Principles of Fluorescence Spectroscopy

776 SINGLE-MOLECULE DETECTION
Figure 23.33. Polarization properties of a single fluorophore with colinear (β = 0) absorption (A) and emission (E) dipoles.
= 0E, even though β = 90E. This surprising result can be
understood as the result of the cos 2 θ dependence on
absorption. Since the fluorophore is observed without an
emission polarizer the maximum signal is seen when the
excitation rate is the highest, at θ = 0. A different result is
obtained if the fluorophore is excited with unpolarized light
and observed through an emission polarizer (Figure 23.34,
bottom panel). Now the cos 2 θ dependence is shifted 90E
due to θ = 90E. The same dependence on cos 2 θ would be
observed for any polarization of the incident light because
the emission intensity through the polarizer is determined
by the position of the emission dipole. The polarization of
the emission is –1.0 because the emission is polarized along
the y-axis.
In a typical ensemble anisotropy measurement one
usually rotates the emission polarizer to perform the measurement
(Chapter 10). However, in Figure 23.33 and 23.34
(top panels) the excitation polarization was rotated. This is
frequently done in polarized fluorescence microscopy
because of the depolarizing effects of the microscope objec-
tive. If linearly polarized light is passed through the aperture
and focused on the sample or molecule, the field is partially
depolarized. 61 This effect is a result of the large
numerical aperture and the different angles of incidence for
light, passing through the center or the outer region of the
objective. Light can be brought to the sample without this
effect if the light is not passed through the objective, but
instead directly to the sample (Figure 23.35). The larger
aperture of the objective does not affect the emission measurements
because the objective simply collects the emission
which depends only on the orientation of the incident polarization
and the fluorophore. The orientation of the incident
polarization defines the parallel component (I || ). This component
can be rotated to be at any angle in the focal plane.
Of course, the single molecule will only be excited if its
absorption dipole has a component along the incident electric
field. The emission is collected by an objective and
passed through a filter to remove the scattered light.
In single-molecule experiments every photon is valuable.
The molecule may display blinking and will be