Analyses of RNA–Ligand Interactions by Fluorescence Anisotropy - Biophysical Approaches to Translational Control of Gene Expression - page 175

Fig. 9.3 Schematic of the
L-format spectro fl uorometer.
This most commonly used
type of fluorometer has only
one emission channel. To
measure anisotropy, the
parallel and perpendicular
components of the emission
are both measured through
this channel, with the
emission polarizer rotating
90° between the readings
and emission channels. These devices are generally configured in one of two different
formats. The most commonly used is called the L-format since it has a single
emission channel oriented at a right angle to the excitation path (Fig. 9.3 ) .
The wavelength limits for excitation and emission are set using monochromators,
or in some cases, optical bandpass filters. Eliminating overlap between excitation
and emission wavelengths minimizes scatter from reflected excitation light. Total
fluorescence of a sample can be read directly from this configuration. However, to
measure anisotropy, light from the excitation plane is first passed through a vertically
oriented polarizer prior to hitting the sample cell. The anisotropy parameter is then
calculated from paired measurements of emission passed through: (1) a vertical
polarizer ( I VV ; intensity from vertically polarized excitation measured through a ver-
tical emission polarizer), and (2) a horizontal polarizer ( I VH ). These intensity mea-
surements are proportional to the parallel ( I ) and perpendicular ( I ) components of
anisotropy, respectively (Jameson and Ross 2010 ) . T-format spectro fl uorometers are
also available, so termed because they are configured with a second emission detec-
tor in the same observational plane but reading from the opposite side of the sample
cuvette. These instruments thus permit both parallel and perpendicular emission to
be read simultaneously, each through its own dedicated detection system. In the past,
T-format instruments were favored since the readings were considered less suscep-
tible to fluctuations in signal intensity, but that bias has been largely minimized as
L-format instruments have become more sophisticated (Lakowicz 1999 ) . However,
for rapid kinetics experiments where cycling of emission polarizers is not practical
(time points in low seconds range or faster), the T-format remains essential.
In L-format instruments, the emission monochromator normally has a sensitivity
bias between vertically and horizontally polarized light (Jameson and Ross 2010 ) .