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5.4.12 Bandpass Selection
The spectral bandpass applied to the monochromators will determine the capability of the
fluorimeter to accurately, or not, record the excitation or emission spectrum shape of a
luminescent sample. The bandpass is determined by the slit width used and also by the diffraction grating parameters in the monochromator. A larger (or wider slits) bandpass means
lower spectral resolution whereas a smaller bandpass (i.e., narrower slits) will result in a
higher spectral resolution. At the same time, the light throughput will change with different
bandpass. As a rule of thumb, doubling the bandpass will increase the signal level by a factor of four. For this reason, many have used the spectral bandpass selection of an instrument
as a means to control the light level presented to the analyzing detector, rather than select
an appropriate neutral density filter. Simplistically, it is quite easy to derive this empirically by adjusting the slit width and observing the resultant spectra in terms of its intensity
and spectral shape. However, great care must be taken when using this approach such that
important spectral information is not lost. For example, if the spectra show structure, such
as multiple peaks or small shoulder effects, then the bandpass should be adjusted to an
appropriate size to accurately record and maintain the integrity of these spectral features
The bandpass appropriate for the features of the spectrum that one wishes to resolve
should be used. This is determined by the photo-physics, chemistry, and biology of the
samples, as well as the closeness (in terms of spectral proximity) of the excitation and
emission peak wavelengths and any scattering caused by the sample. Ideally, the widest
bandpass that does not distort the spectral features should be used to ensure the best possible SNR in the measurement. Finally, it is important to note that bandpass and resolution
are not the same quantity. If the bandpass is equal to the slit width multiplied by the reciprocal linear dispersion and the slit becomes so narrow that no improvement in the bandpass
can be observed, then, in effect, the bandpass is equal to the resolution.
5.4.13 Stray Light
Stray light in a monochromator or spectrometer system is all light that reaches the image
plane of the monochromator from anywhere other than from diffraction by the grating
(according to the grating equation), that is, any light that is passed by the monochromator that is outside of the interval λ 0 ± Δλ, where λ 0 is the wavelength setting and Δλ is
the spectral bandpass. Usually, stray light is expressed as a ratio of the total light passing
through the exit slit at a specified wavelength compared to another wavelength. In some
cases, the stray light is specified by the relative amount of light that is passed x nm from a
specified laser line. Other methods of demonstrating stray light performance can be made
using near-end or far-end methods using either narrow line or broadband spectral sources.
What is clear is that manufacturers of optical systems measure and specify the stray light
performance of their products differently. Thus it is often difficult for users to directly compare the performance of one device against another. In the end, the practical demonstration
of the monochromator stray light performance in the specific illumination and spectral
178
5.4.12 Bandpass Selection
The spectral bandpass applied to the monochromators will determine the capability of the
fluorimeter to accurately, or not, record the excitation or emission spectrum shape of a
luminescent sample. The bandpass is determined by the slit width used and also by the diffraction grating parameters in the monochromator. A larger (or wider slits) bandpass means
lower spectral resolution whereas a smaller bandpass (i.e., narrower slits) will result in a
higher spectral resolution. At the same time, the light throughput will change with different
bandpass. As a rule of thumb, doubling the bandpass will increase the signal level by a factor of four. For this reason, many have used the spectral bandpass selection of an instrument
as a means to control the light level presented to the analyzing detector, rather than select
an appropriate neutral density filter. Simplistically, it is quite easy to derive this empirically by adjusting the slit width and observing the resultant spectra in terms of its intensity
and spectral shape. However, great care must be taken when using this approach such that
important spectral information is not lost. For example, if the spectra show structure, such
as multiple peaks or small shoulder effects, then the bandpass should be adjusted to an
appropriate size to accurately record and maintain the integrity of these spectral features
The bandpass appropriate for the features of the spectrum that one wishes to resolve
should be used. This is determined by the photo-physics, chemistry, and biology of the
samples, as well as the closeness (in terms of spectral proximity) of the excitation and
emission peak wavelengths and any scattering caused by the sample. Ideally, the widest
bandpass that does not distort the spectral features should be used to ensure the best possible SNR in the measurement. Finally, it is important to note that bandpass and resolution
are not the same quantity. If the bandpass is equal to the slit width multiplied by the reciprocal linear dispersion and the slit becomes so narrow that no improvement in the bandpass
can be observed, then, in effect, the bandpass is equal to the resolution.
5.4.13 Stray Light
Stray light in a monochromator or spectrometer system is all light that reaches the image
plane of the monochromator from anywhere other than from diffraction by the grating
(according to the grating equation), that is, any light that is passed by the monochromator that is outside of the interval λ 0 ± Δλ, where λ 0 is the wavelength setting and Δλ is
the spectral bandpass. Usually, stray light is expressed as a ratio of the total light passing
through the exit slit at a specified wavelength compared to another wavelength. In some
cases, the stray light is specified by the relative amount of light that is passed x nm from a
specified laser line. Other methods of demonstrating stray light performance can be made
using near-end or far-end methods using either narrow line or broadband spectral sources.
What is clear is that manufacturers of optical systems measure and specify the stray light
performance of their products differently. Thus it is often difficult for users to directly compare the performance of one device against another. In the end, the practical demonstration
of the monochromator stray light performance in the specific illumination and spectral