Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 785
Figure 23.50. Possible mechanisms for movement of kinesin on actin
filaments. The lower panel shows the time-dependent motion and the
distance for each step. Reprinted with permission from [91].
Copyright © 2000, American Chemical Society.
iments are performed over a limited range of incident intensities,
concentrations, and dye characteristics. At present it
is only possible to observe single molecules above the background
under certain conditions. For SMD the S/N ratio is
given by 16
S/N
DQ ( σ
A ) ( P0 hν ) T
√ ( DQσPP0T ) C
Ahν
BP0T NDT (23.6)
In this expression D is the instrument detection efficiency,
Q is the quantum yield of the fluorophore, σ is its absorption
cross-section, A is the illuminated area, P 0 /hν is the
number of incident photons per second, and T is the data
collection time. In the denominator C B is the number of
background counts per watt of incident power and N D the
number of dark counts per second. At first glance this
expression appears complex but its meaning becomes clear
upon examination. The term in the numerator represents the
signal from the fluorophore. The three terms in the denominator
are the signals from the fluorophore, autofluorescence
from the sample and instrument, and the number of
dark counts recorded by the detector. The denominator is
raised to one-half power because it represents the fluctuations
or noise in the intensity rather than the intensity itself.
Figure 23.51 shows calculations of the S/N ratio for
assumed parameter values that roughly describe R6G in
water. The surfaces show the dependence of S/N on the
incident power and observed area. The middle panel
assumes there is no photobleaching, and the lower panel
assumes a photobleaching quantum yield of 10 –5 . This sur-
Figure 23.51. Calculations of the signal-to-noise (S/N) ratio in singlemolecule
detection using eq. 23.6; 532 nm, 5.6 x 10 3 W/cm 2 , Q = 0.28,
σ = 4 x 10 –16 cm 2 , D = 0.072, N D = 100 cts/s, C B = 2 x 10 8 counts/W.
T = 0.1 s. For the lower panel the photobleaching quantum yield was
taken as 10 –5 . Revised and reprinted with permission from [16],
Copyright © 2003, American Institute of Physics.