Principles of Fluorescence Spectroscopy

36 INSTRUMENTATION FOR FLUORESCENCE SPECTROSCOPY
Figure 2.14. Grating efficiency for a 1800 line/mm halographic grating,
optimized for the UV. The bold line is the average, and the other
lines are for differently (S and P) polarized light, defined in Figure
2.39. Redrawn from [11].
2.3.2. Polarization Characteristics of
Monochromators
For a grating monochromator the transmission efficiency
depends upon the polarization of the light. This is illustrated
in Figure 2.14 for a concave grating. For this reason, the
observed fluorescence intensities can be dependent upon
the polarization displayed by the fluorescence emission.
The emission spectrum can be shifted in wavelength and
altered in shape, depending upon the polarization conditions
chosen to record the emission spectrum. For example,
consider an emission spectrum recorded with the grating
shown in Figure 2.14, and through a polarizer oriented vertically
(||) or horizontally (z). Assume that the dotted transmission
curve corresponds to vertically polarized light. The
spectrum recorded through the vertically oriented polarizer
would appear shifted to shorter wavelengths relative to that
recorded with the polarizer in the horizontal position. This
is because the transmission efficiency for vertically polarized
light is higher at shorter wavelengths. This spectral
shift would be observed irrespective of whether its emission
was polarized or not polarized.
The polarization properties of concave gratings can
have a dramatic effect on the appearance of emission spectra.
This is shown in Figure 2.15 for a probe bound to DNA.
The emission spectrum shows a dramatic decrease near 630
nm when the emission polarizer is in the horizontal position.
This drop is not seen when the emission polarizer is in
a vertical orientation. In addition to this unusual dip in the
spectrum, the emission maximum appears to be different
for each polarizer orientation. These effects are due to the
polarization properties of this particular grating, which dis-
Figure 2.15. Emission spectra of [Ru(bpy) 2 (dppz)] 2+ bound to DNA.
This probe is described in chapter 20. Excitation at 460 nm. Except for
intensity, the same spectral distributions was observed for vertically as
horizontally polarized excitation. From [12].
plays a minimum in efficiency at 630 nm for horizontally
polarized light. Such effects are due to the emission monochromator,
and are independent of the polarization of the
excitation beam.
The polarization characteristics of monochromators
have important consequences in the measurement of fluorescence
anisotropy. Such measurements must be corrected
for the varying efficiencies of each optical component. This
correction is expressed as the G factor (Section 10.4). However,
the extreme properties of the concave gratings (Figure
2.14) can cause difficulties in the measurement of fluorescence
polarization. For example, assume that the polarization
is to be measured at an excitation wavelength of 450
nm. The excitation intensities will be nearly equal with the
excitation polarizers in each orientation, which makes it
easier to compare the relative emission intensities. If the
emission is unpolarized the relative intensities with parallel
(||) and perpendicular (z) excitation intensities will be nearly
equal. However, suppose the excitation is at 340 nm, in
which case the intensities of the polarized excitation will be
very different. In this case it is more difficult to accurately
measure the relative emission intensities because of the
larger difference in the excitation intensities. Measurement
of the G factor is generally performed using horizontally
polarized light, and the intensity of this component would
be low.
2.3.3. Stray Light in Monochromators
The stray light level of the monochromator is a critical
parameter for florescence measurements. Stray light is