Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 39
Figure 2.18. Transmission spectra of colored-glass filters. Top: in
order of increasing wavelength FCG 414, 419, and 423. Bottom: FCG
409, 413, 421, and 425. From [13].
An important consideration in the use of bandpass filters
is the possibility of emission from the filter itself. Many
filters are luminescent when illuminated with UV light,
which can be scattered from the sample. For this reason it is
usually preferable to locate the filter further away from the
sample, rather than directly against the sample. Glass filters
of the type shown in Figures 2.17 and 2.18 are highly versatile,
effective, and inexpensive, and a wide selection is
needed in any fluorescence laboratory. Excitation and emission
filters can be used in all experiments, even those using
monochromators, to reduce the possibility of undesired
wavelengths corrupting the data.
2.4.2. Thin-Film Filters
A wide variety of colored-glass filters are available, but the
transmission curves are not customized for any given application.
During the past ten years there have been significant
advances in the design of thin-film optical filters. 14 Almost
any desired transmission curve can be obtained. Filters are
now being designed for specific applications, rather than
choosing the colored-glass filter that best suits an application.
Long-pass filters are an example of this type filter
(Figure 2.19). These filters have a sharp cut on the transmission
above 325 nm or 488 nm, which are wavelengths available
from a helium–cadmium or argon ion laser, respectively.
The transmission above the cut-on wavelength is close to
100% to provide maximum sensitivity.
Figure 2.19. Long-pass filters designed to reject light from a heliumcadmium
laser at 325 nm or an argon ion laser at 488 nm. Revised
from [15].
Thin-film filters are also available to specifically transmit
or reject laser lines. Laser light can contain additional
wavelengths in addition to the main laser line. This emission
is referred to as the plasma emission, which typically
occurs over a range of wavelengths and is not strongly
directional. The light can be made more monochromatic by
passing the laser beam through a laser line filter, such as the
one shown for a helium–neon laser at 633 nm (Figure 2.20).
Alternatively, it may be necessary to eliminate scattered
light at the laser wavelength. This can be accomplished with
a notch filter which transmits all wavelengths except the
laser wavelengths. These filters are sometimes called
Raman notch filters because of their use in Raman spectroscopy.
Emission can usually be selected using a long-pass filter.
However, there may be additional emission at longer
wavelengths, such as the acceptor emission in an energy
transfer experiment. In these cases it is useful to have a filter
that transmits a selected range of wavelengths or autofluorescence
from the sample. Figure 2.21 shows examples
of bandpass filters that transmit from 460 to 490 nm or from
610 to 700 nm. The width of transmission can be made narrower
or wider. Such filters are often referred to as interference
filters.