Chemistry Reference
In-Depth Information
captured by the detector and low noise ampli
cation or counting of the photoelec-
trons all contribute to increasing the signal. Removal of scattered excitation and
spurious
fluorescence by spectral
filtering and by limitation of the detection volume,
as described above, reduce the background. Interactions of the probe with the optical
excitation and the chemical environment also determine the variability and longevity
of the emission. Organic
fluorescent probes, semiconductor nanocrystals (quantum
dots) and auto-
uorescent proteins (GFP and its variants) all exhibit photophysical
processes in the electronically excited state that lead to reversible and irreversible dark
states, termed blinking,
flickering and photobleaching [23]. Studies of the mechan-
isms of these phenomena are outside the scope of this chapter, but their
consideration is an important aspect of designing all single-molecule imaging
experiments. Photoisomerizations, long-lived triplet states, redox reactions, proton-
ation and breaking of covalent bonds contribute to these
fluorescence changes for
different probes. Various schemes for minimizing the photobleaching rate are
addition of enzymatic oxygen scavenging systems, such as glucose oxidase, catalase
and glucose [24, 25], reducing agents, such as
-mercaptoethanol or dithiothreitol
and triplet state quenchers, such as Trolox [26]. These agents also strongly modulate
the dynamics of
fluorescence blinking and
flickering, making the choice of anti-fade
reagents and even their supplier [27] important considerations.
At a given frame rate and chemical environment of the
fluorescent probe, the only
other parameter that modulates the signal magnitude is the power of the illumina-
tion, which directly controls the emission rate. In a typical
fluorescence microscopy
experiment, the
fluorophores are far from optical saturation (the condition where a
substantial proportion (e.g.
b
20%) of the
fluorophores are excited at any instant), so
increasing the excitation intensity proportionally increases the emission
ux.
The quantum yields for photobleaching and for some of the
flickering mechanisms
are also approximately constant, causing the photobleaching rate to increase
proportionally with incident light intensity as well. An input laser power is used
that achieves a compromise between the magnitude of the
fluorescence signal,
kinetics of
flickering and duration of recording before photobleaching that ends the
experiment.
The emission intensity per camera frame from a single rhodamine
uorophore
under constant TIRF excitation and with effective deoxygenation (Figure 3.4A) is
fairly constant until it decreases suddenly to a much lower background value (at 50
s in this experiment). The
10% frame-to-frame variation of the signal is due to
non-steady excitation rate due to laser noise and mechanical vibrations that shift
fringes in the TIRF illumination,
fluorophore blinking, and the statistics of
counting the photons. The sudden decrease represents the irreversible photo-
bleaching of the
uorophore to a non-
uorescent state, presumably with altered
chemical structure. Approximately 10
5
total photons were collected and registered
by the camera before the photobleaching event. Given the likely collection
ef
ciency of the optics (10%), quantum yield of the camera (0.9) and
uorescence
quantum yield of the rhodamine itself (0.5), this number of detected photons
represents
10
6
electronic excitation/de-excitation cycles of the
uorophore. The
constant emission rate, occupying a relatively narrow distribution and the sudden
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