9 Measurement of Fluorescence

A spectrophotometer is used to measure UV-visible absorption. A spectrofluorimeter is an instrument used to measure fluorescence intensity as a function of wavelength.

Instrument Design

To excite fluorescent molecules in a sample, a bright and broad-spectrum UV-visible light source such as a mercury or xenon arc lamp is commonly used. The excitation wavelength is selected by a grating-based monochromator and optics direct the excitation light through the sample in a cuvette. Whereas only the front and back faces of cuvettes for absorption measurements must be transparent, the front, back and at least one side face of fluorescence cuvettes are transparent.

The optics for collecting fluorescence emission are generally perpendicular to the excitation light. This geometry minimizes the stray excitation light that can interfere with the fluorescence measurement. An important point here is that fluorescence is isotropic—emitted in all directions. A second grating-based monochromator selects the wavelength of fluorescence emission and its intensity is measured by a photodetector, which is most commonly a photomultiplier tube (PMT) because of its high sensitivity to light.

Fluorescence microscopes are also common. The design of these instruments is conceptually similar to the design of spectrofluorimeters, but are usually filter-based rather than monochromator-based, equipped with cameras instead of PMTs, and utilize a greater variety of excitation sources.

Quantitative Analysis

Fluorescence emission intensity can be measured for a single band of excitation wavelengths paired with a single band of emission wavelengths. It can also be measured as one of two types of spectra. For an emission spectrum, the emission wavelength is scanned across a range longer than the excitation wavelength, which is kept constant. For an excitation spectrum, the excitation wavelength is scanned across a range shorter than the emission wavelength, which is kept constant.

Fluorescence intensity is related to the concentration of fluorophore through modification of the Beer-Lambert Law. The intensity will be proportional to the amount of light absorbed by the fluorophore, scaled by its quantum yield (since not all absorbed photons lead to fluorescence). Two other modifications are also needed: (i) a term for the excitation intensity, since more incident photons lead to more photons absorbed and therefore more florescence photons emitted; and (ii) a term to correct for the fraction of total fluorescence collected and detected. The final result is Eqn. 9.1:

(Eqn. 9.1)        [latex]F(λ_{em}) = P_{0}(λ_{exc})ε(λ_{exc})bcΦK(λ_{exc},λ_{em})[/latex]

In Eqn. 9.1, F is the measured fluorescence intensity, ε is the molar absorption coefficient, b is the path length, c is the fluorophore concentration, Φ is the fluorescence quantum yield, P_o is the intensity of excitation light, and K is the correction factor (determined by calibration) for a specific instrument and measurement settings. Many of these parameters have values that depend on the specific excitation and emission wavelengths (λ_exc, λ_em).

The largely dark background for measurements (light-tight sample compartment, excitation and emission at different wavelengths) and the Po term in Eqn. 9.1 make fluorescence far more sensitive than absorbance, which has a bright background and is independent of Po. If more signal is needed for a fluorescence measurement, the excitation intensity can be increased. With laser excitation sources and amplified detectors (e.g. APDs, EM-CCDs, PMTs), even single molecules can be detected by fluorescence.

Connections

• Spectrofluorometer designs use light sources (Ch. 3), wavelength selectors (Ch. 4), and photodetectors (Ch. 5) learned about in previous chapters.
• The monochromators used to measure fluorescence spectra are the same type used to measure absorbance spectra (Ch. 7).
• The polychromators and array detectors used for measuring absorbance spectra (Ch. 7) are also useful for measuring fluorescence emission spectra.
• As with absorbance measurements (Ch. 7), the transmission of the cuvette material must be matched the wavelengths for fluorescence excitation and emission.
• The Beer-Lambert Law (Ch. 6) reappears as part of the quantitative description of fluorescence intensity.
• The excitation spectrum of a fluorophore is also a measurement of how much light is absorbed across different wavelengths. It thus tends to be a copy of the fluorophore’s absorption spectrum (Ch. 7).
• The emission spectrum of a fluorophore tends to be a Stokes-shifted mirror image of its absorbance spectrum because of the similar probabilities of S₀v₀→S₁v_m and S₁v₀→S₀v_m transitions (Ch. 8).
• Fluorescence is usually measured from an angle or position that avoids transmitted or reflected excitation light. Measurements of Raman scattering (Ch. 15) often do the same.

Post-Reading Questions

1. Given that the absorption of excitation light is required for fluorescence emission, predict whether or not a molecule’s fluorescence excitation spectrum will look similar to its absorption spectrum in shape and spectral position.
2. Explain why a spectrofluorometer requires two separate monochromators when a spectrophotometer only requires one.
3. Explain why an L-shaped optical path is used for fluorescence measurements when a straight-line path is used for absorbance measurements.
4. Assuming all instrument settings are kept constant, sketch a generic graph of fluorescence intensity versus fluorophore concentration. What is the trend?
5. List some of the parameters that can be optimized to maximize fluorescence signal.

Topic Learning Objectives

The chapter is a primer for the following learning objectives, which will be covered in lecture and/or with additional assigned reading:

• Draw diagrams of a filter-based fluorometer and a grating-based spectrofluorometer.
• Select light sources, wavelength selectors, and photodetectors to design an instrument capable of a desired type of fluorescence measurement.
• Define the advantage of a double monochromator for excitation light.
• Explain how excitation and emission spectra are obtained.
• Explain why fluorescence is measured in arbitrary units.
• Explain why fluorescence methods are more sensitive than absorption spectroscopy.
• Explain why spectrofluorimeters need to correct for the non-uniform spectral output or non-uniform spectral response of different components.