Chemistry Reference
In-Depth Information
ALEX essentially replaces the multiple excitation methods. There is much to be
gained, with very little to lose. The only issue is whether the alternation period of the
lasers interferes with observations of dynamics which is on that time scale. However,
ALEX has been demonstrated with alternation periods in the microsecond, nano-
second, and millisecond range, so the alternation period can be chosen not to
interfere with the dynamics in question [39].
9.3.3.4 Pulsed Laser Excitation
With a pulsed laser, the laser energy is produced in the form of a train of energy
packets (or pulses) rather than a continuous stream of energy. For short pulses
(
1 ns) the pulsemay be used as a trigger to identify when the
uorescence excitation
occurred. Pulsed lasers are used in three ways: (i) to allow measurement of
uores-
cence lifetime [40]; (ii) to allow two-photon excitation [41]; and (iii) to allow for
stimulated emission depletion (STED) [42]. If these issues are not important to the
question at hand, then continuous wave lasers are suf
cient.
<
9.3.4
What Detection Format should be used?
The
fluorescence obtained from the sample, after passing through the pinhole or
simply passing through the dichroic mirror, must be separated into various
detection channels. For a two-color system, the
uorescence must be separated
by a second dichroic mirror into bands speci
cforthetwocolors.Additional
bandpass
filters are used to exclude stray laser re
ection and other background that
make it through the imperfect dichroic mirrors. Splitting the donor and acceptor
emission by polarization as well requires four detectors [43, 44]. In applications
using FRET, monitoring
fluorescence polarization or anisotropy is desirable since
it allows acquisition of information on the orientation factor
2
(described above).
Additional colors require more dichroic mirrors and bandpass
k
filters to de
ne
detection channels [45
47].
The type of detector used generally depends on the optical isolation method. For
confocal, single point methods, single photon counting avalanche photodiode (APD)
detectors are typically used. A
fluorescence photon impinging on the detector will
trigger an electronic pulse at the output of the detector. The electronic pulses may be
counted in
fixed time intervals to provide a time trace of the number of photon
counting as a function of time. Even better, each electronic pulse may be timed with
high accuracy (depending on the counting/timing electronics,
-
10 ns typically, and
down to 4 ps with a dedicated circuit), providing a list of all detected photons with
their arrival times. If a pulsed laser excitation is used, the time difference between the
arrival of each photon and the laser pulse could be obtained with an accuracy of
several picoseconds (time-correlated single photon counting, TCSPC) constituting a
measurement of the
fluorescence lifetime. The main drawback of APD detection is
that only one point can be monitored at a time.
For TIR applications with a wide
eld of view, theAPDdetectors are not appropriate,
since no spatial information is available. Instead, an intensi
ed CCD camera is used,
