been successfully used in TCSPC. 98–105
APDs are now routinely used for TCSPC, especially in
applications where the emission can be tightly focused,
such as single-molecule detection (SMD) and fluorescencecorrelation
spectroscopy (FCS). TCSPC measurements can
be performed at the same time as SMD and FCS experiments.
APDs have high quantum efficiencies at real wavelengths,
and are the detector of choice for these applications.
4.6.5. Color Effects in Detectors
When performing lifetime measurements, one generally
compares the response of a fluorescent sample with that of
a zero decay time scattering sample. Because of the Stokes
shift of the sample, the wavelengths are different when
measuring the sample and the impulse response function.
The timing characteristics of a PMT can depend on wavelength.
Color effects were significant with the older-style
tubes, such as the linear-focused 56 DVVP and the sidewindow
tubes. 106–111 The time response of PMTs can also
depend on which region of the photocathode is illuminated.
Color effects are almost nonexistent in MCP PMTs 80 and do
not appear to be a problem with the compact PMTs.
Methods are available to correct for such color effects.
There are two general approaches, one of which is to use a
standard with a very short lifetime. 112–113 The standard
should emit at the wavelength used to measure the sample.
Because of the short decay time one assumes that the measured
response is the instrument response function. Because