FLUORESCENCE SPECTROSCOPY: Principle, Instrumentation and Application
Fluorescence spectroscopy is an emission phenomenon where an energy transition from a higher to a lower state is accompanied by radiation. Only molecules in their excited forms are able to emit fluorescence; thus, they have to be brought into a state of higher energy prior to the emission phenomenon.
In these diagrams, energy transitions are indicated by vertical lines. Not all transitions are possible; allowed transitions are defined by the selection rules of quantum mechanics. A molecule in its electronic and vibrational ground state (S1V0) can absorb photons matching the energy difference of its various discrete states. The required photon energy has to be higher than that required to reach the vibrational ground state of the first electronic excited state (S1V0).
The excess energy is absorbed as vibrational energy (v>0), and quickly dissipated as heat by collision with solvent molecules. The molecule thus returns to the vibrational ground state (S1V0). These relaxation processes are non-radiating transitions from one energetic state to another with lower energy, and are called internal conversion (IC). From the lowest level of the first electronic excited state, the molecule returns to the ground state (S0) either by emitting light (fluorescence) or by a non-radiative transition. Upon radiative
transition, the molecule can end up in any of the vibrational states of the electronic ground state (as per quantum mechanical rules).
If the vibrational levels of the ground state overlap with those of the electronic excited state, the molecule will not emit fluorescence, but rather revert to the ground state by non-radiative internal conversion. This is the most common way for excitation energy to be dissipated and is why fluorescent molecules are rather rare. Most molecules are flexible and thus have very high vibrational levels in the ground state. Indeed, most
fluorescent molecules possess fairly rigid aromatic rings or ring systems. The fluorescent group in a molecule is called a fluorophore.
Since radiative energy is lost in fluorescence as compared to the absorption, the fluorescent light is always at a longer wavelength than the exciting light (Stokes shift). The emitted radiation appears as band spectrum, because there are many closely related wavelength values dependent on the vibrational and rotational energy levels attained. The fluorescence spectrum of a molecule is independent of the wavelength of the exciting radiation and has a mirror image relationship with the absorption spectrum. The probability of the transition from the electronic excited to the ground state is proportional to the intensity of the emitted light.
An associated phenomenon in this context is phosphorescence which arises from a transition from a triplet state (T1) to the electronic (singlet) ground state (S0). The molecule gets into the triplet state from an electronic excited singlet state by a process called intersystem crossing (ISC). The transition from singlet to triplet is quantum-mechanically not allowed and thus only happens with low probability in certain molecules where the electronic structure is favourable. Such molecules usually contain heavy atoms. The
rate constants for phosphorescence are much longer and phosphorescence thus happens with a long delay and persists even when the exciting energy is no longer applied.
The fluorescence properties of a molecule are determined by properties of the molecule itself (internal factors), as well as the environment of the protein (external factors). The fluorescence intensity emitted by a molecule is dependent on the lifetime of the excited state. The transition from the excited to the ground state can be treated like a decay process of first order, i.e. the number of molecules in the excited state decreases exponentially with time. In analogy to kinetics, the exponential coefficient Kr is called rate constant and is the reciprocal of the lifetime: τ = kr-1. The lifetime is the time it takes to reduce the number of fluorescence emitting molecules to N0/e, and is proportional to λ3.
The effective lifetime τ of excited molecules, however, differs from the fluorescence lifetime τr since other, non-radiative processes also affect the number of molecules in the excited state τ is dependent on all processes that cause relaxation: fluorescence emission, internal conversion, quenching, fluorescence resonance energy transfer, reactions of the excited state and intersystem crossing.
The ratio of photons emitted and photons absorbed by a fluorophore is called quantum yield Φ (equation 12.3). It equals the ratio of the rate constant for fluorescence emission kr and the sum of the rate constants for all six processes mentioned above.
The quantum yield is a dimensionless quantity, and, most importantly, the only absolute measure of fluorescence of a molecule. Measuring the quantum yield is a difficult process and requires comparison with a fluorophore of known quantum yield. In biochemical applications, this measurement is rarely done. Most commonly, the fluorescence emissions of two or more related samples are compared and their relative
Fluorescence spectroscopy works most accurately at very low concentrations of emitting fluorophores. UV/Vis spectroscopy, in contrast, is least accurate at such low concentrations. One major factor adding to the high sensitivity of fluorescence applications is the spectral selectivity. Due to the Stokes shift, the wavelength of the emitted light is different from that of the exciting light. Another feature makes use of the fact that fluorescence is emitted in all directions. By placing the detector perpendicular to the excitation pathway, the background of the incident beam is reduced.
The schematics of a typical spectrofluorimeter are shown in Fig. Two monochromators are used, one for tuning the wavelength of the exciting beam and a second one for analysis of the fluorescence emission. Due to the emitted light always having a lower energy than the exciting light, the wavelength of the excitation monochromator is set at a lower wavelength than the emission monochromator. The better fluorescence
spectrometers in laboratories have a photon-counting detector yielding very high sensitivity. Temperature control is required for accurate work as the emission intensity of a fluorophore is dependent on the temperature of the solution.
Two geometries are possible for the measurement, with the 90° arrangement most commonly used. Pre- and post-filter effects can arise owing to absorption of light prior to reaching the fluorophore and the reduction of emitted radiation. These phenomena are also called inner filter effects and are more evident in solutions with high concentrations.
As a rough guide, the absorption of a solution to be used for fluorescence experiments should be less than 0.05. The use of microcuvettes containing less material can also be useful. Alternatively, the front-face illumination geometry can be used which obviates the inner filter effect. Also, while the 90° geometry requires cuvettes with two neighbouring faces being clear (usually, fluorescence cuvettes have four clear faces), the front-face illumination technique requires only one clear face, as excitation and emission occur at the same face. However, front-face illumination is less sensitive than the 90° illumination.
There are many and highly varied applications for fluorescence despite the fact that relatively few compounds exhibit the phenomenon. The effects of pH, solvent composition and the polarisation of fluorescence may all contribute to structural elucidation.
Measurement of fluorescence lifetimes can be used to assess rotation correlation coefficients and thus particle sizes. Non-fluorescent compounds are often labelled with fluorescent probes to enable monitoring of molecular events. This is termed extrinsic fluorescence as distinct from intrinsic fluorescence where the native compound exhibits the property.
Some fluorescent dyes are sensitive to the presence of metal ions and can thus be used to track changes of these ions in in vitro samples, as well as whole cells.
Since fluorescence spectrometers have two monochromators, one for tuning the excitation wavelength and one for analysing the emission wavelength of the fluorophore, one can measure two types of spectra: excitation and emission spectra.
For fluorescence excitation spectrum measurement, one sets the emission monochromator at a fixed wavelength (λem) and scans a range of excitation wavelengths which are then recorded as ordinate (x-coordinate) of the excitation spectrum; the fluorescence emission at λem is plotted as abscissa.
Measurement of emission spectra is achieved by setting a fixed excitation wavelength (λexc) and scanning a wavelength range with the emission monochromator. To yield a spectrum, the emission wavelength λem is recorded as ordinate and the emission intensity at λem is plotted as abscissa.