Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 123
Figure 4.32. Intensity decays and recovered lifetimes of four labeled nucleotides used for DNA sequencing. Revised from [142].
the 16 anodes is sent to a multiplexing TCSPC board that
measures the time delays between the start and stop pulses.
The system keeps track of the anode that sent the signal, so
the counts can be loaded into different regions of memory,
which correspond to different wavelengths.
Sixteen decay curves were measured simultaneously
for the fluorescein–rhodamine 6G mixture (Figure 4.30).
The different decay times are seen from the slopes, which
change with emission wavelength. In this case the lifetime
of fluorescein was probably decreased by resonance energy
transfer to R6G. It is easy to imagine such measurements
being extended to time-dependent processes such as resonance
energy transfer or solvent relaxation.
4.7.1. Multidimensional TCSPC and
DNA Sequencing
An important application of multichannel detection will be
DNA sequencing. Because of the large number of bases the
sequencing must be performed as inexpensively as possible.
Sequencing was initially done using four lanes, one for each
base. It would be an advantage to identify the four bases in
a single lane. Because of the limited range of available
wavelengths and overlap of emission spectra, there is ongoing
research to perform sequencing using the decay times of
the labeled bases. 137–142 Using four detectors the intensity
decays are at four different excitation, and/or emission
wavelengths could be measured in a single lane of a
sequencing gel. If the decay times of the labeled bases were
different, the decay times could be used to identify the
bases.
The possibility of DNA sequencing based on lifetime is
shown in Figure 4.32. TCSPC data are shown for four
probes attached to DNA. These dyes are typical of those
used for sequencing. They can be excited with two NIR
laser diodes, which can easily occur at different times by
turning the laser diodes on and off. The four probes show
different lifetimes, and can be identified based on the lifetimes
recovered from a reasonable number of counts. Each
of the decays in Figure 4.32 were collected in about 7.5 s.