Optical Spectroscopy Instrumentation Design
157
5.3.1 The Ideal Fluorescence Spectrometer System
Obtaining meaningful and quantitative fluorescence spectral intensity data from a given
sample requires an understanding of instrumentation, design, operation, and calibration
requirements. The ideal fluorescence spectrometer is one that allows the true spectra of the
sample to be measured; in other words, it should possess the following qualities:
Have high sensitivity and ideally no noise signals
•
Measure excitation and emission spectra, that is, the photon flux emitted at each wavelength
•
Be nonresponsive to interfering signals such as Raman and Rayleigh scattering, stray
•
light, fluorescence from solvents, and so forth
Measure the “true sample spectra” that are fully corrected for the nonuniform spectral
•
output of light sources and the wavelength-dependent efficiencies of monochromators
and detectors.
Thus, from an instrument design perspective, the ideal fluorimeter should possess the following attributes:
A light source that yields constant photon output at all wavelengths
•
Monochromators that pass photons at all wavelengths with equal efficiency
•
Monochromators that are not sensitive to polarization effects, and
•
Detectors that must detect photons of all wavelengths with equal efficiency
•
Unfortunately, there are so many variables in the optical path of any fluorimeter, or any
other spectrometer system, that such systems simply do not exist and it is for this reason
that careful consideration to instrumental system design, calibration, and correction methodologies is essential.
Table 5.4. Most common fluorescence scanning techniques
Excitation scan
Select emission wavelength and bandpass, λ em , Δλ em , and hold fixed.
Select excitation bandpass, Δλ exc .
Scan excitation monochromator across excitation spectral region of
interest.
Emission scan
Select excitation wavelength and band, λ exc , Δλ exc , and hold fixed.
Select emission bandpass, Δλ em .
Scan emission monochromator across emission spectral region of interest.
Synchronous scan
Select excitation and emission bandpasses, Δλ exc and Δλ em .
Select wavelength offset between excitation and emission wavelengths, Δλ.
Scan excitation and emission monochromators in synchronization with the
offset Δλ.
Excitation–emission
matrices
Select excitation and emission bandpasses, Δλ exc and Δλ em .
Scan emission spectra (over a spectral range) as a function of excitation
wavelength.
157
5.3.1 The Ideal Fluorescence Spectrometer System
Obtaining meaningful and quantitative fluorescence spectral intensity data from a given
sample requires an understanding of instrumentation, design, operation, and calibration
requirements. The ideal fluorescence spectrometer is one that allows the true spectra of the
sample to be measured; in other words, it should possess the following qualities:
Have high sensitivity and ideally no noise signals
•
Measure excitation and emission spectra, that is, the photon flux emitted at each wavelength
•
Be nonresponsive to interfering signals such as Raman and Rayleigh scattering, stray
•
light, fluorescence from solvents, and so forth
Measure the “true sample spectra” that are fully corrected for the nonuniform spectral
•
output of light sources and the wavelength-dependent efficiencies of monochromators
and detectors.
Thus, from an instrument design perspective, the ideal fluorimeter should possess the following attributes:
A light source that yields constant photon output at all wavelengths
•
Monochromators that pass photons at all wavelengths with equal efficiency
•
Monochromators that are not sensitive to polarization effects, and
•
Detectors that must detect photons of all wavelengths with equal efficiency
•
Unfortunately, there are so many variables in the optical path of any fluorimeter, or any
other spectrometer system, that such systems simply do not exist and it is for this reason
that careful consideration to instrumental system design, calibration, and correction methodologies is essential.
Table 5.4. Most common fluorescence scanning techniques
Excitation scan
Select emission wavelength and bandpass, λ em , Δλ em , and hold fixed.
Select excitation bandpass, Δλ exc .
Scan excitation monochromator across excitation spectral region of
interest.
Emission scan
Select excitation wavelength and band, λ exc , Δλ exc , and hold fixed.
Select emission bandpass, Δλ em .
Scan emission monochromator across emission spectral region of interest.
Synchronous scan
Select excitation and emission bandpasses, Δλ exc and Δλ em .
Select wavelength offset between excitation and emission wavelengths, Δλ.
Scan excitation and emission monochromators in synchronization with the
offset Δλ.
Excitation–emission
matrices
Select excitation and emission bandpasses, Δλ exc and Δλ em .
Scan emission spectra (over a spectral range) as a function of excitation
wavelength.